CN111057154B

CN111057154B - Preparation and application of immunogen based on camel source Fc fragment

Info

Publication number: CN111057154B
Application number: CN201911333414.5A
Authority: CN
Inventors: 苏志鹏; 孟巾果; 姚尧; 周耿
Original assignee: Nanjing Rongjiekang Biotechnology Co ltd
Current assignee: Nanjing Rongjiekang Biotechnology Co ltd
Priority date: 2019-12-23
Filing date: 2019-12-23
Publication date: 2023-06-06
Anticipated expiration: 2039-12-23
Also published as: CN111057154A

Abstract

The invention relates to the technical field of biotechnology or immunology, and relates to preparation and application of an immunogen based on a camel source Fc fragment, wherein the immunogen is formed by fusing a target protein and the camel source Fc fragment, the camel source Fc fragment is shown in any one of SEQ ID NO. 17-22, and coding gene sequences are shown in SEQ ID NO. 23-28 respectively. The invention discloses a method for immunizing camel source animals by fusion construction of a target gene and a camel source immunoglobulin Fc fragment coding sequence, and taking the target gene and camel source immunoglobulin Fc fragment coding sequence as an immunogen after expression and purification. The camel source Fc fusion protein ensures the expression quantity and stability of the antigen, ensures the immunogenicity of the antigen, and provides great convenience for developing single-domain antibodies.

Description

Preparation and application of immunogen based on camel source Fc fragment

Technical Field

The invention relates to the technical field of biotechnology or immunology, and relates to preparation and application of an immunogen based on a camel source Fc fragment.

Background

Antibodies (abs), i.e., immunoglobulins (igs), are a class of glycoproteins in blood and tissue fluids, produced by plasma cells generated by proliferation and differentiation of B cells after antigen stimulation, and are important effector molecules that mediate fluid immunity, mainly in body fluids such as serum, and specifically bind to the corresponding antigen. In addition to antibodies mediating specific humoral immune responses as important effector molecules, antibodies play an important role in the control of diseases, in particular infectious diseases, behring creates a serum therapy and thus will gain medical and physiological nobel. Thereafter, antibodies artificially prepared by polyclonal, monoclonal, and genetically engineered antibody techniques are gradually applied to the clinic. Only 2011 has the drug-resistant drug lead the global drug market at the sales of dollars of 480 billions, accounting for 34.4% of the whole biopharmaceutical market. By 2017, 76 therapeutic antibody drugs approved by the us FDA and eu EMA are mainly used for the treatment of tumors, autoimmune diseases, and the like.

The antibody is used as a molecule capable of specifically recognizing a certain target protein (antigen), and is obtained by taking the protein as an immunogen, immunizing animals and then adopting immunological, cell biological and genetic engineering means. The molecular weight of an antibody molecule is typically around 150kDa, with two identical heavy chains and two identical light chains constituting the multi-stage structure of the antibody molecule. The antibody molecule can be changed into Fab fragments and Fc fragments under the hydrolysis of papain or pepsin in vitro, the Fab fragments have antigen recognition capability, and the Fc fragments are free. In vivo, fc fragments on intact IgG molecules can also mediate antibody-dependent cytotoxicity, thereby killing target cells.

In the subsequent researches, researchers find that, after subcloning the Fc fragment of IgG and fusing and expressing the target gene, the expression quantity of the target gene, the solubility after purification and the stability during protein storage can be improved before unfused, and because the two Fc fragments have cysteine, disulfide bonds can be generated on the Fc fragment, when the Fc fusion protein is expressed in vitro in a recombination mode, the dimer form of the fusion protein is generally obtained, and the monomer or dimer form of the Fc fusion protein in vitro in a recombination mode can be changed by mutating or not mutating the cysteine at the corresponding position of the Fc fragment or changing the charges of other amino acids, so that a very convenient tool is provided for the researches of researchers on the target protein. The advantages of Fc fusion proteins are of fundamental importance, but they have the disadvantage that Fc fusion proteins present little trouble to researchers when used as immunogens to immunize animals. The size of the monomer Fc fragment is 25kDa, and the immunogenicity of the monomer Fc fragment is very strong for a allogeneic animal, so that after the Fc fusion protein is used as the immunogen for immunizing the animal, a researcher can obtain a large amount of antibodies aiming at the Fc protein when carrying out antibody screening and identification, but the antibodies aiming at the target protein are almost the same, and the antibodies are difficult to obtain. The reason for the selection of Fc fusion expression for these immunogens is often: 1. expression of the protein of interest alone is difficult, yields are low, storage is unstable, 2. The protein of interest is too poorly autoimmune and immunization of animals alone is insufficient to elicit its humoral immune response. Therefore, in the process of developing antibodies, finding a better production method for the immunogen with higher difficulty in single preparation is a problem to be solved.

In the process of developing antibodies, the most commonly used host for immunization of the antigen is mice, the use cost of the mice is lower, the period of the development of the antibodies can be controlled to be short by combining with phage display technology, and for the immunization of the mice, the dosage of the immunogens is very small due to the small weight of animals, so the development of the antibodies from the mice has low requirements on the total preparation amount of the immunogens and can be realized by injecting the form of inclusion bodies of prokaryotic expression products. In recent decades, single domain antibodies of camel origin have been increasingly touted in antibody drug development, but camel animals naturally require more amount of immunogen due to the large weight, and injection of the inclusion body form of immunogen to camel animals is not a judicious choice because camel animals use more costly and antibodies to conformational epitopes are more easily produced in camel animals, so the quality and quantity of immunogen are a very important factor for the immunization of camel animals. Therefore, researchers have again directed their eyes toward Fc fusion proteins.

Disclosure of Invention

In order to overcome the defects, the invention aims to provide preparation and application of an immunogen based on a camel source Fc fragment, and aims to solve the problems that a target protein cannot be expressed or the immunogenicity is insufficient, and select the target protein to be fused with the camel source Fc and immunize a camel, but the immunized camel can generate a great number of antibodies aiming at human source Fc instead of protein A.

The Fc fusion protein has the defects that important factors for restricting the acquisition of preferred antibodies are overcome, camel source Fc fusion target protein is selected, and then the fusion protein is used for immunizing the homologous animals, so that the expression quantity and stability of the antigen are ensured, the immunogenicity of the antigen is ensured, and antibodies aiming at the Fc protein are not generated in the process of antibody screening. Provides great convenience for the development of single-domain antibodies. The single domain antibody is used as a monoclonal antibody, has higher affinity generally and better stability than the traditional monoclonal antibody, and has the advantages of the traditional monoclonal antibody. Therefore, the invention utilizes the advantages to obtain targets which are difficult to obtain for some antigens, and finally obtains the antibodies for target proteins by injecting camel animals.

In order to solve the technical problems, the invention adopts the following technical scheme: an immunogen based on camel source Fc fragment is formed by fusing target protein and camel source Fc fragment, wherein the camel source Fc fragment is shown in any one of SEQ ID NO. 17-22.

Specifically, the coding sequences of the camel source Fc fragments are shown in any one of SEQ ID NO. 23-28 respectively. The camelid Fc fragment herein includes camelid Fc and a linker sequence.

The invention also provides an expression vector of immunogen based on camel source Fc fragment, which comprises a 5 '-Multiple Cloning Site (MCS) -connecting region (linker) coding sequence, camel source Fc coding sequence, and a sequence of 6 histidine (6 XHis) coding sequences-3'; the connecting region (linker) coding sequence-camel source Fc coding sequence is shown in any one of SEQ ID NO. 23-28. The camelid Fc coding sequence herein is an Fc sequence only, excluding the linker sequence.

Preferably, the coding sequence of the protein of interest is inserted into the multiple cloning site of a camel source Fc protein expression vector.

It is another object of the present invention to provide a method for constructing an expression vector for an immunogen based on a camelid Fc fragment comprising the steps of:

1) Obtaining a camel source Fc protein coding sequence;

2) Constructing a camel source Fc protein expression vector;

3) Inserting a coding sequence of a target protein into a multiple cloning site of a camel source Fc protein expression vector;

the step 1) is specifically as follows: isolating PBMCs in the peripheral blood of the inner Mongolian alashan llama; extracting total RNA, and carrying out reverse transcription on the total RNA into cDNA by a reverse transcription kit; carrying out PCR reaction on the cDNA subjected to reverse transcription through gene specific primers SEQ ID NO. 1-SEQ ID NO. 10 to obtain 5 PCR products;

the step 2) is specifically as follows: carrying out PCR amplification on the 5 PCR products obtained in the step 1) through primers SEQ ID NO. 11 and 16, SEQ ID NO. 12 and 16, SEQ ID NO. 13 and 16, SEQ ID NO. 14 and 16, SEQ ID NO. 15 and 16 respectively to obtain 6 DNA sequences shown in SEQ ID NO. 23-28, and then carrying out enzyme digestion on the 6 DNA sequences through proper enzyme digestion sites; the cleavage product with the structure [ 5 '-Multiple Cloning Site (MCS) -linker (linker) -camel Fc-6 histidine (6 XHis) -3' ] was cloned into a vector.

It is a further object of the present invention to provide a method for the preparation of an immunogen based on a camelid Fc fragment comprising the steps of:

1) Constructing an expression vector of an immunogen based on a camel source Fc fragment by adopting the method;

2) The eukaryotic cells are transfected by the expression vector to realize the expression of fusion proteins consisting of target proteins and camel source Fc proteins.

The invention also discloses application of the camel source Fc fragment in preparation of an immunogen, wherein the immunogen is formed by fusing a target protein and the camel source Fc fragment, and the camel source Fc fragment is shown in any one of SEQ ID NO. 17-22. The immunogen is used for immunizing camels.

Use of a camel source Fc fragment in the construction of an immunogenic expression vector comprising a 5 '-Multiple Cloning Site (MCS) -linker (linker) coding sequence-a camel source Fc coding sequence-a sequence of 6 histidine (6 xhis) coding sequence-3'; the connecting region (linker) coding sequence-camel source Fc coding sequence is shown in any one of SEQ ID NO. 23-28.

The invention also provides application of the immunogen based on camel source Fc fragment in preparing a single domain antibody aiming at target protein.

The camel source Fc sequence is from an inner Mongolia alashan camel; all the sequences of SEQ ID NOS.17-22 described above may be replaced with a sequence having "at least 80% homology" with the sequence or with a sequence having only one or a few amino acid substitutions; preferably "at least 85% homology", more preferably "at least 90% homology", more preferably "at least 95% homology", and most preferably "at least 98% homology".

The coding sequence of the camel source Fc sequence is shown in any one of SEQ ID NOs 23-28. The camel source Fc sequence has a structure of linker-CH2-CH3.

It is another object of the present invention to provide a nucleotide molecule encoding the aforementioned camel source Fc fragment, the nucleotide sequence of which is shown in any one of SEQ ID NOs 23 to 28. It is a further object of the present invention to provide a host cell which can express the aforementioned camelid Fc protein or camelid Fc fusion protein, or which comprises the aforementioned expression vector.

Compared with the prior art, the invention has the beneficial effects that: the camel source Fc fusion protein ensures the expression quantity and stability of the antigen and the immunogenicity of the antigen, and antibodies aiming at the camel source Fc protein can not be generated in the process of antibody screening. Provides great convenience for the development of single-domain antibodies.

The invention discloses a method for fusing a target gene (such as human IL-13, or other genes which can not or hardly realize eukaryotic expression) and a camel immunoglobulin Fc fragment coding sequence, and expressing and purifying the target gene to be used as an immunogen for immunizing camel animals, wherein the camel immunoglobulin Fc fragment has an amino acid sequence shown as SEQ ID NO. 17-22, the coding gene sequences of the camel immunoglobulin Fc fragment are shown as SEQ ID NO. 23-28 respectively, and the target gene fragment can be expressed efficiently and applied to camel animal immunization through the camel immunoglobulin Fc fragment gene sequence, an expression vector and a host cell disclosed by the invention.

Drawings

FIGS. 1-2 are schematic representations of expression vectors into which camelid Fc protein sequences may be fused;

FIG. 3 is an SDS-PAGE identification of the expressed products before and after fusion of the target protein with camel source Fc protein, M: the standard molecular weight of the protein,

lanes 1-8 are all purified or cell lysis supernatant assays for non-Fc fusion proteins (i.e., post-expression products of the negative control plasmid for IL-13); wherein 1, 2,5,6 are derived from SDS-PAGE results after purification of supernatant obtained after transfection of suspension 293F cells with the negative control plasmid of example 4, and 5,6 are derived from SD of supernatant obtained after lysis of cell pellet obtained after collection of supernatant obtained after transfection of suspension 293F cells with the negative control plasmid of example 4S-PAGE analysis results; wherein 3, 4,7,8 are from examples 5,3, 4 are negative control plasmid transfection of ExpiCHO-S in example 5 ^TM SDS-PAGE results of supernatant after cell purification, 7,8 are negative control plasmid transfection of ExpiCHO-S in example 5 ^TM The supernatant after the cells is collected and the residual cell sediment is cracked, and then the result of SDS-PAGE analysis is carried out on the supernatant; 9. 10 are all SDS-PAGE analysis of IL-13-camelid Fc fusion proteins purified by the method of example 6, wherein 9 is derived from the supernatant of example 5 (which was transfected with ExpiCHO-S from the plasmid extracted in example 3 ^TM Cells obtained), 10 from the supernatant of example 4 (obtained by transfecting suspension 293F cells with the plasmid extracted in example 3);

FIG. 4 is a specific result of biopanning a constructed single domain antibody library against IL13-CbFc, wherein P/N = the number of monoclonal bacteria grown after infection of TG1 bacteria with phage removed positive Kong Xi in biopanning/the number of monoclonal bacteria grown after infection of TG1 bacteria with phage removed negative Kong Xi, which parameter increases gradually after enrichment has occurred; I/E = total phage added to positive wells per round of biopanning/total phage removed from positive Kong Xi per round of biopanning, which parameter gradually approaches 1 after enrichment has occurred.

Detailed Description

The present invention is described in further detail below with reference to examples to enable those skilled in the art to practice the same by referring to the description.

The invention adopts pcDNA3.4 purchased from Thermo Fisher company as a template to construct a carrier RJK-V4-CH containing camel source Fc coding sequences, constructs camel source Fc fusion proteins aiming at a plurality of target proteins on the basis of the carrier, transfects 293F cells with the recombinant carriers, expresses and purifies the camel source Fc fusion proteins, immunizes an inner Mongolian alpine with the fusion proteins with SDS-PAGE analysis purity of more than 90 percent, extracts the alpine peripheral blood lymphocytes after 7 times of immunization, and establishes a single domain antibody library aiming at single fusion protein. Then, a single-domain antibody aiming at the target protein is screened by utilizing a phage display technology, and finally, the phage library aiming at the target protein is obviously enriched, and the nanobody aiming at the target protein is obtained.

And (3) expressing the single-domain antibody obtained by screening in escherichia coli, purifying by using nickel ion coupled agarose, and analyzing the purified nano antibody.

The invention will be further illustrated with reference to specific examples.

Example 1: obtaining a camel source Fc protein coding sequence:

(1) Randomly selecting 3-4 inner Mongolian alashan Bactrian camels, and collecting 5mL of peripheral blood through jugular vein;

(2) Separating PBMCs from the collected peripheral blood by lymphocyte separation liquid;

(3) Extracting total RNA from the obtained PBMCs, and then carrying out reverse transcription on the total RNA into cDNA by a reverse transcription kit;

(4) Carrying out PCR reaction on the cDNA subjected to reverse transcription through a gene specific primer SEQ ID NO. 1-SEQ ID NO. 10;

(5) The PCR products of five pairs of primers of SEQ ID NO. 1-SEQ ID NO.2, SEQ ID NO. 3-SEQ ID NO. 4, SEQ ID NO. 5-SEQ ID NO. 6, SEQ ID NO. 7-SEQ ID NO. 8 and SEQ ID NO. 9-SEQ ID NO. 10 are respectively subjected to agarose gel electrophoresis analysis. Wherein SEQ ID NO.1 and SEQ ID NO.2 are named as FC-IgG2c-FP and FC-IgG2c-RP respectively and are used for preliminary amplification of the FC-IgG2c fragment; SEQ ID NO.3, 4 are designated as FC-IgG1a-FP, FC-IgG1a-RP, respectively, for preliminary amplification of the FC-IgG1a fragment; SEQ ID No.5, 6 are designated as FC-IgG2a-FP, FC-IgG2a-RP, respectively, for preliminary amplification of the FC-IgG2a fragment; SEQ ID NO.7, 8 are named FC-IgG gamma 1a-FP and FC-IgG gamma 1a-RP, respectively, and are used for preliminary amplification of the FC-IgG gamma 1a fragment; SEQ ID NO.9, 10 are designated as FC-IgG1b-FP, FC-IgG1b-RP, respectively, for preliminary amplification of the FC-IgG1b fragment;

example 2: construction of camel source Fc protein expression vector:

(1) The vectors involved in the patent are all modified on the basis of pcDNA3.4, and can be purchased at a Thermo Fisher website;

(2) Selecting unique restriction enzyme cutting sites XbaI and AgeI on pcDNA3.4;

(3) Introducing a polyclonal site at the 5' end of the camel source Fc protein coding sequence, so as to facilitate the transformation of a later-stage vector and the subcloning of a fusion protein sequence;

(4) Introducing a coding sequence of a connecting region (Linker) behind the multiple cloning site in the step (3), wherein the protein sequence of the Linker is GGGGSGGGGSGGGGS;

(5) Fusing the 3' -end of the camel source Fc protein coding sequence with a coding sequence of 6 histidines;

(6) Primers SEQ ID NO. 11 and 16 (PCR products obtained by SEQ ID NO. 1-2 are used as templates, FC-IgG2c coding sequence and CfR coding sequence are obtained by amplification), SEQ ID NO. 12 and 16 (FC-IgG 1a coding sequence is obtained by amplification by taking PCR products obtained by SEQ ID NO. 3-4 as templates), SEQ ID NO. 13 and 16 (FC-IgG 2a coding sequence is obtained by amplification by taking PCR products obtained by SEQ ID NO. 5-6 as templates), SEQ ID NO. 14 and 16 (FC-IgG gamma 1a coding sequence is obtained by amplification by taking PCR products obtained by SEQ ID NO. 7-8 as templates), and SEQ ID NO. 15 and 16 (FC-IgG 1b coding sequence is obtained by amplification by taking PCR products obtained by SEQ ID NO. 9-10), PCR amplification is performed on 5 PCR products in example 1, and bands are analyzed by electrophoresis; wherein, the PCR products obtained by SEQ ID NO. 1-2 in example 1 are amplified by primers SEQ ID NO. 11 and 16 to obtain two products with different sequences, including FC-IgG2c coding sequence (SEQ ID NO. 23) and CfR coding sequence (SEQ ID NO. 28). Wherein SEQ ID NO. 11-16 is named as FP-2c, FP-1a, FP-2a, FP-gamma 1a, FP-1b and RP in sequence.

(7) The purified DNA product is connected to a T vector for sequencing, the sequencing result shows that 6 sequences shown as SEQ ID NO. 23-28 are shared, and then 6 DNA sequences are digested through digestion sites XbaI and AgeI; simultaneously, carrying out XbaI and AgeI double enzyme digestion on pcDNA3.4;

(8) Cloning the digestion product with the structure of [ 5 '-Multiple Cloning Site (MCS) -connecting region (linker) -camel source Fc coding sequence-6 histidine (6 XHis) coding sequence-3' ] in the step (7) into a vector pcDNA3.4 in a connecting mode;

(9) Finally, a recombinant vector RJK-V4-CH1, RJK-V4-CH2, RJK-V4-CH3, RJK-V4-CH4, RJK-V4-CH5, RJK-V4-CH6 (corresponding to FC-IgG2c, FC-IgG1a, FC-IgG2a, FC-IgG gamma 1a, FC-IgG1b, and CfR respectively) was constructed, and the relative positions of the multiple cloning sites, the linking region, and the Fc fragment of the vector are shown in FIGS. 1-2. Wherein CfR is an abbreviation for Camelus ferus Fc region.

Table 1 primers, camel source Fc fragment amino acid sequence and coding sequence

Example 3: construction of a fusion expression vector of a target protein and camel source Fc (RJK-V4-CH 1 is taken as an example):

the purpose of this example was to add the expression sequence of the target protein to the constructed RJK-V4-CH1 vector to allow fusion expression of the target protein and camel Fc.

(1) Selecting an expression sequence of a target protein, such as IL-13 (NCBI, NM_ 002188.2), and selecting two enzyme cutting sites of XbaI and EcoRI on a recombinant vector polyclonal site and a protein coding sequence;

(2) Specific primer PCR was amplified using the coding sequence of IL-13 protein purchased (from the university of Harvard medical college, cat# HsCD00346103, linked https:// plasma. Med. Harvard. Edu/PLASMID/GetCloneDetail. Docronid=346103 & species=) as template; an upstream primer: 5'-gcTCTAGAGGATCGAACCCTTATGCATCCGCTCCTC-3' (SEQ ID NO. 29), downstream primer: 5'-gagaGAATTCCACCACCACCGTTGAACTGTCCCTC-3' (SEQ ID NO. 30).

(3) Respectively carrying out restriction enzyme digestion on the amplified IL-13 coding sequence and the RJK-V4-CH1 vector;

(4) Connecting the products after enzyme digestion by T4 ligase;

(5) Converting the ligation product into escherichia coli DH5 alpha, amplifying, extracting plasmids, and carrying out enzyme digestion identification;

(6) Sequencing the recombinant vector with the correct identified band size;

(7) And (5) extracting a large amount of recombinant vector with correct sequencing for standby.

Example 4: expression of IL-13-camelid Fc fusion proteins in suspension 293F cells

Recombinant fusion protein expression experimental procedure (500 ml shake flask for example):

(1) 3 days before transfection at 2.5X10 ⁵ The cells/ml were passaged and expanded 293F cells and the calculated desired cell volume was transferred to 500ml shake flasks with fresh pre-warmed 120ml (final volume) OPM-293 CD05 Medium. To achieve a cell concentration of about 2X 10 ⁶ -3×10 ⁶ Living cells/mL;

(2) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 2X 10 before transfection ⁶ -3×10 ⁶ Living cells/mL.

(3) Dilution of cells to 1X 10 with pre-warmed OPM-293 CD05 Medium ⁶ Each living cell/mL. The calculated cell volume was transferred to a 500ml shake flask containing fresh pre-warmed 100ml (final volume) of medium;

(4) Diluting PEI (1 mg/ml) reagent with 4ml Opti-MEM culture medium, and stirring or blowing to mix uniformly; the large amount of extracted plasmid DNA obtained in example 3 was diluted with 4ml of Opt-MEM medium, mixed back and forth, and filtered with a 0.22um filter head. Incubate at room temperature for 5min. In addition, a plasmid containing a target protein coding gene and containing no coding sequence of camel source Fc fragment (namely IL-13 coding sequence is directly cloned into pcDNA3.4 vector to form IL13 recombinant vector fused with non-camel source Fc) is used as a negative control plasmid, and the negative control plasmid is also transfected into the suspension 293F cell; the 3' -end of the IL13 coding sequence of the non-camel Fc fusion IL13 recombinant vector is introduced with a 6 XHis tag during construction, so that the subsequent purification is facilitated, and SDS-PAGE detection is carried out after the non-camel Fc fusion IL13 passes through Ni-NTA resin affinity chromatography (figure 3);

(5) Diluted PEI reagent was added to the diluted DNA and mixed upside down. Incubating the PEI/plasmid DNA complex for 15-20 minutes at room temperature, then lightly adding the PEI/plasmid DNA complex into the prepared cell suspension, and lightly agitating the shake flask during the adding process;

(6) The cells were incubated at 37℃with 5% CO ₂ Shake culturing at 120 rpm;

(7) 5ml OPM-CHO PFF05 feed was added 24h and 72h after transfection;

(8) Supernatants were collected about 7 days after transfection (cell viability below 70%).

Example 5: IL-13-camel Fc fusion protein in ExpiCHO-S ^TM Expression in cells

3 days before transfection at 2.5X10 ⁵ ExpiCHO-S cell passage and expansion culture/ml ^TM The cells, calculated desired cell volume, were transferred to an ExpiCHO containing fresh pre-warmed 120ml (final volume) ^TM 500ml shake flask of expression medium; to achieve a cell concentration of about 4X 10 ⁶ -6×10 ⁶ Living cells/mL;

(1) One day prior to transfection, expiCHO-S was used ^TM Cell dilution concentration to 3.5X10 ⁶ Living cells/mL, allowing the cells to incubate overnight;

(2) The day of transfection, cell density and percent viable cells were determined. The cell density should reach about 7X 10 before transfection ⁶ -10×10 ⁶ Living cells/mL;

(3) Fresh ExpiCHO preheated to 37 ℃ ^TM Dilution of cells to 6X 10 in expression Medium ⁶ Each living cell/mL. The calculated desired cell volume was transferred to an ExpiCHO containing fresh pre-warmed 100ml (final volume) ^TM 500ml shake flask of expression medium;

(4) Gently mixing the mixture with the mixture of the Expifectamine in a reverse manner ^TM CHO reagent with 3.7ml OptiPRO ^TM Dilution of Expifectamine in Medium ^TM CHO testOscillating or mixing uniformly;

(5) With refrigerated 4ml OptiPRO ^TM Diluting a large amount of extracted plasmid DNA obtained in the example 3 by a culture medium, and mixing the plasmid DNA in a stirring way; in addition, a plasmid containing a target protein coding gene and no coding sequence of camel source Fc fragment (namely IL-13 coding sequence is directly cloned into pcDNA3.4 vector to form IL13 recombinant vector fused with non-camel source Fc) is used as a negative control plasmid, and the negative control plasmid is also transfected with the ExpiCHO-S ^TM A cell; the 3' -end of the IL13 coding sequence of the non-camel Fc fusion IL13 recombinant vector is introduced with a 6 XHis tag during construction, so that the subsequent purification is facilitated, and SDS-PAGE detection is carried out after the non-camel Fc fusion IL13 passes through Ni-NTA resin affinity chromatography (figure 3);

(6) Incubating the ExpiFectamine CHO/plasmid DNA complex for 1-5 minutes at room temperature, then gently adding to the prepared cell suspension, gently agitating the shake flask during the addition;

(7) The cells were incubated at 37℃with 8% CO ₂ Shake culturing in humidified air;

(8) 600ul of Expiectamine was added on day 1 (18-22 hours post transfection) ^TM CHO enhancement and 24ml of expi CHO feed.

(9) Supernatants were collected about 8 days after transfection (cell viability below 70%).

Example 6: purification of camel source Fc recombinant proteins

(1) The protein expression supernatants obtained in examples 4 and 5 were filtered with a disposable filter head of 0.45 μm to remove insoluble impurities;

(2) Purifying the filtrate by using a Protein purifier to carry out affinity chromatography, and purifying by using agarose filler coupled with Protein A by utilizing the binding capacity of camel source Fc and Protein A;

(3) Passing the filtrate through a Protein A pre-packed column at a flow rate of 1 mL/min, wherein the target Protein in the filtrate is combined with the packing;

(4) Washing the column-bound impurity proteins with a low-salt and high-salt buffer;

(5) Eluting the target protein bound on the column with a low pH buffer;

(6) Rapidly adding the eluent into Tris-HCl solution with pH of 9.0 for neutralization;

(7) After the neutralized protein solution was dialyzed, SDS-PAGE analysis (FIG. 3) was performed to confirm that the protein purity was 95% or higher and the concentration was 0.5mg/mL or higher, and the protein solution was stored at a low temperature for later use. From FIG. 3, it can be seen that human IL-13 cannot be expressed in eukaryotic cells without camelid Fc fragment; IL-13-camel Fc fusion proteins can be successfully expressed in eukaryotic cells.

Example 7: construction of Single-Domain antibody library against IL-13-camel Fc fusion protein

(1) Mixing 1mg of camel source Fc recombinant protein prepared in example 6 with Freund's adjuvant in equal volume, immunizing an inner Mongolian Alshan Bactrian camel once a week for 7 times in total, and stimulating B cells to express specific nanobodies during the immunization process; (2) After the immunization is finished, 100ml of camel peripheral blood lymphocytes are extracted and total RNA is extracted; (3) synthesizing cDNA and amplifying VHH by using nested PCR; (4) Cutting 20ug pMECS phage display vector and 10ug VHH by restriction enzymes PstI and Not I, and connecting the two fragments; (5) Transforming the connection product into electrotransformation competent cell TG1, constructing IL-13-camel source Fc fusion protein phage display library and measuring the library capacity, wherein the library capacity is about 2 multiplied by 10 ⁸ The method comprises the steps of carrying out a first treatment on the surface of the Meanwhile, the correct insertion rate of the target fragment in the built library is detected by colony PCR, and FIG. 1 shows colony PCR results, and 24 clones are randomly selected as colony PCR, so that the insertion rate reaches 95.8%.

Example 8: single domain antibody screening process for IL-13-camel Fc fusion protein

(1) 200uL of recombinant TG1 cells are taken and cultured in a 2 XTY culture medium, 40uL of helper phage VCSM13 is added during the period to infect the TG1 cells, and the culture is carried out overnight to amplify phage, the phage is precipitated by PEG/NaCl the next day, and the amplified phage is collected by centrifugation; (2) NaHCO diluted at 100mM pH 8.3 ₃ The neutral avidin 500ug is coupled on an ELISA plate, and is placed at 4 ℃ overnight, and a negative control hole is formed; (3) The following day 100uL of biotin-labeled IL-13-camel source Fc fusion protein was added, incubated at room temperature for 2 hours, and 100uLPBS was added to the negative control wells; (4) After 2 hours, 100ul of 3% BSA was added at room temperatureClosing for 2h; (5) After blocking was completed, 100ul of amplified phage library (approximately 2X 10 ¹¹ Individual phage particles), 1h at room temperature; (6) After 1 hour of action, wash 5 times with PBS+0.05% Tween-20 to wash away unbound phage; (7) Phage specifically binding to IL-13-camel Fc fusion protein was dissociated with trypsin at a final concentration of 25mg/ml, and E.coli TG1 cells in the logarithmic growth phase were infected, cultured at 37℃for 1h, phage were generated and collected for the next round of screening, and the same screening procedure was repeated for 3 rounds to gradually effect enrichment, and the results are shown in FIG. 4.

Example 9: screening of specific positive clones for IL-13 protein by phage display

(1) Screening IL-13-camel source Fc fusion protein for 3 rounds according to the single domain antibody screening method, wherein after screening, phage enrichment factors aiming at the IL-13-camel source Fc fusion protein reach more than 10, selecting 100 single colonies from positive clones obtained by screening, respectively inoculating the single colonies into 96 deep well plates containing 100ug/mL ampicillin TB culture medium, setting blank control, culturing at 37 ℃ to a logarithmic phase, adding IPTG with a final concentration of 1mM, and culturing at 28 ℃ overnight; (2) obtaining a crude extract antibody by using a permeation swelling method; dilution of neutravidin protein to 100mM NaHCO pH 8.3 ₃ Coating 100ug of neutravidin protein in an ELISA plate at 4 ℃ overnight, and adding 100ug of V5-Biotin protein into the ELISA plate the next day; (3) Transferring 100uL of the crude antibody extract obtained in the steps to an ELISA plate added with antigen, and incubating for 1h at room temperature; (4) Washing off unbound antibody with PBST, adding 100ul of Mouse anti-HA tag anti-body (Mouse anti-HA antibody, thermo Fisher) diluted 1:2000, and incubating for 1h at room temperature; (5) Unbound antibody was washed off with PBST, 100ul of Anti-Rabbit HRP conjugate (goat Anti-rabbit horseradish peroxidase labeled antibody, available from Thermo Fisher) diluted 1:20000 was added and incubated for 1h at room temperature; (6) Washing off unbound antibody with PBST, adding horseradish peroxidase chromogenic solution, reacting at 28deg.C for 15min, and reading the absorption value at 405 wavelength on an enzyme-labeled instrument; (7) When the OD value of the sample hole is more than 5 times that of the control hole, judging that the sample hole is a positive cloning hole; (8) The positive clone well was transformed into a strain containing 100uIn LB medium of g/ul ampicillin to extract plasmids and to sequence.

Example 10: purification and expression of specific single domain antibody of IL-13 protein in host bacterium escherichia coli

(1) Plasmids (pMECS-VHH) of the different clones obtained by the sequencing analysis were respectively electrotransformed into E.coli WK6, and spread on LB+amp+glucose-containing culture plates, and cultured overnight at 37 ℃; (2) Selecting single colony to inoculate in 5ml LB culture solution containing shore penicillin, shaking culture at 37 deg.C overnight; (3) Inoculating 1mL of overnight culture strain into 330mL of TB culture solution, shake culturing at 37deg.C to OD _600nm When the value reaches 0.6-0.9, 1M IPTG is added, and shaking culture is carried out at 28 ℃ for overnight; (4) Centrifuging, collecting escherichia coli, and obtaining an antibody crude extract by using a osmotic bursting method; (5) Purifying the antibody by nickel column affinity chromatography, and purifying the single domain antibody.

Although embodiments of the present invention have been disclosed above, it is not limited to the use of the description and embodiments, it is well suited to various fields of use for the invention, and further modifications may be readily apparent to those skilled in the art, and accordingly, the invention is not limited to the particular details without departing from the general concepts defined in the claims and the equivalents thereof.

Sequence listing

<110> Nanjing Rongjiekang biotechnology Co., ltd

<120> preparation and use of camel Fc fragment-based immunogen

<130> GY-03-2019-004

<141> 2019-12-20

<160> 30

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

gcgcaccacc ccgaagaccc 20

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

ttacccgaag actgggtgat g 21

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gcctccacca aggccccatc gg 22

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

tttacccgaa gactgggtga tgg 23

<210> 5

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

gaacccaaga taccccaacc ac 22

<210> 6

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

tttacccgaa gactgggtga tgg 23

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

gcccctgagc tcccgggag 19

<210> 8

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

tttacccgaa gactgggtga tg 22

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gtctccacca aggccccatc ggtc 24

<210> 10

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tttacccgaa gactgggtga tgg 23

<210> 11

<211> 71

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gagagaattc ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttctgcgca 60

ccaccccgaa g 71

<210> 12

<211> 71

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

gagagaattc ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttctgcctc 60

caccaaggcc c 71

<210> 13

<211> 71

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

gagagaattc ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttctgaacc 60

caagataccc c 71

<210> 14

<211> 72

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

gagagaattc ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttctgcccc 60

tgagctcccg gg 72

<210> 15

<211> 72

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

gagagaattc ggtggtggtg gttctggtgg tggtggttct ggtggtggtg gttctgtctc 60

caccaaggcc cc 72

<210> 16

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

tctcaccggt ttaatggtga tggtgatgat gtttacccga agactg 46

<210> 17

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 17

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

1 5 10 15

His His Pro Glu Asp Pro Ser Ser Gln Cys Pro Lys Cys Pro Ala Pro

20 25 30

Glu Leu Leu Gly Gly Pro Thr Val Phe Ile Phe Pro Pro Lys Pro Lys

35 40 45

Asp Val Leu Ser Ile Ser Gly Arg Pro Glu Val Thr Cys Val Val Val

50 55 60

Asp Val Gly Lys Glu Asp Pro Glu Val Asn Phe Asn Trp Tyr Ile Asp

65 70 75 80

Gly Val Glu Val Arg Thr Ala Asn Thr Lys Pro Lys Glu Glu Gln Phe

85 90 95

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Ile Gln His Gln Asp

100 105 110

Trp Leu Thr Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Ala Leu

115 120 125

Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala Lys Gly Gln Thr Arg

130 135 140

Glu Pro Gln Val Tyr Thr Leu Ala Pro His Arg Glu Glu Leu Ala Lys

145 150 155 160

Asp Thr Val Ser Val Thr Cys Leu Val Lys Gly Phe Tyr Pro Pro Asp

165 170 175

Ile Asn Val Glu Trp Gln Arg Asn Arg Gln Val Glu Ser Glu Gly Ala

180 185 190

Tyr Ala Thr Thr Leu Pro Gln Leu Asp Asn Asp Gly Thr Tyr Phe Leu

195 200 205

Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp Gln Arg Gly Glu Thr

210 215 220

Phe Thr Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

225 230 235 240

Lys Ser Ile Thr Gln Ser Ser Gly Lys

245

<210> 18

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 18

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

1 5 10 15

His His Pro Glu Asp Pro Ser Ser Gln Cys Pro Lys Cys Pro Ala Pro

20 25 30

Glu Leu Pro Gly Gly Pro Ser Val Phe Val Phe Pro Pro Lys Pro Lys

35 40 45

Asp Val Leu Ser Ile Ser Gly Arg Pro Glu Val Thr Cys Val Val Val

50 55 60

Asp Val Gly Lys Glu Asp Pro Glu Val Asn Phe Asn Trp Tyr Ile Asp

65 70 75 80

Gly Val Glu Val Arg Thr Ala Asn Thr Lys Pro Lys Glu Glu Gln Phe

85 90 95

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Ile Gln His Gln Asp

100 105 110

Trp Leu Thr Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Ala Leu

115 120 125

Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala Lys Gly Gln Thr Arg

130 135 140

Glu Pro Gln Val Tyr Thr Leu Ala Pro His Arg Glu Glu Leu Ala Lys

145 150 155 160

Asp Thr Val Ser Val Thr Cys Leu Val Lys Gly Phe Tyr Pro Pro Asp

165 170 175

Ile Asn Val Glu Trp Gln Arg Asn Arg Gln Pro Glu Ser Glu Gly Ala

180 185 190

Tyr Ala Thr Thr Leu Pro Gln Leu Asp Asn Asp Gly Thr Tyr Phe Leu

195 200 205

Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp Gln Arg Gly Glu Thr

210 215 220

Phe Thr Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

225 230 235 240

Lys Ser Ile Thr Gln Ser Ser Gly Lys

245

<210> 19

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 19

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

1 5 10 15

His His Pro Glu Asp Pro Ser Ser Gln Cys Pro Lys Cys Pro Ala Pro

20 25 30

Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro Lys

35 40 45

Asp Val Leu Ser Ile Ser Gly Arg Pro Glu Val Thr Cys Val Val Val

50 55 60

Asp Val Gly Gln Glu Asp Pro Glu Val Ser Phe Asn Trp Tyr Ile Asp

65 70 75 80

Gly Val Glu Val Arg Thr Ala Asn Thr Lys Pro Lys Glu Glu Gln Phe

85 90 95

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Ile Gln His Gln Asp

100 105 110

Trp Leu Thr Gly Lys Glu Leu Lys Cys Lys Val Asn Asn Lys Ala Leu

115 120 125

Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala Lys Gly Gln Thr Arg

130 135 140

Glu Pro Gln Val Tyr Thr Leu Ala Pro His Arg Glu Glu Leu Ala Lys

145 150 155 160

Asp Thr Val Ser Ile Thr Cys Leu Val Lys Gly Phe Tyr Pro Ala Asp

165 170 175

Ile Asn Val Glu Trp Gln Arg Asn Gly Arg Pro Glu Ser Glu Gly Ala

180 185 190

Tyr Ala Thr Thr Leu Pro Gln Leu Asp Asn Asp Gly Thr Tyr Phe Leu

195 200 205

Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp Gln Arg Gly Glu Thr

210 215 220

Phe Thr Cys Val Val Met His Glu Ala Leu His Asn His Ser Thr Gln

225 230 235 240

Lys Ser Ile Thr Gln Ser Ser Gly Lys

245

<210> 20

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 20

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

1 5 10 15

His His Pro Glu Asp Pro Ser Ser Gln Cys Pro Lys Cys Pro Ala Pro

20 25 30

Glu Leu Pro Gly Gly Pro Ser Val Phe Val Phe Pro Pro Lys Pro Lys

35 40 45

Asp Val Leu Ser Ile Ser Gly Arg Pro Glu Val Thr Cys Val Val Val

50 55 60

Asp Val Gly Gln Glu Asp Pro Glu Val Asn Phe Asn Trp Tyr Ile Asp

65 70 75 80

Gly Val Glu Val Arg Thr Ala Asn Thr Lys Pro Lys Glu Glu Gln Phe

85 90 95

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Ile Gln His Gln Asp

100 105 110

Trp Leu Thr Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Ala Leu

115 120 125

Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala Lys Gly Gln Thr Arg

130 135 140

Glu Pro Gln Val Tyr Thr Leu Ala Pro His Arg Glu Glu Leu Ala Lys

145 150 155 160

Asp Thr Val Ser Val Thr Cys Leu Val Lys Gly Phe Tyr Pro Ala Asp

165 170 175

Ile Asn Ile Glu Trp Gln Arg Asn Arg Gln Pro Glu Ser Glu Gly Ala

180 185 190

Tyr Ala Thr Thr Leu Pro Gln Leu Asp Asn Asp Gly Thr Tyr Phe Leu

195 200 205

Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp Gln Arg Gly Glu Thr

210 215 220

Phe Thr Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

225 230 235 240

Lys Ser Ile Thr Gln Ser Ser Gly Lys

245

<210> 21

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 21

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

1 5 10 15

His His Pro Glu Asp Pro Ser Ser Gln Cys Pro Lys Cys Pro Glu Pro

20 25 30

Glu Leu Pro Gly Gly Pro Ser Val Phe Val Phe Pro Pro Lys Pro Lys

35 40 45

Asp Val Leu Ser Ile Ser Gly Arg Pro Glu Val Thr Cys Val Val Val

50 55 60

Asp Val Gly Lys Glu Asp Pro Glu Val Asn Phe Asn Trp Tyr Ile Asp

65 70 75 80

Gly Val Glu Val Arg Thr Ala Asn Thr Glu Pro Lys Glu Glu Gln Phe

85 90 95

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Ile Gln His Gln Asp

100 105 110

Trp Leu Thr Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Ala Leu

115 120 125

Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala Lys Gly Gln Thr Arg

130 135 140

Glu Pro Gln Val Tyr Thr Leu Ala Pro His Arg Glu Glu Leu Ala Lys

145 150 155 160

Asp Thr Val Ser Val Thr Cys Leu Val Lys Gly Phe Tyr Pro Ala Asp

165 170 175

Ile Asn Val Glu Trp Gln Arg Asn Gly Gln Pro Glu Ser Glu Gly Asp

180 185 190

Tyr Ala Thr Thr Leu Pro Gln Leu Asp Asn Asp Gly Thr Tyr Phe Leu

195 200 205

Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp Gln Gln Gly Glu Thr

210 215 220

Phe Thr Cys Val Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

225 230 235 240

Lys Ser Ile Thr Gln Ser Ser Gly Lys

245

<210> 22

<211> 249

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 22

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala

1 5 10 15

His His Pro Glu Asp Pro Ser Ser Gln Cys Pro Lys Cys Pro Ala Pro

20 25 30

Glu Leu Leu Gly Gly Pro Thr Val Phe Ile Phe Pro Pro Lys Pro Lys

35 40 45

Asp Val Leu Ser Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val

50 55 60

Asp Val Gly Lys Glu Asp Pro Glu Ile Glu Phe Ser Trp Ser Val Gly

65 70 75 80

Asp Lys Glu Val His Thr Ala Glu Thr Lys Pro Lys Glu Glu Gln Phe

85 90 95

Asn Ser Thr Tyr Arg Val Val Ser Ile Leu Thr Ile Lys His Gln Asp

100 105 110

Trp Leu Thr Gly Glu Glu Phe Lys Cys Lys Val Asn Asn Lys Ala Leu

115 120 125

Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala Lys Gly Gln Thr Arg

130 135 140

Glu Pro Gln Val Tyr Thr Leu Ala Pro His Arg Glu Glu Leu Ala Lys

145 150 155 160

Asp Thr Val Ser Val Thr Cys Leu Val Lys Gly Phe Tyr Pro Pro Asp

165 170 175

Ile Asn Val Glu Trp Gln Arg Asn Gly Gln Pro Glu Ser Glu Gly Ala

180 185 190

Tyr Ala Thr Thr Leu Pro Gln Gln Asp Asn Asp Gly Thr Tyr Phe Leu

195 200 205

Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp Gln Gln Gly Glu Thr

210 215 220

Phe Thr Cys Val Val Met His Glu Ala Leu His Asn His Ser Thr Gln

225 230 235 240

Lys Ser Ile Thr Gln Ser Ser Gly Lys

245

<210> 23

<211> 747

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 23

ggaggtggag gttcaggtgg aggtggatct ggtggaggtg gatctgcgca ccaccccgaa 60

gaccccagct cccagtgtcc caaatgccca gcccctgagc tccttggagg gcccacggtc 120

ttcatcttcc ccccgaaacc caaggacgtc ctctccattt ctgggaggcc cgaggtcacg 180

tgcgttgtgg tggacgtggg taaggaagac cccgaggtca atttcaactg gtacattgat 240

ggcgttgagg tgcgaacggc caacacgaag ccaaaggagg aacagttcaa cagcacgtac 300

cgcgtggtca gcgtcctgac catccagcac caggactggc tgacggggaa ggagttcaag 360

tgcaaggtca acaacaaagc tctcccggcc cccatcgaga ggaccatctc caaggccaaa 420

gggcagaccc gggagccgca ggtgtacacc ctggccccac accgggaaga gctggccaag 480

gacaccgtga gcgtaacctg cctggtcaaa ggcttctacc cacctgacat caacgttgag 540

tggcagagga accgacaggt agagtcagag ggcgcctacg ccaccacgct gccccagctg 600

gacaacgacg ggacctactt cctctacagc aagctctcgg tgggaaagaa cacgtggcag 660

cggggagaaa ccttcacctg tgtggtgatg cacgaggccc tgcacaacca ctacacccag 720

aaatccatca cccagtcttc gggtaaa 747

<210> 24

<211> 747

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 24

ggaggtggag gttcaggtgg aggtggatct ggtggaggtg gatctgcgca ccaccccgaa 60

gaccccagct cccagtgtcc caaatgccca gcccctgagc tcccgggagg gccctccgtc 120

ttcgtcttcc ccccgaaacc caaggacgtc ctctccattt ctgggaggcc cgaggtcacg 180

tgcgttgtgg tggacgtggg taaggaagac cccgaggtca atttcaactg gtacattgat 240

ggcgttgagg tgcgaacggc caacacgaag ccaaaggagg aacagttcaa cagcacgtac 300

cgcgtggtca gcgtcctgac catccagcac caggactggc tgacggggaa ggagttcaag 360

tgcaaggtca acaacaaagc tctcccggcc cccatcgaga ggaccatctc caaggccaaa 420

gggcagaccc gggagccgca ggtgtacacc ctggccccac accgggaaga gctggccaag 480

gacaccgtga gcgtaacctg cctggtcaaa ggcttctacc cacctgacat caacgttgag 540

tggcagagga accgacagcc agagtcagag ggcgcctacg ccaccacgct gccccagctg 600

gacaacgacg ggacctactt cctctacagc aagctctcgg tgggaaagaa cacgtggcag 660

cggggagaaa ccttcacctg tgtggtgatg cacgaggccc tgcacaacca ctacacccag 720

aaatccatca cccagtcttc gggtaaa 747

<210> 25

<211> 747

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 25

ggaggtggag gttcaggtgg aggtggatct ggtggaggtg gatctgcgca ccaccccgaa 60

gaccccagct cccagtgtcc caaatgccca gcccctgagc tcctgggagg gccctctgtc 120

ttcatcttcc ccccgaaacc caaggacgtc ctctccattt ctgggaggcc cgaggtcaca 180

tgtgttgtgg tggacgtggg ccaggaagac cccgaggtca gtttcaactg gtacattgat 240

ggcgttgagg tgcgaacggc caacacgaag ccaaaggagg aacagttcaa cagcacgtac 300

cgcgtggtca gcgtcctgac catccagcac caggactggc tgacggggaa ggagttaaaa 360

tgcaaggtca acaacaaagc tctcccggcc cccatcgaga ggaccatctc caaggccaaa 420

gggcagaccc gggagccgca ggtgtacacc ctggccccac accgggaaga gctggccaag 480

gacaccgtga gcataacctg cctggtcaaa ggcttctacc cagctgacat caacgttgag 540

tggcagagga acgggcggcc ggagtcagag ggcgcctacg ccaccacgct gccccagctg 600

gacaatgacg ggacctactt cctctacagc aagctctcgg tgggaaagaa cacgtggcag 660

cggggagaaa ccttcacctg tgtggtgatg cacgaggccc tgcacaacca ctccacccag 720

aaatccatca cccagtcttc gggtaaa 747

<210> 26

<211> 747

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 26

ggaggtggag gttcaggtgg aggtggatct ggtggaggtg gatctgcgca ccaccccgaa 60

gaccccagct cccagtgtcc caaatgccca gcccctgagc tcccgggagg gccctccgtc 120

ttcgtcttcc ccccgaaacc caaggacgtc ctctccattt ctgggaggcc cgaggtcaca 180

tgtgttgtgg tggacgtggg ccaggaagac cccgaggtca atttcaactg gtacattgat 240

ggcgttgagg tgcgaacggc caacacgaag ccaaaggagg aacagttcaa cagcacgtac 300

cgcgtggtca gcgtcctgac catccagcac caggactggc tgacggggaa ggagttcaag 360

tgcaaggtca acaacaaagc tctcccggcc cccatcgaga ggaccatctc caaggccaaa 420

gggcagaccc gggagccgca ggtgtacacc ctggccccac accgggaaga gctggccaag 480

gacaccgtga gcgtaacctg cctggtcaaa ggcttctacc cagctgacat caacattgag 540

tggcagagga accgacagcc agagtcagag ggcgcctacg ccaccacgct gccccagctg 600

gacaacgacg ggacctactt cctctacagc aagctctcgg tgggaaagaa cacgtggcag 660

aggggagaaa ccttcacctg tgtggtgatg cacgaggccc tgcacaacca ctacacccag 720

aaatccatca cccagtcttc gggtaaa 747

<210> 27

<211> 747

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 27

ggaggtggag gttcaggtgg aggtggatct ggtggaggtg gatctgcgca ccaccccgaa 60

gaccccagct cccagtgtcc caaatgccca gagcctgagc tcccgggagg gccctccgtc 120

ttcgtcttcc ccccaaaacc caaggacgtc ctctccattt ctgggaggcc cgaggtcacg 180

tgcgttgtgg tggacgtggg taaggaagac cccgaggtca atttcaactg gtacattgat 240

ggcgttgagg tgcgaacggc caacacggag ccaaaggagg aacagttcaa cagcacgtac 300

cgcgtggtca gcgtcctgac catccagcac caggactggc tgacggggaa ggagttcaag 360

tgcaaggtca acaacaaagc tctcccagcc cccatcgaga ggaccatctc caaggccaaa 420

gggcagaccc gggagccgca ggtgtacacc ctggccccac accgggaaga gctggccaag 480

gacaccgtga gcgtaacctg cctagtcaaa ggcttctacc cagctgacat caacgttgag 540

tggcagagga atgggcagcc ggagtcagag ggcgactacg ccaccacgct gccccagctg 600

gacaacgacg ggacctactt cctctacagc aagctctcgg tgggaaagaa cacgtggcag 660

cagggagaaa ccttcacctg tgtggtgatg catgaggccc tgcacaacca ctacacccaa 720

aaatccatca cccagtcttc gggtaaa 747

<210> 28

<211> 747

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 28

ggaggtggag gttcaggtgg aggtggatct ggtggaggtg gatctgcgca ccaccccgaa 60

gaccccagct cccagtgtcc caaatgccca gcccctgagc tccttggagg gcccacggtc 120

ttcatcttcc ccccgaaacc caaggacgtc ctctccatca ccctaacacc taaggtcacg 180

tgcgttgtgg tggacgtggg taaggaagac cctgagatag agttcagctg gtccgtgggt 240

gacaaagagg tacacacggc tgagacaaag ccaaaggagg aacagttcaa cagcacgtac 300

cgcgtggtca gcatcctgac aatcaagcac caggactggc tgacggggga ggagttcaag 360

tgcaaggtca acaacaaagc tctcccggcc cccatcgaga ggaccatctc caaggccaaa 420

gggcagaccc gggagccgca ggtgtacacc ctggccccac accgggaaga gctggccaag 480

gacaccgtga gcgtaacctg cctggtcaaa ggcttctacc cacctgacat caacgttgag 540

tggcagagga acgggcagcc ggagtcagag ggcgcctacg ccaccacgct gccccagcag 600

gacaacgacg ggacctactt cctctacagc aagctctcgg tgggaaagaa cacgtggcag 660

cagggagaaa ccttcacctg tgtggtgatg cacgaggccc tgcacaacca ctccacccag 720

aaatccatca cccagtcttc gggtaaa 747

<210> 29

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 29

gctctagagg atcgaaccct tatgcatccg ctcctc 36

<210> 30

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 30

gagagaattc caccaccacc gttgaactgt ccctc 35

Claims

1. An immunogen based on camel source Fc fragment, which is formed by fusing target protein and camel source Fc fragment, is characterized in that the camel source Fc fragment is shown in any one of SEQ ID NO. 17-22; the target protein is a human IL-13 protein.

2. An immunogen based on a camelid Fc fragment according to claim 1 wherein the coding sequences of said camelid Fc fragment are shown in any one of SEQ ID NOs 23-28, respectively.

3. An expression vector based on an immunogen of a camel source Fc fragment, characterized in that said expression vector comprises a 5 '-Multiple Cloning Site (MCS) -linker coding sequence-camel source Fc coding sequence-6 histidine (6 xhis) coding sequence-3' sequence; the connecting region (linker) coding sequence-camel source Fc coding sequence is shown in any one of SEQ ID NO. 23-28; the coding sequence of the human IL-13 protein is inserted into the multiple cloning site of the camel Fc protein expression vector.

4. A method of constructing an expression vector for an immunogen based on a camelid Fc fragment of claim 3, comprising the steps of:

1) Obtaining a camel source Fc protein coding sequence;

2) Constructing a camel source Fc protein expression vector;

3) Inserting a coding sequence of a human IL-13 protein into a multiple cloning site of a camel source Fc protein expression vector;

the step 2) is specifically as follows: PCR amplifying the 5 PCR products obtained in the step 1) by primers SEQ ID NO. 11 and 16, SEQ ID NO. 12 and 16, SEQ ID NO. 13 and 16, SEQ ID NO. 14 and 16 and SEQ ID NO. 15 and 16 respectively to obtain 6 DNA sequences shown in SEQ ID NO. 23-28, and then carrying out enzyme digestion on the 6 DNA sequences by a proper enzyme digestion site; the cleavage product with the structure 5 '-Multiple Cloning Site (MCS) -linker (linker) -Fc coding sequence of camel origin-6 histidine (6 XHis) coding sequence-3' was cloned into a vector.

5. A method for the preparation of an immunogen based on a camelid Fc fragment according to claim 1 or 2 comprising the steps of:

1) Constructing an expression vector for an immunogen based on a camelid Fc fragment using the method of claim 4;

2) The eukaryotic cells are transfected by the expression vector to realize the expression of fusion proteins consisting of human IL-13 protein and camel Fc protein.

6. The application of the camel source Fc fragment in preparing the immunogen is characterized in that the immunogen is formed by fusing a human IL-13 protein and the camel source Fc fragment, and the camel source Fc fragment is shown as any one of SEQ ID NO. 17-22; the immunogen is used for immunizing camels.

7. Use of an immunogen based on a camelid Fc fragment according to claim 1 or 2 for the preparation of a single domain antibody directed against a human IL-13 protein.