CN104017080B - A kind of dog source single-chain antibody and construction method thereof and application - Google Patents

Abstract

The invention relates to the field of biotechnology, and specifically discloses a canine single-chain antibody and its construction method and application. The invention successfully amplifies the canine heavy chain variable region VH and light chain variable region VL genes by designing degenerate primers for the variable region of the canine antibody. On this basis, the canine single-chain antibody scFv library was successfully constructed using phage display technology. The constructed scFv single-chain antibody library has diversity and large enough library capacity. The present invention uses the dog DEA1.1 blood group antigen to screen the scFv single-chain antibody library to obtain 3 single-chain antibodies capable of binding to the dog DEA1.1 blood group antigen through the Dot-ELISA method. Among them, Clone16 has strong binding properties. The pET‑32a‑Clone16 recombinant protein was constructed based on the Clone16 gene for prokaryotic expression, and the purified antibody had binding activity. It provides a solid foundation for the preparation of canine DEA1.1 blood group identification reagent.

Description

A kind of canine single-chain antibody and its construction method and application

technical field

The present invention relates to the field of biotechnology, more specifically, to a canine single-chain antibody and its construction method and application.

Background technique

Traditional antibody preparation, including hybridoma-monoclonal antibody technology, needs to immunize the body with antigen first, and the performance of the obtained antibody depends on the effectiveness of in vivo immunization. Therefore, it is difficult to prepare antibodies against weak antigens, self-antigens, toxic antigens, and human antibodies. The phage antibody library technology developed in the 1990s is a major advance in the field of antibody engineering, and it has become an important way to develop human monoclonal antibodies. The phage antibody library technology simulates the process of antibody production in vivo, which provides the possibility to prepare antibodies without immunization, and has gradually become one of the important means of preparing human antibodies.

A blood type is a genetic shape expressed in the form of blood antigens. The blood group system is a system for typing blood according to the different types of antigens on the red blood cell membrane. At present, more than 12 canine blood group systems have been discovered, and each blood group system is named by DEA+digital method. At present, there are 8 blood types recognized internationally, namely DEA1.1, DEA1.2, DEA3, DEA4, DEA5, DEA6, DEA7 and DEA8. Since the information of canine blood group antigens is unknown, it is not possible to obtain relatively large amounts of canine blood group antigens through gene expression, biosynthesis and antigen purification. It is very difficult to prepare monoclonal antibodies of canine blood type by the conventional method of preparing monoclonal antibodies by hybridoma method, because more antibodies to other red blood cell antigens will be produced due to the impurity of antigens, which poses a great challenge to the preparation and screening of specific antibodies challenge. However, the method of immunizing canine red blood cells between dogs can avoid the influence of a large number of non-blood group antigens. Most of the polyclonal antisera produced are specific to canine blood types. At the same time, phage display technology can extract antibody gene information from peripheral blood lymphocytes of immunized animals.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiency of antibody research technology for canine blood group antigen in the prior art, and provide a canine single-chain antibody.

The second object of the present invention is to provide a primer set for constructing a canine single-chain antibody library.

The third object of the present invention is to provide a canine single-chain antibody library.

The fourth object of the present invention is to provide a method for constructing the canine single-chain antibody library.

The fifth object of the present invention is to provide the application of the primer set or canine single-chain antibody or single-chain antibody library in the preparation of canine blood group identification reagents.

The purpose of the present invention is achieved through the following technical solutions:

A canine single-chain antibody Clone 16, characterized in that the scFv sequence of the antibody is 870bp in length, and the sequence is shown in SEQ ID NO: 48; wherein the heavy chain variable region VH is 435bp in length, and the sequence is in SEQ ID NO: 49 As shown; the light chain variable region VK is 390bp long, and the sequence is shown in SEQ ID NO:50; the connecting peptide sequence is 45bp long, and the sequence is shown in SEQ ID NO:51.

The amino acid sequence of the canine single-chain antibody Clone 16 is shown in SEQ ID NO:52.

A primer for amplifying the gene sequence of the single-chain antibody Clone 16 is provided, and the sequence is shown in SEQ ID NO: 53-54.

A primer set for constructing a canine single-chain antibody library is provided, including antibody heavy chain variable region primers, light chain variable region primers and connecting peptide sequence primers, and the primer sequences are shown in SEQ ID NO: 1-47.

Provide a canine single-chain antibody library, the antibody library contains canine blood type antibody heavy chain variable region VH and light chain variable region VL, and the middle linker sequence (Gly4Ser) ₃ , with a complete antigen binding site, With pCANTAB-5E as the vector, the library capacity reaches 7×10 ⁵ .

A method for constructing the canine single-chain antibody library is provided, comprising the following steps:

S1. Extraction of total RNA and synthesis of cDNA: take peripheral blood from immunized dogs, separate lymphocytes, extract total RNA, reverse transcribe into cDNA by reverse transcription PCR;

S2. PCR amplification of antibody heavy chain and light chain variable region genes: According to the canine antibody gene sequence, respectively design primers for PCR amplification of antibody heavy chain and light chain variable regions. The sequences of the primers are shown in SEQ ID NO: 1 ～33; use the cDNA obtained from S1 as a template to amplify the variable region genes VH and VL;

S3. Amplification of single-chain antibody scFv gene: design primers containing linker peptide sequence linker and SfiI, NotI restriction sites, and then amplify on the basis of heavy chain VH and light chain VL variable region genes obtained from S2 obtain the scFv gene;

S4. Construction of single-chain antibody scFv library: scFv gene fragment was cut with double enzymes, and inserted into pACNTAB-5E to construct a recombinant plasmid, transformed into competent E.coli TG1, and a preliminary single-chain antibody library was obtained. After repeated panning, the canine source was obtained Single-chain antibody library.

The present invention utilizes the pCANATB-5E carrier of Amerhsma Company to display the scFv antibody on the g3 protein. Therefore, most of the expressed proteins will be read through, express the scFv-g3 fusion protein, and display on the phage capsid through phage assembly.

There are generally three sources of cells for constructing a phage antibody library: hybridoma cells, sensitized B lymphocytes of the immune body, and non-sensitized B lymphocytes. Among them, non-sensitized B lymphocytes, that is, B lymphocytes of patients who have been naturally immunized, are a good source of the target antibody gene. Because natural immune lymphocytes in the human body undergo antigen pressure selection and affinity maturation, the mRNA abundance of the target antibody gene is relatively high, which is conducive to the screening of the built library and the cloning of high-affinity antibodies (Ichiyoshi et al., 1995; Kasaian et al., 1994). Therefore, in this study, canine DEA1.1 blood group antigen red blood cells were selected as the source of immunity, and dogs negative for DEA1.1 were immunized. This can minimize other non-blood group antigen substances on canine red blood cells to participate in immune stimulation of canine immune system. And it is hoped that the anti-canine DEA1.1 blood group antibody library constructed in this way can reflect the true state of the blood group antibody in the dog as much as possible.

Preferably, the immunization method for the immunized dogs described in S1 is as follows: select canine DEA1.1 blood group antigen red blood cells as the source of immunization, and immunize DEA1.1-negative dogs. In the present invention, the canine peripheral blood lymphocytes immunized with canine DEA1.1 blood group antigen are selected to construct the antibody gene library, which is beneficial to increase the specificity of the antibody library. When the canine peripheral blood lymphocyte mRNA is reverse-transcribed into cDNA, the present invention selects Oligo dT18 for reverse transcription. Since this primer needs to reverse-transcribe the full length of the mRNA to be effective, the extraction of the total mRNA in the peripheral blood Higher requirements.

Although degenerate primers designed to amplify the VH and VL genes of antibody variable regions in mouse, human, rabbit, chicken and other species have been reported (Chiang et al., 1989; Coloma et al., 1991; Orlandi et al. , 1989; Wang et al., 2000), but so far there is no report on degenerate primers for canine antibody variable region genes. The antibody variable region (V region) is composed of 4 framework regions (FR) and 3 CDR regions spaced apart, that is (N-terminal) "FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4" ( C terminal) arrangement. Relatively speaking, the framework region FR coding sequence is relatively conserved. Therefore, it is possible to obtain a complete set of antibody variable region sequences by PCR by designing primers for relatively conserved DNA base sequences on the upper and lower reaches of the antibody variable region gene. The present invention designs and verifies two sets of different degenerate primers for VH and VL genes, and each primer matches the end sequence of the variable region of the heavy and light chains of the canine antibody respectively. In order to increase the diversity of antibody genes, the present invention randomly combines all designed primers in pairs to amplify antibody gene fragments respectively. Avoid situations due to mutual inhibition between primer pairs or low gene template copies. The VH gene of about 430bp and the VL gene of 390bp can be amplified respectively by PCR amplification.

In this study, the traditional three-fragment SOE-PCR method was used to assemble VH, VL and linker. VH and VL are assembled into a scFv Linker with a full length of _45bp through a Linker containing the coding (Gly ₄ Ser) ₃ sequence _. Connect belly. The addition of equal amounts of VH and VL gene fragments is the key to efficient assembly and PCR amplification. When scFv was amplified and assembled by PCR, SfiI and NotI restriction sites were introduced at the 5' and 3' ends of the sFcv, respectively, and the scFv gene was inserted into the pCANATB-5E vector through these two restriction sites. The frequency of SfiI and NotI restriction sites in antibody genes is very low, which can ensure that most scFv genes can be cloned correctly when constructing antibody libraries.

Preferably, the connecting peptide sequence linker of S3 and the primer sequences of SfiI and NotI restriction sites are shown in SEQ ID NO: 34-47.

There are many screening methods for phage antibody library, including pure antigen screening, impure antigen screening, functional screening, selective infection screening, etc. Impurity antigen screening includes cell surface screening, tissue screening, animal in vivo screening, etc. For antigens that cannot be purified or whose properties are uncertain, traditional solid-phase and liquid-phase screening methods have many limitations. In this study, the Dot-ELISA method constructed by ourselves was used to immobilize the dog DEA1.1 blood group antigen on the nitrocellulose membrane, and to screen the antibody by immobilizing the antigen. The cell surface of canine erythrocytes is known to be of indeterminate nature. Its surface not only contains blood group-related specific antigens, but also contains a large number of non-specific antigens, such as proteins, sugars and lipids. Because phage antibodies are easy to combine with these non-specific antigens, it is possible to screen a large number of non-specific antibodies when using a single canine DEA1.1 positive cell for screening. At the same time, a small number of phages expressing highly specific antibodies on the surface are lost, and the screening efficiency is low. In this study, canine DEA1.1 negative canine erythrocytes were used for negative screening of the antibody library and canine DEA1.1 blood group positive antigens were used for positive screening, which greatly improved the screening efficiency.

Preferably, the panning method described in S4 is: using canine blood group antigen-positive and negative erythrocyte membranes, immobilized on nitrocellulose membranes, and performing "adhesion-elution-amplification" screening of single-chain antibody scFv libraries; After each round of screening, the titer of the amplified phage was measured to detect the enrichment result of specific phage.

The input/output ratio of phage antibody is continuously increased through the continuous "adsorption-elution-amplification" process, so that phage clones that bind tightly to canine DEA1.1 blood group antigen can be obtained. In the pCANATB-5E vector, the scFv gene was inserted between the g3 signal peptide and the g3 protein, and an E-Tag tag was added to the 3' end, and connected to the g3 gene through a succinic acid stop codon. The TG1 strain expressed the E-Tag tag at the C-terminus, and the anti-E-Tag antibody was used as the primary antibody to verify the binding activity of the phage antibody. From the bacterial colonies obtained after the third round of screening, 100 clones were randomly selected for Dot-ELISA activity identification, and it was confirmed that the phages resistant to canine DEA1.1 blood type were screened. After gene sequencing and merging of repeated antibody genes, 3 positive clones against canine DEA1.1 blood group antigen were screened out in the experiment. As verified by ELISA method, Clone16 can bind dog DEA1.1 blood group antigen very well with high affinity. The present invention selects the vector pET-32a to construct the recombinant protein particle pET-32a-Clone16, and there is a His-Tag tag downstream of the cloning site, which is beneficial to the high expression of Clone16 and antibody purification. After verification and analysis, the constructed recombinant protein can be well expressed in a soluble manner. The purified Clone16 single-chain antibody protein was verified by Dot-ELISA method in clinical samples, and it has good binding activity.

The application of the primer set in preparing canine blood group identification reagents is provided.

The application of the single-chain antibody in the preparation of canine blood group identification reagent is provided.

The application of the single-chain antibody library in preparing canine blood group identification reagents is provided.

Compared with prior art, the beneficial effect of the present invention:

Based on the premise that the dog DEA1.1 blood group antigen cannot be purified, the present invention uses DEA1.1 positive canine erythrocytes as immunogens to immunize DEA1.1 negative dogs. After multiple immunizations, a good immune effect is obtained and a large amount of Anti-canine DEA1.1 polyclonal antiserum.

The present invention successfully constructs degenerate primers for dogs, successfully establishes a canine phage display antibody library through the degenerate primers, and finds through identification that the designed primers can amplify diversified single-chain antibody scFv gene combinations. Calculated by the library capacity, the canine DEA1.1 blood group antigen immune library established in this study has a capacity greater than 7×10 ⁵ , which can be considered as a large enough immune library. The sequences obtained by random selection and sequencing showed that the constructed canine single-chain antibody library has diversity.

Based on the Dot-ELISA method, the present invention immobilizes the canine DEA1.1 blood group antigen on the nitrocellulose membrane, and uses the phage antibody immune library to carry out three rounds of screening, and finally screens three strains that can combine with the canine DEA1.1 blood group antigen single-chain antibody. Clinical samples verified that Clone16 has strong binding properties. The pET-32a-Clone16 recombinant protein was constructed based on the Clone16 gene for prokaryotic expression, and the purified antibody had binding activity. It provides a solid foundation for the preparation of canine DEA1.1 blood group identification reagent.

Description of drawings

Figure 1 is the gel electrophoresis diagram of canine VH, _Vκ and _Vλ PCR amplification;

Figure 2 is the amplification result of the heavy chain variable region primer pair; 1-7 upstream primers are JH1-2, and 8-14 upstream primers are JH3;

Figure 3 is the verification of the amplification of the light chain variable region kappa primer pair; 1 to 9 are the amplification of the kappa For 1 to 9 primer combination;

Figure 4 is the amplification verification of lambda primer pairs in the light chain variable region; (A): 1-8 are JL1 and VL1-8; 9-17 are JL2 and VL1-8; (B) 1-8 are JL3 and VL1-8 8; 9-17 are JL4 and VL1-8; (C): 1-8 are JL5 and VL1-8; 9-13 are JL1-5 and VL9;

Fig. 5 is the fusion PCR amplification gel electrophoresis picture of canine scFv;

Figure 6 is the identification of scFv phage library plasmid digestion;

Figure 7 is the panning Dot-ELISA identification of the phage scFv library;

Figure 8 is the amino acid sequence of the dog-derived scFv gene against dog DEA1.1 blood group antigen;

Figure 9 shows Clone16 gene cloning and identification; M: Marker DL2000; 1: PCR identification of positive Clone16 bacteria solution; 2-3: PCR identification of pET-32a-Clone16 positive bacteria; 4: Negative control;

Figure 10 is the double enzyme digestion identification of recombinant plasmid pET-32a-Clone16;

Figure 11 is the SDS-PAGE results of fusion protein expression induced by IPTG at different times; M: protein molecular mass standard; 1: before vector pET-32a induction; 2: 6h after vector pET-32a induction; 3 recombinant plasmid pET-32a-Clone16 Before induction; 4: 1h after induction with recombinant plasmid pET-32a-Clone16; 5: 2h after induction with recombinant plasmid pET-32a-Clone16; 6: 3h after induction with recombinant plasmid pET-32a-Clone16; 7: recombinant plasmid pET-32a- 4h after induction of Clone16; 8: 5h after induction of recombinant plasmid pET-32a-Clone16; 9: 6h after induction of recombinant plasmid pET-32a-Clone16;

Figure 12 shows the solubility analysis of recombinant protein; M: protein molecular weight standard; 1: lysis precipitate 4 hours after pET-32a-Clone16 induction; 2: lysis supernatant 4 hours after pET-32a-Clone16 induction;

Figure 13 is the result of Western-Blot identification of Clone16 single chain antibody; M: protein marker; 1: lysed precipitate; 2: lysed supernatant; 2: negative control;

Figure 14 is the Dot-ELISA activity analysis of clinical samples of single-chain antibody Clone16;

Figure 15 is a map corresponding to the gene sequence and amino acid sequence of the single-chain antibody Clone16;

Figure 16 is the map corresponding to the gene sequence and amino acid sequence of the single-chain antibody Clone1;

Figure 17 is a map corresponding to the gene sequence and amino acid sequence of the single-chain antibody Clone5.

detailed description

The present invention will be further elaborated below in conjunction with the accompanying drawings and specific embodiments of the specification. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not specify specific conditions in the following examples, usually follow the conventional conditions or the conditions suggested by the manufacturer. Unless otherwise defined, the molecular cloning techniques used in the specific embodiments are well-disclosed conventional techniques in the art, and all professional and scientific terms used herein have the same meanings as are familiar to those skilled in the art.

Example 1 Amplification of scFv gene

(1) Isolation of Canine Peripheral Blood Lymphocytes

Immune DEA1.1-negative dogs with canine DEA1.1-positive antigen red blood cells, and immunize 5ml of 5% red blood cells through the abdominal cavity and vein each time. or heparin) anticoagulant. Whole blood was diluted with an equal volume of PBS or 0.9% NaCl (normal saline). Add a certain volume of separation liquid into the centrifuge tube, spread the diluted blood sample above the liquid surface of the separation liquid, and keep the interface between the two liquid surfaces clear. The volume ratio of separation medium, anticoagulated undiluted whole blood, and PBS (or normal saline) is 1:1:1. At room temperature, centrifuge with a horizontal rotor at 700-800g (2000-2500rpm) for 20-30min. After centrifugation, the bottom of the centrifuge tube is red blood cells, the middle layer is the separation solution, the top layer is the plasma/tissue homogenate layer, and between the plasma layer and the separation solution layer is a thin and dense white film, which is mononuclear cells (including lymphocytes and monocytes) layer. Carefully pipette the buffy coat into another centrifuge tube. Dilute to a certain volume with PBS and mix by inversion. At room temperature, centrifuge at 250g (1000rpm) with a horizontal rotor for 10min, and discard the supernatant. Repeat washing 1 to 2 times.

(2) Extraction of total RNA from canine lymphocytes

Count the isolated canine lymphocytes, divide them into about 10 ⁷ tubes and put them into 1.5mL centrifuge tubes, add 750 μL Trizol Ls Reagent to the tubes to directly lyse the cells, vibrate vigorously, and let stand at room temperature for 5 minutes; add 200 μL Chloroform, mix well at room temperature and let stand for 1-2min, then centrifuge at 10,000r/min for 15min at 2-8°C, then take out the centrifuge tube and carefully absorb 500μL of the supernatant and add another new DEPC (diethyl pyrocarbonate) Add 500 μL of isopropanol to the treated 1.5mL centrifuge tube (mix the supernatant with isopropanol in equal proportions) and mix well; then centrifuge at 12000g for 10min at 2-8°C, take out the centrifuge tube carefully Pour off the supernatant, then add 1000μL 75% ethanol, then centrifuge at 8000g for 5min at 2-8°C, take out the centrifuge tube and pour off the supernatant carefully, invert the centrifuge tube on the filter paper to make it fully dry at room temperature; finally add 11.5 μL of DEPC-treated water was used to obtain total RNA from canine lymphocytes, which was directly reverse-transcribed or frozen at -20°C for later use.

(3) First strand cDNA synthesis

Add the following reagents to one sterile Eppendorf tube: 1 µL Oligo(dT)18 (0.5 µg/µL), 10 µL total RNA, 1 µL DEPC water for a total volume of 12 µL. Immediately place on ice after reacting at 70°C for 5 minutes. Add the following reagents: 4 μL 5×buffer (first strand), 1 μL RNase inhibitor, 1 μL 10 mM dNTP. Mix gently and incubate at 37°C for 5min. Add 1 μL of reverse transcriptase and mix gently by pipetting up and down to avoid generating air bubbles. Incubate at 42°C for 60 minutes, terminate the reaction at 70°C for 10 minutes, and place on ice. The product can be used for second strand synthesis or stored at -20°C.

The total RNA of canine peripheral blood lymphocytes extracted by agarose gel electrophoresis showed that two bands of 18s RNA and 25s RNA were clearly visible, indicating that the obtained total RNA was in good integrity. After being reverse-transcribed into the first-strand cDNA by the random primer Oligo(dT)18, the heavy chain variable region primer was used to verify whether the gene was available. As shown in Figure 1, a 430bp fragment can be amplified according to the heavy chain primer, and a 390bp fragment can be amplified by the light chain variable region primer, indicating that the extracted RNA can be used for the next experiment.

(4) Primer design

According to the published antibody gene sequences of dogs, the sequences were classified, and the gene primers for amplifying the heavy chain variable region gene and the light chain variable region gene primers of the antibody were designed in the conserved region. Design primers containing restriction sites and linker sequences according to the light and heavy chain primers (Table 1, 2). The primer list is as follows:

Table 1 Primers for variable regions of canine heavy and light chain antibodies

Table 2 Construction primers of canine scFv gene

Since there is no mature primer set for the canine phage single-chain antibody library, the present invention designed multiple sets of primer pairs, heavy chain variable region primer pairs, and primer sequence comparison results based on the published canine antibody gene sequence analysis. The heavy chain variable region FR1 was divided into two regions. The FR4 region is divided into 7 regions, and the primers are designed respectively, the primer set is verified by cross combination, and the availability of the primer pair is verified by amplification. The verified results are shown in Figure 2. The combination of upstream and downstream heavy chain primers can amplify a specific band of 430bp respectively. According to the results of primer sequence alignment, the kappa variable region FR1 was divided into 9 regions, and FR4 was divided into 1 region, primers were designed respectively, and the primer set was cross-combined to verify. The verified results are shown in Figure 3. The pairwise combination of upstream and downstream light chain kappa primers can amplify a specific band of 390bp respectively. According to the sequence alignment results of the primers, the lambda variable region FR1 was divided into five regions. The FR4 region was divided into 9 regions, primers were designed respectively, and the primer set was cross-combined to verify. The verified results are shown in Figure 4. The combination of upstream and downstream of light chain lambda primers can amplify a specific band of 390bp respectively.

(5) Antibody variable region gene amplification

The VH and VL gene amplification system is as follows:

VH and VL gene reaction procedure:

(6) Amplification of scFv gene

VH and VL amplified gene product fragments were recovered according to the Tiangen gum recovery kit. The purified fragment was used as a template, and the corresponding light and heavy chain fragments were amplified with linker-added primers. The reaction system and procedure are as follows:

reaction system:

Reaction procedure:

The amplified VLL (the product amplified by adding a linker and restriction site to the light chain) and VHH (the product amplified by adding a linker and restriction site to the heavy chain) were used to construct the scFv gene through the following reaction procedure. The reaction system is as follows:

The reaction procedure is:

After the reaction is completed, add upstream and downstream primers with restriction sites to each tube, 0.5 μL each;

Using the purified variable region gene fragments VH, V _λ and V _κ gene fragments as templates, carry out secondary PCR amplification, at the 5' end of the heavy chain variable region VH and the light chain variable region V _λ , V _κ The 3' ends have been inserted into SfiI and NotI restriction sites, respectively. A linker fragment has been inserted at the 3' end of the heavy chain variable region VH and the 5' end of the light chain variable region V _λ and V _κ to make the middle of the heavy chain and the light chain complementary, and the middle 45bp code (Gly ₄ Ser ) ₃ connecting peptide, this soft and easy to bend connecting peptide can guarantee not to affect the activity of single chain antibody. The amplification result after fusion PCR is shown in Figure 5, and the gene fragment is about 850bp. It is consistent with the theoretical calculation value.

Example 2 Construction and panning of phage antibody library

(1) scFv linked to pCANTAB-5E

Proceed as follows:

Ligate at 16°C for 12-16 hours, and extinguish T4 ligase at 70°C for 10 minutes.

(2) Preparation of electrotransformation competent bacteria TG1

Resuspend the ligated vector in 1 mL LB medium. Incubate overnight at 37°C, 250rpm. Inoculate into mmp (minimal medium plate) medium and culture overnight (12h) at 37°C. Pick a single colony and culture in 5mL LB medium, 37°C, 250rpm, 10h. Take 900 μL of Ecoli. TG1 cells in the stable growth phase, add 250 μL of sterilized 80% glycerol, mix well and store at -70°C. Specifically include the following steps:

A. Pick a single colony from the micro-medium plate, inoculate it in 10mL 2×YT medium, and cultivate it overnight at 37°C with shaking;

B. Dilute the above-mentioned overnight bacteria 1:100 and inoculate them into 1000mL fresh 2×YT culture solution, culture with shaking at 37°C until OD600=0.5-0.7, and incubate for about 2.5-3 hours;

C. Place the above-mentioned Erlenmeyer flask with the bacterial solution in an ice bath for 15-30 minutes;

D. Centrifuge at 4000g for 20min at 4°C. Remove the supernatant, and resuspend the cell pellet with 1000 mL of pre-cooled sterile 1 mmol/L Hepes (pH 7.0) solution;

E. Centrifuge at 4000g for 20min at 4°C. Remove the supernatant, and resuspend the cell pellet with 500 mL of pre-cooled sterile 1 mmol/L Hepes (pH 7.0) solution;

F. Centrifuge at 4000g for 20min at 4°C. Resuspend the cells with 20 mL of 1 mmol/L Hepes (pH70) solution containing 10% glycerol;

G. Centrifuge at 4000g at 4°C, and finally resuspend the cell pellet with 1mL 10% glycerol, the total amount is about 2mL;

H. Aliquot into small tubes, 100 μL per tube, put in an ice bath for later use, or immediately freeze in dry ice and store at -70°C.

(3) Construction of scFv library

Add 10 μL of the purified scFv gene ligation product to 40 μL of Ecoli. TG1 competent, and transform by electric shock at 25uF, 2.5kV, 200 ohms, immediately add 1 mL of pre-warmed LB to mix well, and shake gently on a shaker at 37°C for 1 hour. The bacterial solution was coated on the SOBAG culture plate, and the bacterial solution was diluted to measure the storage capacity, and all the culture plates were incubated overnight at 30°C. The next day, the colonies were eluted from the plate with 2×YT medium, added glycerol to a concentration of 20%, and frozen at 70°C.

(4) Detection of library capacity

For the scFv library established by electrotransformation, take 10 μL of the transformed bacterial liquid, dilute it according to the 10-fold ratio, and take a certain amount to coat the SOBAG plate, and make the other two transformed bacterial liquids as a control. Cultivate at 30°C for 20-24 hours. Calculate and estimate the library capacity based on the number of colonies on the plate coated with different dilutions: library capacity (cfu) = number of colonies × dilution factor, store the plate at 4°C.

After electroporation, the product was diluted 10 times, and the conversion efficiency was detected, and the conversion efficiency was found to be 10 ⁶ . 20 colonies were randomly picked from the plate for PCR detection. The results showed 17 positive results, and the actual storage capacity of the constructed scFv library was calculated at 7×10 ⁵ . The positive colonies detected by PCR were identified by NotI and SfiI double enzyme digestion after amplifying and extracting the plasmid. The results are shown in Figure 6. After electrotransformation, the positive bacteria extracted the plasmid and identified it by double enzyme digestion. As a result, a specific band was obtained at the position of 850 bp.

Add an appropriate amount of 2×YT medium to the coated plate of the experimental group, scrape the bacterial liquid into the medium with a spreading rod, collect the medium and mix it evenly, and at this time the antibody library is preserved in the form of bacterial liquid. Part of the bacterial liquid was taken for phage antibody rescue experiment, and the remaining bacterial liquid was stored at -70°C by adding glycerol with a final concentration of 30%.

(5) Rescue phage antibody library

A. Take 900 μL of undiluted electroporated scFv library cells and add 9.1 mL of 2×YT-AG medium;

B. Shake culture at 30°C and 250rpm for 1 hour until OD600=0.5, so that the scFv library cells are in the logarithmic growth phase and cannot overgrow;

C. Add 50μL 20mg/mL Amp ⁺ and 4×10 ¹⁰ pfu auxiliary M13K07, let stand at 37°C for 10min, and incubate on a shaker at 250rpm for 1h;

D. Then centrifuge at 1500g for 10min, discard the supernatant and keep the cells. Add 10mL (200mL) 2×YT-AK medium to resuspend the bacteria. Shake at 200rpm in a shaker and culture overnight at 37°C;

E. Centrifuge at 1500g for 20min, take the supernatant into a new centrifuge tube, and store at 4°C. And the supernatant was taken out to infect TG1 in logarithmic growth phase, coated with SOBAG culture plate, and the titer of prepared phage was measured.

(6) Enrichment and panning of single-stranded phage library

Canine DEA1.1 negative and positive antigen erythrocytes were coated on the nitrocellulose membrane. Then block and wash. Specifically include the following steps:

A. To prepare the purified phage antibody, completely immerse the prepared canine DEA1.1 negative antigen nitrocellulose membrane in the phage antibody solution. Incubate at 37°C for 2 hours, and shake gently 3 to 5 times during the period;

B. Take out the canine DEA1.1 negative antigen nitrocellulose membrane and discard it. Completely immerse the prepared canine DEA1.1 positive antigen nitrocellulose membrane in the phage antibody solution. Incubate at 37°C for 2 hours, and shake gently 3 to 5 times during the period;

C. Wash the nitrocellulose membrane with PBS 20 times, each time rinsed;

D. Elute the nitrocellulose membrane with glycine-hydrochloric acid (pH2.2) eluent, incubate at room temperature for 10 minutes, blow and blow the nitrocellulose membrane repeatedly without interruption, add Tris-HCl (pH7.0) neutralizing solution, and collect all liquids;

E. Add 100 μL of phage to 1 mL of freshly prepared TG1 bacteria in logarithmic growth phase, incubate at 37°C for 1 hour, take 100 μL of culture solution, and dilute the bacteria solution 10 times with 2×YT medium (1:10, 1:100, 1 : 1000, 1: 10000), coated SOBAG plates, cultured overnight at 30°C, and calculated the colony forming units, and at the same time coated the culture plates with TG1 empty bacteria in the logarithmic growth phase as a negative control;

F. Collect the colonies of the SOBAG plate with 2×YT medium, resuspend in 2×YT-AG medium containing 15% glycerol, that is, the phage antibody selected in the first round of affinity selection, and freeze in -70°C;

G. Rescue the phage antibody culture liquid obtained in the first round of affinity selection. And do the second round of screening, in the second round of screening, increase the canine DEA1.1 negative antigen nitrocellulose membrane by 2 times and increase the action time by 2 times.

Canine DEA1.1 antigen-positive and negative erythrocyte membranes were immobilized on nitrocellulose membranes, and the phage single-chain antibody variable region antibody library was screened for three rounds of "adhesion-elution-amplification". After each round of screening, the amplified phages were titered to detect the enrichment results of specific phages (Table 3). It was shown that with the increase of panning times, the number of eluted phages also increased, and the output rate after the third round of affinity screening was about 1.125×10 ³ times higher than before. It shows that the phages that can combine with DEA1.1 positive antigen erythrocytes are highly enriched.

Table 3 Enrichment results of specific phages

Example 3 Detection of Positive Phage Single-Chain Antibody

(1) Positive phage antibody gene amplification and sequencing

A. Add 400 μL of 2×YT-AG medium to 96 tubes, randomly pick 96 single clones from the SOBAG plate after the third round of elutriation, and culture them overnight at 200 rpm at 30°C;

B. Add 400 μL of 2×YT-AG medium to a new tube, take 40 μL of overnight cultured cells into a new tube, and save the remaining bacterial solution in glycerol for later use;

C. Take a new tube and incubate at 30°C for 2 hours at 200 rpm. Centrifuge at room temperature at 1500g for 20min, discard the supernatant;

D. Centrifuge to prepare 50mL 2×YT-AI medium, transfer 400μL to each tube;

E. Cultivate at 30°C for 3-4 hours, 200rpm. Centrifuge at 1500 g for 20 min at room temperature;

F. Carefully transfer 320 μL of supernatant to a new tube. Add 80 μL blocking solution to block 320 μL supernatant, incubate at room temperature for 10 min, and use Dot-ELISA to detect recombinant phage antibody (HRP/Anti-ETag conjugate).

From the phage-infected E.coli TG1 in the third round of screening, 96 clones of a single colony were randomly selected, superinfected to prepare the phage antibody supernatant, and the binding activity of the anti-DEA1.1 antibody contained in the supernatant was detected by Dot-ELISA method. DEA1.1-positive polyclonal antibody, positive phage antigen, DEA1.1-negative antigen, and PBS were used as negative and positive controls, respectively. Among them, the Dot-ELISA detection results of the supernatants of 12 strains were darker, and were judged as positive binding (Figure 7). It shows that these phage antibodies have the activity of specifically binding DEA1.1 blood group antigen.

Add the positive phage antibody samples detected by Dot-ELISA to 2 mL of 2×YT-AG medium in a 10 mL centrifuge tube, culture at 200 rpm at 30°C overnight. Plasmids were extracted and sequenced to analyze the gene sequence.

The obtained positive clones were verified by sequencing, and several clones had the same sequence. Among them, 1, 13, 27, 33 and 51 clones have the same sequence, 5, 28, 34 and 36 clones have the same sequence, 16 and 55 clones have the same sequence, and they are divided into 3 different gene types. There are differences in their CDR sequences. The three clones were named Clone1, Clone5 and Clone16 respectively (Figure 8). Clone1 and Clone5 use the combination of VH and V _K , and _Clone16 uses the combination of VH and Vλ. Clone1 sequencing obtained 873bp scFv, encoding 291 amino acids, the heavy chain variable region VH was 429bp long, encoding 143 amino acids, and the light chain variable region VK was 399bp long, encoding 133 amino acids. Clone5 sequencing obtained 858bp scFv, encoding 286 amino acids, the heavy chain variable region VH was 414bp long, encoding 138 amino acids, and the light chain variable region VK was 399bp long, encoding 133 amino acids. Clone16 sequencing obtained 870bp scFv, encoding 290 amino acids, the heavy chain variable region VH is 435bp long, encoding 145 amino acids, the light chain variable region _VK is 390bp long, encoding 130 amino acids, and the connecting peptide sequence is 45 long bp, encoding 15 amino acids. Using Megalign software to compare the obtained clones, it was found that the variation of CDR1 and CDR2 regions of VH was relatively small, and the variation of CDR3 was relatively large. The VL variation of Clone1 and Clone5 was small.

(2) Canine blood group antigen ELISA detection

In this study, the blood of 56 dogs was collected, and the red blood cell membrane was extracted. The DEA1.1 blood types of 56 dogs were detected by antiglobulin test and polybrene medium detection reagent. The test results showed that 30 dogs were positive for DEA1.1 blood type and 26 dogs were negative for DEA1.1 blood type. Since the scFv single-chain antibody initially obtained in this study is a monovalent antibody, it cannot agglutinate red blood cells. Therefore, the ELISA using the collected and extracted canine DEA1.1 red blood cells as the antigen was used to initially detect the binding activity of the antibody in the induced supernatant to the membrane antigen of DEA1.1 positive red blood cells.

According to the results of ELISA detection, it was found that the single-chain antibodies obtained from the three clones had very different binding activities with 56 canine blood erythrocyte membrane samples. The results of Clone1 and Clone5 are relatively similar, and the test takes the P/N value greater than 2 as the positive judgment standard. As a result, only 28 positive results were detected. The detection P/N of sample 3 and sample 17 were both less than 2, and the positive binding activity obtained by the detection was lower than that of Clone16 (Table 4). The single-chain antibody cloned by Clone16 has the same detection results as the antiglobulin and polybrene medium reagents, and has good binding activity.

Table 4 ELISA activity analysis of clinical samples of single chain antibody Clone16

(3) Amplification and purification of positive phage antibody genes

According to phage single-chain antibody screening, gene amplification sequencing results and ELISA identification results. Use the positive sample bacterial liquid as a template to carry out PCR amplification of the bacterial liquid, and the PCR reaction amplifies 898 bp of the Clone16 gene for the next step of the experiment. The upstream primer is: 5'-AGC GGATCC ATGGAGTCTGTGCTCAGC-3' contains Hind III site. The downstream primer is: 5'-ATA TGTCGA AAGGACGGTCAGTGGGGTTCC-3' contains Xho I restriction site.

The reaction system is:

The PCR amplification conditions are:

PCR products were purified and detected by 1% agarose gel electrophoresis. There is an amplified fragment at about 898bp (Figure 9), which is consistent with the expected fragment size.

Example 4 Expression and purification of canine single-chain antibody Clone16

(1) Construction of expression plasmid pET-32a-Clone16

Both the PCR product obtained above and the empty vector pET-32a were subjected to a double enzyme digestion reaction with Hind III and Xho I. The enzyme digestion system is as follows:

After mixing, place in a 37°C water bath for 4 hours. Afterwards, the digested products were detected on 1.5% agarose gel electrophoresis. The digested product was purified with a DNA purification kit.

T4 DNA ligase was used to perform a ligation reaction on the obtained double digestion product of the vector pET-32a and the double digestion product of the Clone16 gene target fragment. The concentrations of both were measured with a spectrophotometer. The ratio of the number of Clone16 gene fragments to the number of pET-32a expression vector molecules in the ligation system is about 3-10:1.

The reaction system is as follows:

After mixing, set the connection instrument at 16°C for more than 15 h.

After the positive colonies picked by the recombinant plasmid pET-32a-Clone16 were identified by PCR, the plasmids extracted from the expansion culture were identified by double enzyme digestion. After enzyme digestion, a fragment with a size of 892bp and a vector fragment with a size of 5600bp can be obtained, and the fragment obtained by double digestion is consistent with the expected size, as shown in Figure 10. The sequencing results were consistent with expectations, and no mutation occurred, indicating that the plasmid was constructed correctly.

(2) Screening and preparation of recombinant expression plasmids

Follow the steps below to transform the ligation product into competent DH5α:

A. Add 5 μL of the ligation product to 50 μL DH5α competent cells under sterile conditions, flick and mix well, and place in ice bath for 30 minutes;

B. Quickly take the transformation system out of the ice bath, put it in a water bath at 42°C for 90 s, and be careful not to shake the centrifuge tube;

C. Quickly place the centrifuge tube in the ice bath for 5 minutes;

D. Add 700 μL LB liquid medium, shake and culture at 37°C 150 r/min for 45 min;

E. Centrifuge the culture at 4000g for 4 min, discard part of the supernatant under sterile conditions, mix the precipitate, and spread the suspension evenly on the LB agar plate containing Amp ⁺ (100μg/mL);

F. Leave the plate at room temperature until the liquid is completely absorbed;

G. Place the plate in a 37°C incubator and incubate for 12-18 hours. When the colony grows well, take out the plate and store it at 4°C for later use.

PCR was performed on the colonies for preliminary identification. The identified positive bacterial liquid was expanded and cultured, and the plasmid was extracted and identified. The recombinant plasmids that were positive in the identification of plasmid PCR and double enzyme digestion were selected and sent to Huada Gene Co., Ltd. for sequencing. This plasmid was named pET-32a-Clone16.

(3) Induced expression of pET-32a-Clone16 single-chain antibody

The correct plasmid identified by sequencing was transformed into competent cells of expression bacteria BL21 (DE3) according to the above steps, cultured overnight at 37°C, and a single bacterial liquid was picked for culture, and part of the bacterial liquid identified as positive recombinant bacterial liquid was added to a final concentration of 10 % sterilized glycerol and stored at -80°C. At the same time, do pET-32a empty vector control.

Pick positive colonies and inoculate them into 5 mL of LB liquid medium containing Amp ⁺ , shake and culture overnight at 37°C as seed liquid, and inoculate 1% of the seed liquid into 3 mL of LB liquid medium containing Amp ⁺ , at 200 r /min Shaking culture at 37°C until OD600 was 0.4-0.6, induced Escherichia coli BL21 (DE3) to express the fusion protein with IPTG. 4 h after induction, take 1 mL of the bacterial solution, centrifuge at 12,000 g for 1 min, discard the supernatant, resuspend the bacterial cells with 100 µL of 1×SDS-PAGE loading buffer, boil in boiling water for 5-10 min, and place at 12,000 g at 4°C After centrifugation for 2 min, the supernatant was taken for SDS-PAGE analysis, and samples of uninduced transformed bacteria, BL21 (DE3) bacteria, and pET-32a empty vector transformed bacteria were set as controls.

Set the final concentration of the inducer IPTG to 2mM, 1mM, 0.8mM, 0.6mM, 0.4mM and 0.2mM to optimize the concentration of the inducer, and place them at 37°C for 1h, 2h, 3h, 4h, 5h, and 6h to optimize the induction time .

After the recombinant plasmid pET-32a-Clone16 transformed (DE3) competent cells, the set time was 1h, 2h, 3h, 4h, 5h, 6h, induced by IPTG, and compared with the non-induced positive bacteria after SDS-PAGE electrophoresis, it was found that Obvious specific protein bands appeared at about 34KDa respectively, which was consistent with the expected recombinant protein size. For the SDS-PAGE analysis of the induced products of the recombinant bacterial solution at different time periods, the induction time of the recombinant protein was selected as 4 h (Figure 11).

Add IPTG with final concentrations of 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 mmol/L, respectively, and induce expression at 37°C for 4 hours. According to SDS-PAGE electrophoresis analysis, there is no significant difference between the concentration of IPTG and the amount of induced expression. The induction concentration of IPTG was selected 1.0mmol/L.

(4) Analysis of antibody expression and solubility

Protein purification was performed according to the instruction manual of Ni Sepharose 6 Fast Flow from GE Healthcare. Specific steps are as follows:

Centrifuge the supernatant at 12,000 g at 4°C for 20 min, collect the supernatant, filter it with a 0.45 μm microporous membrane, and store it at 4°C; wash off the ethanol in the column with 10 column bed volumes of double-distilled water; use 10 Double the volume of Binding Buffer to equilibrate the column bed; put the sample filtered through a 0.45 μm microporous membrane on the column; wash with 10 times the volume of Binding Buffer; collect the eluate after standing for 10 min with 4 mLElution Buffer, and use a UV spectrophotometer After the protein content of the sample was determined, it was frozen at -20°C; the column was equilibrated with 10 times the volume of Binding Buffer; the column bed was washed with 10 times the volume of double distilled water; the column bed was filled with 20% ethanol.

(5) Western blot analysis of Clone16 antibody

Take an appropriate amount of protein samples and perform SDS-PAGE electrophoresis. After electrophoresis, take out the gel, cut a piece of nitrocellulose membrane with the same size as SDS-PAGE, and six pieces of 3mm filter paper. First equilibrate the filter paper and nitrocellulose membrane in the pre-cooled transfer solution for 15 minutes, and prepare according to the following structural hierarchy: 3 layers of filter paper-nitrocellulose membrane-gel-3 layers of filter paper are stacked in order from bottom to top, avoiding up and down filter paper contact. Install electrotransfer and transfer with 15V voltage for 30min.

After the transfer is complete, remove the nitrocellulose membrane and mark it for the next step:

A. Blocking: Take out the transfer membrane, block with 5% skimmed milk powder TBST buffer, overnight at 4°C or shake gently at room temperature for 2h. The blocking solution can be reused;

B. Membrane washing: Discard the blocking solution, wash the membrane with TBST buffer at 37°C 56r/min for 3 times, 5min each time;

C. Primary antibody incubation: Discard the TBST buffer, add canine anti-DEA1.1 blood group antigen polyclonal serum, and incubate at 37°C 50r/min for 2h;

D. Membrane washing: Discard the primary antibody, wash the membrane with TBST buffer at 37°C 56r/min for 3 times, 5min each time;

E. Secondary antibody incubation: Discard the TBST buffer, add goat anti-dog HRP-labeled secondary antibody, and incubate at 37°C 50r/min for 1h;

F. Membrane washing: Discard the secondary antibody, wash the membrane with TBST buffer at 37°C 56r/min for 3 times, 5min each time;

G. TMB staining: add precipitation-type TMB chromogenic solution, and then react at 37°C for 5-15min. Stop the reaction and rinse the plate with distilled water;

H. Record the results: Take pictures to record the experimental results.

After the bacteria were induced to express at the optimal induction time, they were ultrasonically lysed, and an equal volume of supernatant and precipitate were taken for SDS-PAGE electrophoresis detection (as shown in Figure 12). The results showed that there were more target proteins in the supernatant, indicating that The target protein is soluble.

After the protein on the gel after SDS-PAGE electrophoresis was transferred to the NC membrane, the expressed protein was verified by Western blot with a His-tag monoclonal antibody, and it was found that the recombinant protein could display specific binding bands (Figure 13). The expression level was higher in the supernatant.

(6) Canine blood group antigen Dot-ELISA to verify antibody activity

Red blood cell membranes collected and extracted from 56 dogs were used. The Dot-ELISA method was used to detect the binding activity of the purified Clone16 single-chain antibody to the DEA1.1 positive erythrocyte membrane antigen. Canine DEA1.1 negative and positive red blood cells were used as antigens, single-chain antibody was used as primary antibody, and anti-His antibody was used as secondary antibody. To verify the detection of canine DEA1.1 blood group erythrocyte binding activity.

The binding activity of purified Clone16 single-chain antibody was detected by 56 clinical samples of canine blood erythrocyte membrane, and verified by Dot-ELISA method. The results obtained by Clone16 single-chain antibody are shown in Figure 14. A total of 30 canine blood type DEA1.1 positives were detected. 26 dogs were negative for DEA1.1 blood type. The results were consistent with those detected by antiglobulin test and polybrene medium detection reagent. It shows that Clone16 can be well combined with DEA1.1 blood group antigen and can be used in the development of blood group detection reagents.

SEQUENCE LISTING

<110> South China Agricultural University

<120> A canine single-chain antibody and its construction method and application

<130>

<160> 54

<170> PatentIn version 3.3

<210> 1

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aataatatga cctccaccag ggcctggtcc cctc 34

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<213> K9 Vl For-1

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aaggacggtc agttgggttc 20

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<213> K9 Vl For-2

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aataatgagg acggccaggc gggtgccttt g 31

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<213> K9 VH G6 Back SfiI

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<213> K9 VH G102 Back SfiI

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<213> K9 VH Back SfiI

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<212>DNA

<213> K9 JH For linker 1-2

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<212>DNA

<213> K9 JH For linker 3

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<212>DNA

<213> K9 Vk Back linker

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ggcggaggtg gctctggcgg tggcggatcc atgaggttcc cwtctcagct cct 53

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<211> 45

<212>DNA

<213> K9 Jk For NotI-1

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<212>DNA

<213> K9 Jk For NotI-2

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<212>DNA

<213> K9 Vl Back linker-1

<400> 42

ggcggaggtg gctctggcgg tggcggatcc atggsctggt yccctctcmt 50

<210> 43

<211> 50

<212>DNA

<213> K9 Vl Back linker-2

<400> 43

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<210> 44

<211> 52

<212>DNA

<213> K9 Vl Back linker-3

<400> 44

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<211> 51

<212>DNA

<213> K9 Vl Back linker-4

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<212>DNA

<213> K9 Jl For NotI-1

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<212>DNA

<213> K9 Jl For NotI-2

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<210> 48

<211> 870

<212>DNA

<213> Clone16 gene sequence

<400> 48

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tgcgtggcct ctggattcaa cttcagtaac tatgacatga actgggtccg ccaggctcca 180

gggaaggggc tgcagtgggt cgcatacatt agcagtggtg ggatcaacac atactatgca 240

gatgttgtgc agggccggtt caccatctcc agagacaacg ccaagagtat gttgtatctt 300

cagatggaca ggctgagagt cgaggacacg gccatgtatt actgtgcggg tgagggccct 360

tattctagag gttcgggctt attcggttac ggtatggact actggggtcc tggcacctca 420

ctcttcgtgt cctcaggtgg aggcggttca ggcggaggtg gctctggcgg tggcggatcc 480

atgggctggt tccctctcct cctcaccctc cttgctcatt tcacagggtc ctgggcccag 540

cctgtgctga ctcagccacc ctccgtgtct gggtccctgg accagagggt caccattcc 600

tgcactggaa gcagctccaa cgttggctat agcagtagtg tgggctggta ccaacagttt 660

ccaggaagag gccccagaac catcatctat tttgatacta gtcgaccctc gggggtcccc 720

gatcgattct ctggctccaa gtctggcaac acagccaccc tgactatctc tggactccgg 780

actgaggatg gggctcatta ttactgctca tcttgggaca gcggtgtcag agctccggta 840

ttcggctcgg gaacccccact gaccgtccctt 870

<210> 49

<211> 435

<212>DNA

<213> clone16 heavy chain coding sequence

<400> 49

atggagtctg tgctcagctg ggtttccctt gtcgctattt taaaaggtgt ccagagtgag 60

gtgcaactgg tggagtctgg gggagacctg gtgaagcctg ggggatccct gagactctcc 120

tgcgtggcct ctggattcaa cttcagtaac tatgacatga actgggtccg ccaggctcca 180

gggaaggggc tgcagtgggt cgcatacatt agcagtggtg ggatcaacac atactatgca 240

gatgttgtgc agggccggtt caccatctcc agagacaacg ccaagagtat gttgtatctt 300

cagatggaca ggctgagagt cgaggacacg gccatgtatt actgtgcggg tgagggccct 360

tattctagag gttcgggctt attcggttac ggtatggact actggggtcc tggcacctca 420

ctcttcgtgtcctca 435

<210> 50

<211> 390

<212>DNA

<213> clone16 light chain coding sequence

<400> 50

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tgcactggaa gcagctccaa cgttggctat agcagtagtg tgggctggta ccaacagttt 180

ccaggaagag gccccagaac catcatctat tttgatacta gtcgaccctc gggggtcccc 240

gatcgattct ctggctccaa gtctggcaac acagccaccc tgactatctc tggactccgg 300

actgaggatg gggctcatta ttactgctca tcttgggaca gcggtgtcag agctccggta 360

ttcggctcgg gaacccccact gaccgtccctt 390

<210> 51

<211> 45

<212>DNA

<213> clone16 linked peptide coding sequence

<400> 51

ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcc 45

<210> 52

<211> 290

<212> PRT

<213> clone16 amino acid sequence

<400> 52

Met Glu Ser Val Leu Ser Trp Val Ser Leu Val Ala Ile Leu Lys Gly

1 5 10 15

Val Gln Ser Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys

20 25 30

Pro Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Asn Phe

35 40 45

Ser Asn Tyr Asp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Gln Trp Val Ala Tyr Ile Ser Ser Gly Gly Ile Asn Thr Tyr Tyr Ala

65 70 75 80

Asp Val Val Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser

85 90 95

Met Leu Tyr Leu Gln Met Asp Arg Leu Arg Val Glu Asp Thr Ala Met

100 105 110

Tyr Tyr Cys Ala Gly Glu Gly Pro Tyr Ser Arg Gly Ser Gly Leu Phe

115 120 125

Gly Tyr Gly Met Asp Tyr Trp Gly Pro Gly Thr Ser Leu Phe Val Ser

130 135 140

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

145 150 155 160

Met Gly Trp Phe Pro Leu Leu Leu Thr Leu Leu Ala His Phe Thr Gly

165 170 175

Ser Trp Ala Gln Pro Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ser

180 185 190

Leu Asp Gln Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Val

195 200 205

Gly Tyr Ser Ser Ser Val Gly Trp Tyr Gln Gln Phe Pro Gly Arg Gly

210 215 220

Pro Arg Thr Ile Ile Tyr Phe Asp Thr Ser Arg Pro Ser Gly Val Pro

225 230 235 240

Asp Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile

245 250 255

Ser Gly Leu Arg Thr Glu Asp Gly Ala His Tyr Tyr Cys Ser Ser Trp

260 265 270

Asp Ser Gly Val Arg Ala Pro Val Phe Gly Ser Gly Thr Pro Leu Thr

275 280 285

Val Leu

290

<210> 53

<211> 27

<212>DNA

<213> Clone16 upstream primer

<400> 53

agcggatcca tggagtctgt gctcagc 27

<210> 54

<211> 30

<212>DNA

<213> clone16 downstream primer

<400> 54

atatgtcgaa aggacggtca gtggggttcc 30

Claims (5)

1. A canine single-chain antibody Clone 16, characterized in that the coding sequence of the scFv of the antibody is 870 bp long, and the sequence is as shown in SEQ ID NO: 48; wherein the coding sequence of the heavy chain variable region VH is 435 bp long, The sequence is shown in SEQ ID NO: 49; the coding sequence of the light chain variable region VK is 390 bp long, and the sequence is shown in SEQ ID NO: 50; the coding sequence of the connecting peptide is 45 bp long, and the sequence is shown in SEQ ID NO: 51. 2. The canine single-chain antibody Clone 16 according to claim 1, wherein its amino acid sequence is shown in SEQ ID NO:52. 3. A pair of primers for amplifying the gene sequence of the single-chain antibody Clone 16 according to claim 1, characterized in that the sequence is as shown in SEQ ID NO: 53-54. 4. A primer set for constructing a canine single-chain antibody library, characterized in that it includes antibody heavy chain variable region primers, light chain variable region primers and connecting peptide sequence primers, and its primer sequence is as shown in SEQ ID NO: 1～ 47. 5. The application of the single-chain antibody according to claim 1 or 2 or the primer set according to claim 4 in the preparation of canine blood group identification reagents.