A mouse-derived single-chain antibody against porcine IgA and its application

Abstract

The invention belongs to the field of biotechnology, and specifically relates to a mouse-derived anti-swine IgA single-chain antibody and an application thereof. The single-chain antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.3. The gene sequence of the mouse-derived antibody variable region is compared with the gene sequence of the prepared single-chain antibody, and the result shows that the gene sequence of the single-chain antibody is basically consistent with the gene sequence of the mouse-derived antibody variable region, proving that the constructed antibody gene sequence is correct. The single-chain antibody fills the gap of the anti-swine IgA single-chain antibody on the current market, provides new materials for the early diagnosis and prevention and control of pig-derived IgA antibodies, and provides new technical support for controlling the epidemic spread of pig diarrhea diseases as soon as possible.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a mouse-derived anti-porcine IgA single-chain antibody and an application thereof. Background Art

[0002] In pig production practice, various intestinal diseases occur frequently, seriously affecting pig production performance and causing huge losses to the pig industry. A common pig intestinal disease is diarrhea, which is caused by various causes, such as bacterial diarrhea, viral diarrhea, typhoid fever, infectious gastroenteritis, etc. Among them, the pathogens of viral diarrhea mainly include porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV) and porcine delta coronavirus (PDCoV). At present, there are few effective vaccines against the above viruses, and the occurrence of the disease cannot be effectively prevented. The only way is "early diagnosis and early treatment". Therefore, early diagnosis is a crucial step in preventing and controlling the development of such diseases.

[0003] As the immunoglobulin with the largest production in animals, IgA has important immune and physiological regulatory functions, especially in local mucosal immunity. IgA has multiple polymeric forms, including monomers, dimers, and multimers. Most of them exist in serum in the form of monomers, called serum type IgA, accounting for 10% to 15% of the total serum immunoglobulins. The basic structure is "Y" type, consisting of two identical heavy chains and two identical light chains. The single chain and the light chain are connected by disulfide bonds to assemble into a symmetrical molecule with light and heavy chains paired. The J chain synthesized by plasma cells connects two monomeric IgAs into IgA (SIgA). Compared with monomeric IgA, SIgA has an additional secretory piece (SP), which is also called secretory component (SC). It is synthesized by mucosal epithelial cells and is the extracellular segment of the polymeric immunoglobulin receptor pIgR. It binds to SIgA, assists its secretion to the mucosal surface through intestinal epithelial cells, and protects the hinge region of SIgA from hydrolysis. Secretory IgA (SIgA) is a major component of the body's mucosal defense system. A large amount of SIgA is present in mammalian colostrum, tears, saliva and other secretions. It can inhibit the attachment of microorganisms to the respiratory epithelium and slow down the reproduction of viruses. It is an important barrier to the mucosa and has antibody activity against certain viruses, bacteria and general antigens. It is the first line of defense to prevent pathogens from invading the body. At the same time, it is also the most abundant type of antibody in the intestines of humans and other mammals. Porcine IgA plays an important role in the diagnosis of swine infectious diseases. It can help detect whether pigs are infected with a certain pathogen in the early stage of infection, evaluate the mucosal immune status, and achieve specific diagnosis of specific pathogens. However, the content of IgA in pigs is relatively low, and there are differences in different stages of infection and individuals, which may lead to false negative or false positive results during the detection process, affecting the accuracy of diagnosis. In addition, the similarity of antigenic structures between different pathogens may cause cross-reactions and lead to misdiagnosis. When collecting and processing samples, care should also be taken to avoid sample contamination or loss because IgA has poor stability in vitro. In order to improve the accuracy and reliability of diagnosis, it is necessary to continuously optimize detection methods and means, and combine them with other detection indicators for comprehensive diagnosis.

[0004] Single chain antibody (scFv) is a recombinant antibody modified by genetic engineering. It retains the targeting of antibodies and obtains characteristics that complete antibodies do not have, such as better permeability, easy diffusion, weak immunogenicity, short in vivo residence time, easy expression, etc. It is an ideal antibody for disease diagnosis and treatment. It can also be fused with fluorescent proteins for disease diagnosis and connected with chemical toxins to form immunotoxins for cancer treatment. When scFv is connected to T cells, it constructs chimeric antigen receptor T cells, which is a very promising cancer treatment technology. With the increasing development and maturity of antibody library screening technology and protein expression technology, scFv will make breakthrough progress in the fields of tumor treatment, virus detection and prevention, and drug delivery, and become a hot spot for new antibody research and development.

[0005] The present invention targets IgA and screens out a mouse-derived single-chain antibody against porcine IgA, which has porcine IgA specificity, good biological activity, strong antigen binding ability, and potential neutralizing function, and is of great significance for the early clinical diagnosis method of porcine diarrhea diseases and the subsequent prevention and control thereof. Summary of the invention

[0006] The primary purpose of the present invention is to provide a mouse anti-porcine IgA single-chain antibody, which includes a heavy chain variable region and a light chain variable region. The amino acid sequence of the heavy chain variable region is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.3.

[0007] Preferably, the heavy chain variable region and the light chain variable region are connected by a connecting peptide having an amino acid sequence as shown in SEQ ID No.5.

[0008] Preferably, the single-chain antibody comprises a light chain variable region, a connecting peptide and a heavy chain variable region in sequence, or comprises a heavy chain variable region, a connecting peptide and a light chain variable region in sequence.

[0009] The second object of the present invention is to provide a gene sequence encoding the single-chain antibody, wherein the nucleotide sequence of the heavy chain variable region is shown in SEQ ID No.2, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.4.

[0010] Preferably, the nucleotide sequence encoding the connecting peptide is shown as SEQ ID No.6.

[0011] The third object of the present invention is to provide a vector capable of expressing the single-chain antibody gene sequence.

[0012] The fourth object of the present invention is to provide a host cell containing the vector.

[0013] The fifth object of the present invention is to provide the use of the single-chain antibody in the preparation of diagnostic reagents for porcine intestinal diseases.

[0014] The sixth object of the present invention is to provide the use of the single-chain antibody in the preparation of a diagnostic kit for porcine intestinal diseases.

[0015] The present invention has the following beneficial effects: the present invention provides a mouse-derived single-chain antibody against porcine IgA, the single-chain antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.3. The gene sequence of the mouse antibody variable region is compared with the gene sequence of the prepared anti-porcine IgA ScFv antibody (VH-VLκ), and the result shows that the gene sequence of the antibody is basically consistent with the gene sequence of the mouse antibody variable region, proving that the constructed antibody gene sequence is correct. The mouse-derived single-chain antibody against porcine IgA prepared by the present invention fills the gap of the porcine IgA ScFv antibody on the current market, provides a small molecule antibody with low immunogenicity and strong specificity, provides new materials for the early diagnosis and prevention and control of porcine IgA antibodies, and provides new technical support for controlling the epidemic spread of porcine diarrhea diseases as soon as possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0017] Figure 1 It is the agarose gel electrophoresis diagram of the second round amplification product of ds cDNA of positive hybridoma cell line;

[0018] Note: M: DL2000 relative molecular mass standard DETAILED DESCRIPTION

[0019] The present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the following embodiments, and any technical solutions that can be thought of by any technician in the field based on the present invention and combined with the common knowledge in the field belong to the protection scope of the present invention.

[0020] The light chain is divided into a κ chain or a λ chain according to the different constant regions. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known to those skilled in the art. In the present invention, VH is the heavy chain variable region, and VL is the light chain variable region, which is divided into κ type and λ type.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] Example 1. Preparation of anti-mouse IgAScFv antibody

[0023] 1 Materials

[0024] 1.1 Experimental animals: 6-8 week old female BALB / c mice were purchased from the Experimental Animal Center of Lanzhou Veterinary Research Institute.

[0025] 1.2 Strains and reagents

[0026] Polymerase, RNase-free Water, DL2,000DNAMarker, and 6×Loading Buffer were all purchased from Takara Biotechnology Co., Ltd.; Trizol, nickel affinity chromatography resin, DNA fragment recovery kit, and plasmid extraction kit were OMEGA products; molecular biology reagents were from Sigma; and other biochemical reagents were all domestically produced analytical grade.

[0027] 2 Methods

[0028] 2.1 Immunization of mice

[0029] Dilute the purified porcine IgA to 300 μg / mL with sterile PBS, take 1.5 mL of the diluted porcine IgA and mix it with an equal amount of Freund's complete adjuvant to emulsify it. Select 6 healthy 6-week-old female BALB / c mice, and inject 200 μL of emulsified porcine IgA subcutaneously on the back. Use Freund's incomplete adjuvant to emulsify an equal amount of protein for booster immunization twice on the 15th and 30th days after the first immunization, respectively. The immunization site and method are the same as the first immunization. One week after the third immunization, blood was collected from the tail vein, and the serum was separated. The antibody level of each mouse was determined by indirect ELISA. The serum OD 450nm The mice with a Gamma-Glutathione Oxygenase Index (GMO) greater than 2.0 were boosted once again three days before fusion by intraperitoneal injection of 0.5 mL of porcine IgA (300 μg / mL) without adjuvant.

[0030] 2.2 Cell fusion

[0031] Take BALB / c mice that were boosted 3 days ago, collect blood and separate serum as positive control; dislocate the cervical vertebra of the mice and kill them, soak them in 75% alcohol for disinfection for 5 minutes, fix them on the plastic plate in the clean bench, lift the abdominal skin with sterilized tweezers, cut the abdominal skin and peritoneum with scissors, find the spleen, remove it with sterile tweezers, put it in a sterile plate containing incomplete RPMI-1640 culture medium for a little cleaning, and peel off the surface fat and connective tissue. Then put it on a high-pressure 200-mesh copper mesh and cut it into pieces, grind the spleen with a high-pressure syringe core, and add incomplete RPMI-1640 culture medium while grinding until the spleen is completely ground, and then rinse the mesh with about 20mL of incomplete RPMI-1640 culture medium to allow the spleen cells to completely pass through the mesh and enter the beaker, and centrifuge at 1000r / m for 10min to collect the spleen cells for use.

[0032] Spleen cells and myeloma cells Sp2 / 0 were mixed evenly according to the cell number of 1:5-1:10, centrifuged at 1000r / m for 10min, the supernatant was discarded, and the bottom of the tube was flicked to loosen it. 37℃ distilled water was added to the beaker, and the centrifuge tube containing the mixed cells was placed in a water bath. Within 90s, 800μL of PEG 2000 preheated at 37℃ was added, and the centrifuge tube was rotated while adding dropwise, and it was allowed to stand for 1min. Then, within 5min, incomplete RPMI-1640 culture medium preheated at 37℃ was added, and it was dripped slowly at first, and then accelerated. The cells at the bottom should not be washed up. After the PEG was diluted, centrifuged at 1500r / min for 10min, and the supernatant was discarded.

[0033] The cell pellet was resuspended in HAT selection medium containing 20% ​​fetal bovine serum and 10% growth factor, mixed evenly, and added to a 96-well cell culture plate using a multichannel pipette, 200 μL per well, and cultured at saturated humidity, 5% CO2, and 37°C.

[0034] 2.3 Screening and subcloning of positive hybridoma cells

[0035] After fusion, the culture medium of the cells is replaced every 2 to 4 days. When replacing, half-replacement is adopted, that is, 100 μL of culture medium is discarded and 100 μL of new HAT selection culture medium is added. 7 to 8 days after fusion, the cells are observed and the cell wells with surviving hybridoma cells are marked. When the hybridoma cells grow more than 1 / 10 of the bottom of the well, 100 μL of supernatant is taken as the primary antibody, and purified porcine IgA is used as the coating antigen to screen the positive clone wells by indirect ELISA detection method. Blow up the hybridoma cells in the cell wells that are positive for indirect ELISA detection and move them into a 24-well cell culture plate. After they grow, blow up again to mix and count the cells. After counting, dilute it to 10 cells / mL with HT culture medium containing 20% ​​fetal bovine serum, and add this concentration of cell suspension to a 96-well cell culture plate pre-laid with feeder cells at 100 μL / well for culture. The screening method is the same as above. At the same time, the remaining cells after each cloning should be expanded and frozen to avoid loss. After three subclonings, when the supernatants in all cloned cell wells are tested positive, it can be determined that a cell line secreting anti-porcine IgA monoclonal antibodies has been obtained.

[0036] 2.4 Extraction of total mRNA from positive hybridoma cells

[0037] 400 μL of the above positive hybridoma cell solution was taken, 1 mL TRizol was added, mixed, and allowed to stand at 4°C for 5 min; 250 μL chloroform was added, mixed, allowed to stand at 4°C for 10 min, and centrifuged at 12000 r / min at 4°C for 15 min; 450 μL of supernatant was taken, an equal amount of isopropanol was added, allowed to stand at -20°C for 30 min, and centrifuged at 12000 r / min at 4°C for 15 min; the supernatant was discarded, 1 mL 75% ethanol was added to the precipitate, and centrifuged at 12000 r / min at 4°C for 5 min; the precipitate was placed in a fume hood for 10 min to dry; the precipitate dissolved in 25 μL RNase-free water was the total mRNA harvested, and the concentration of Total mRNA measured by ND2000 was 356 ng / μL, and the purity OD 260 / OD 280 It is 1.87.

[0038] 2.5 Primer design

[0039] First round amplification primers:

[0040] (1) Primers for amplifying VH sequences

[0041] I: 5'-AGGAACTGCAGGTGTCC-3' (SEQ ID No. 9)

[0042] II: 5'-CAGCTACAGGTGTCCACTCC-3' (SEQ ID No. 10)

[0043] III: 5’-TGGCAGCARCAGCTACAGG-3’ (SEQ ID No.11)

[0044] IV: 5’-CTGCCTGGTGACATTCCCA-3’ (SEQ ID No.12)

[0045] V: 5’-CCAAGCTGTGTCCTGTC-3’ (SEQ ID No.13)

[0046] VI: 5’-TTTTAAAAGGTGTCCAGKGT-3’ (SEQ ID No.14)

[0047] VII: 5’-CCTGTCAGTAACTRCAGGTGTCC-3’ (SEQ ID No.15)

[0048] VIII: 5’-TTTTAAAAGGGGTCCAGTGT-3’ (SEQ ID No.16)

[0049] IX: 5’-CGTTCCTGGTATCCTGTCT-3’ (SEQ ID No.17)

[0050] X: 5’-ATGAAGTTGTGGYTRAACTGG-3’ (SEQ ID No.18)

[0051] XI: 5’-TGTTGGGGCTKAAGTGGG-3’ (SEQ ID No.19)

[0052] XII: 5’-AGAAGGTGTGCACACCGCTGGAC-3’ (SEQ ID No.20)

[0053] (2) Primers for amplifying the VL sequence

[0054] I: 5’-RGTGCAGATTTTCAGCTTCCTGCT-3’ (SEQ ID No.21)

[0055] II: 5’-TGGACATGAGGGCYCCTGCTCAGT-3’ (SEQ ID No.22)

[0056] III: 5’-CTSTGGTTGTCTGGTGTTGAYGGA-3’ (SEQ ID No.23)

[0057] IV: 5’-GTTGCTGCTGCTGTGGCTTACA-3’ (SEQ ID No.24)

[0058] V: 5’-GTATCTGGTACCTGTGG-3’ (SEQ ID No.25)

[0059] VI: 5’-TGCCTGTTAGGCTGTTGGTGCT-3’ (SEQ ID No.26)

[0060] VII: 5’-GCTCAGTTCCTTGGTCTCCTGTTGC-3’ (SEQ ID No.27)

[0061] VIII: 5’-TGGGTGCTGCTGCTCTGGGT-3’ (SEQ ID No.28)

[0062] IX: 5’-CAGTTCCTGTTTCTGTTARTGCTCTGG-3’ (SEQ ID No.29)

[0063] X: 5’-TGCTCTGGTTATATGGTGCTGATGGG-3’ (SEQ ID No.30)

[0064] Ⅺ: ’-ACTGAGGCACCTCCAGATGTT-3’ (SEQ ID No.31)

[0065] Second-round amplification primers:

[0066] Ⅰ: 5’-GGGAATTCGAGGTGCAGCTGCAGGAGTCTGG-3’ (SEQ ID No.32)

[0067] Ⅱ: 5’-GCTCAGGGAARTAGCCCTTGAC-3’ (SEQ ID No.33)

[0068] Ⅲ: 5’-GAYATTGTGMTSACMCARWCTMCA-3’ (SEQ ID No.34)

[0069] Ⅳ: 5’-TGGGAAGATGGATACAGTT-3’ (SEQ ID No.35)

[0070] Ⅴ: 5’-CAGGCTGTTGTGACTCAG-3’ (SEQ ID No.36)

[0071] Ⅵ: 5’-CAACTTGTGCTCACTCAG-3’ (SEQ ID No.37)

[0072] Ⅶ: 5'-CTCYTCAGRGGAAGGTGGRAACA-3' (SEQ ID No. 38)

[0073] 2.6 cDNA double-strand synthesis and purification

[0074] (1) Whole-gene cDNA synthesis

[0075] Add the following components in a 0.2 mL PCR amplification tube:

[0076] Reagents and Materials volume DEPC water 4.5μL 5×FirststrandBuffer 4μL DTT(0.1M) 2μL Oligo(dT) 1μL RRI 1μL dATP 1μL Radomprimers 0.5μL M-MLV reverse transcription (200u / ul) 1μL mRNA 5μL Total volume 20μL

[0077] After mixing, place the PCR amplification tube in a PCR instrument, and the reaction program is: 25℃10min, 37℃60min, 72℃15min. The obtained product is the whole genome cDNA, which is stored at -20℃ for future use.

[0078] (2) First round of amplification

[0079] Add the following components in a 0.2 mL PCR amplification tube:

[0080] ①Used to amplify VH sequences:

[0081] Reagents and Materials volume 5×PCR buffer 10μL dNTPMixture 4μL Full-length cDNA 2.5μL VH primer mix (10pmol / μL) 4μL DNA Polymerase Mix 0.5μL DEPC water 29μL Total volume 50μL

[0082] ②Used to amplify VL sequence:

[0083] Reagents and Materials volume 5×PCR buffer 10μL dNTPMixture 4μL Full-length cDNA 2.5μL VL primer mix (10pmol / μL) 4μL DNA Polymerase Mix 0.5μL DEPC water 29μL Total volume 50μL

[0084] The above amplification procedures are as follows: ①: 98℃2min, 98℃10s, 52℃5s, 72℃30s, 30 cycles, 72℃10min; electrophoresis of the amplified product, purification and collection of products of about 300bp, which is the amplified target product; ②: 98℃2min, 98℃10s, 54℃5s, 72℃30s, 30 cycles, 72℃10min. Electrophoresis of the amplified product, purification and collection of products of about 300bp, which is the amplified target product. ③: 98℃2min, 98℃10s, 53℃5s, 72℃30s, 30 cycles, 72℃10min. Electrophoresis of the amplified product, purification and collection of products of about 300bp, which is the amplified target product. The amplified product was stored at -20℃ for future use.

[0085] (3) Second round of amplification

[0086] Add the following components to four 0.2 mL PCR amplification tubes:

[0087] Reagents and Materials quantity 5×PCR buffer 10μL dNTPMixture 4μL Primer mix1 / 2 / 3 / 4 (10pmol / μL) 4μL The first round of amplification products 0.5μL DNA Polymerase Mix 0.5μL DEPC water 31μL Total volume 50μL

[0088] The above amplification procedures are as follows: ①: 98℃2min, 98℃10s, 56℃5s, 72℃30s, 35 cycles, 72℃10min; electrophoresis of the amplified product, purification and collection of products of about 320bp, which is the amplified target product; ②: 98℃2min, 98℃10s, 68℃30s, 30 cycles, 72℃10min. Electrophoresis of the amplified product, purification and collection of products of about 320bp, which is the amplified target product; ③: 98℃2min, 98℃10s, 68℃30s, 35 cycles, 72℃10min. Electrophoresis of the amplified product, purification and collection of products of about 320bp, which is the amplified target product; the amplified product is sent to the company for sequencing.

[0089] 2.7 Gene sequence alignment

[0090] The sequencing results were compared with the mouse antibody heavy chain variable region sequence, light chain Lambda chain, and light chain Kappa chain published on the NCBI website.

[0091] 3 Results

[0092] 3.1 Agarose gel electrophoresis of cDNA synthesis double strands

[0093] The ds cDNA product of the second round of amplification was subjected to electrophoresis, and bands of about 300 bp were collected.

[0094] Figure 1 The figure is an agarose gel electrophoresis diagram of the first round amplification products of ds cDNA of positive hybridoma cells. In the figure, M represents the DL2000 relative molecular mass standard. The results show that the sizes of VH and VLκ are both around 300 bp, but no specific band is amplified for VLλ.

[0095] 3.2 PCR results sequencing

[0096] After sequencing, it was found that both VH and VLκ were sequenced correctly, while the VLλ sequencing results did not show a single peak. The measured sequence was basically consistent with the framework region of the mouse immunoglobulin variable region sequence, and there were differences in the CDR region, which was consistent with the gene structure of the mouse light and heavy chain variable regions. The VH part was 342bp (SEQ ID No. 2), the VLκ part was 315bp (SEQ ID No. 4), and the linker base sequences between the heavy chain and the light chain were all correct.

[0097] Among them, the size of VH-Linker-VLκ is 678bp, and the nucleotide sequence is as follows, and the underlined sequence is the Linker sequence (SEQ ID No.6):

[0098] 5'-GTGCAGCTGCAGGAGTTCTGGGCCTCAGCTGGTTAGGCCTGGGGCTTCAGTGAAGA TATCCTGCAAGGCTTCTGGTTACTCATTCATCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGTCTTGAGTGGATTGGCATGATTGATACTTCCGATAGTGATACTAGGTTAAATCAGAAGTTCAAGGACAAG GCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATACAACTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGACCGATCTACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA GGTGGCGGAGGGTCAGGCGGC GATATTGTGTTGACGCATATTCTAGATTCTGTACCTGTTAATCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTGTATTGTTTGGGGCAGAGGACAGAACAGTGTCCTCAGCTATTGATA TATCGGATGAGCAACCTTGCCTCAGGACAGGCAGCCAGGTTTCAGTGGCAGTGGGTCAGGAACTCAGTTCACTCTGAGAATCAGTAGAGGGGAGGGAGAGGATGTGGGCGGGTATTACAGTATGCAAAATGTAGAATATCCGTACCCGTTCGGAGGA-3'.

[0099] 3.3 ScFv antibody amino acid sequence alignment

[0100] The correctly sequenced gene sequence was compared with the mouse antibody variable region sequence published on the NCBI website, and the comparison result was displayed in the form of amino acid sequence. The amino acid sequence of VH is shown in SEQ ID No.1, the amino acid of VLκ is shown in SEQ ID No.3, and the amino acid sequence of Linke is shown in SEQ ID No.5. The amino acid sequence is as follows: VQLQESGPQLVRPGASVKISCKASGYSFISYWMHWVKQRPGQGLEWIGMIDTSDSDTRLNQKFKDKATLTVDKSSSTAYIQLSSPTSEDSAVYYCARPIYFDYWGQGTTLTVSS GGGGSGG DIVLTHILDSVPVNPGESVSISCRSSKSLLHSNGNTYLYCLGQRTEQCPQLLIYRMSNLASGQAARFSGSGSGTQFTLRISRGEGEDVGGYYSMQNVEYPYPFGG

[0101] (1) VH sequence alignment of VH-Linker-VLκ

[0102] Mouse antibody VH sequence:

[0103] VQLQQPGAELVKPGASVKISCKASGYTFTSYWMNWVKQRPGQGLEWIGEIDPSDSYTN NNQKFKDKATLTVDKSSSTAYMQLSSSLTSEDSAVYYCAR(SEQ ID No.7)

[0104] VH sequence of ScFv antibody:

[0105] VQLQESGPQLVRPGASVKISCKASGYSFISYWMHWVKQRPGQGLEWIGMIDTSDSDTRL NQKFKDKATLTVDKSSSTAYIQLSSPTSEDSAVYYCARPIYFDYWGQGTTLTVSS(SEQ ID No.1)

[0106] (2) VLκ sequence alignment of VH-Linker-VLκ

[0107] Mouse antibody VLκ sequence:

[0108] DIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLYWFLQRPGQSPQLLIYRMSNLAS GVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYP(SEQ ID No.8)

[0109] Sequence of VLκ of ScFv antibody:

[0110] DIVLTHILDSVPVNPGESVSISCRSSKSLLHSNGNTYLYCLGQRTEQCPQLLIYRMSNLAS GQAARFSGSGSGTQFTLRISRGEGEDVGGYYSMQNVEYPYPFGG(SEQ ID No.3)

[0111] 3.4 VH-Linker-VLκ Sequence Alignment

[0112] The above sequence was compared with the mouse antibody variable region sequence published on the NCBI website. The comparison results showed that the homology of the heavy chain and light chain sequences was more than 80%.

[0113] The results showed that the constructed ScFv antibody gene sequence was basically consistent with the framework region of the mouse antibody variable region sequence published on the NCBI website, with differences in the CDR region.

[0114] In summary, the present invention provides a mouse-derived single-chain antibody against porcine IgA, the single-chain antibody comprising a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No. 3. The gene sequence of the mouse antibody variable region was compared with the gene sequence of the prepared anti-porcine IgA ScFv antibody (VH-VLκ), and the results showed that the gene sequence of the antibody was basically consistent with the gene sequence of the mouse antibody variable region, proving that the constructed antibody gene sequence was correct; the mouse-derived single-chain antibody against porcine IgA prepared by the present invention fills the gap in the current anti-porcine IgA ScFv antibody on the market, provides a small molecule antibody with low immunogenicity and strong specificity, provides new materials for the early diagnosis and prevention and control of porcine IgA antibodies, and provides new technical support for controlling the epidemic spread of porcine diarrhea diseases as soon as possible.

Claims

1. A mouse anti-porcine IgA single-chain antibody, characterized in that: The single-chain antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.

3.

2. The single-chain antibody according to claim 1, characterized in that The heavy chain variable region and the light chain variable region are connected by a connecting peptide with an amino acid sequence as shown in SEQ ID No.

5.

3. The single-chain antibody according to claim 2, characterized in that The single-chain antibody sequentially comprises a light chain variable region, a connecting peptide and a heavy chain variable region, or sequentially comprises a heavy chain variable region, a connecting peptide and a light chain variable region.

4. A nucleotide encoding the single-chain antibody according to claim 1, characterized in that: The nucleotide sequence of the heavy chain variable region is shown in SEQ ID No.2, and the nucleotide sequence of the light chain variable region is shown in SEQ ID No.

4.

5. The single-chain antibody according to claim 2, characterized in that The nucleotide sequence encoding the connecting peptide is shown as SEQ ID No.

6.

6. A vector capable of expressing the single-chain antibody gene sequence of claim 4.

7. A host cell containing the vector according to claim 6.

8. Use of the single-chain antibody according to any one of claims 1 to 3 in the preparation of diagnostic reagents for porcine intestinal diseases.

9. Use of the single-chain antibody according to any one of claims 1 to 3 in the preparation of a diagnostic kit for porcine intestinal diseases.

Images