African swine fever virus CD2v protein single-chain antibody, gene, primer, device, drug and preparation method

Abstract

The present invention provides a single-chain antibody, gene, primer, device, drug and preparation method of African swine fever virus CD2v protein. The preparation method comprises the following steps: S1, constructing a recombinant plasmid using the nucleotide sequence as described in SEQ ID NO.1, and expressing it through an Escherichia coli expression system; S2, obtaining inclusion body precipitate from the expressed bacteria; incubating the inclusion body precipitate with an inclusion body lysis buffer, and collecting the supernatant; the inclusion body lysis buffer contains urea; S3, dialyzing and renaturing the supernatant to obtain a single-chain antibody of African swine fever virus CD2v protein. The present invention obtains a single-chain antibody against African swine fever virus CD2v protein by urea denaturation and dialysis renaturation.

Description

Technical Field

[0001] The present invention belongs to the field of antibodies of African swine fever virus, and in particular relates to single-chain antibodies, genes, primers, devices, drugs and preparation methods of African swine fever virus CD2v protein. Background Art

[0002] Single-chain antibody (scFv) is a genetically engineered antibody molecule that is usually composed of the variable domain of heavy chains (VH) and the variable domain of light chains (VL) connected by a connecting peptide. It is one of the smaller functional structural units with antibody activity. scFv has a small molecular weight and good tissue penetration. scFv lacks the Fc (Fragment crystallizable) fragment, so its immunogenicity is weak. However, scFv has unique advantages in molecular modification, such as adding specific tag proteins [4], constructing trimers [5] and bispecific antibodies. Currently, scFv has been applied in the fields of molecular diagnosis, targeted drugs, tumor treatment and diagnosis. Compared with the preparation of monoclonal antibodies, scFv can be prepared in prokaryotes such as Escherichia coli (E. coli) or Brevibacillus choshinensis at low cost and high yield.

[0003] African swine fever (ASF) is an acute, febrile and extensive hemorrhagic disease caused by African swine fever virus (ASFV). It mainly infects domestic pigs and wild boars. Virulent strains usually cause acute clinical symptoms in pigs, such as high fever, lethargy, loss of appetite, skin and organ bleeding, etc., with a mortality rate of 100%. ASFV has spread widely around the world since it was first reported in Kenya in 1921.

[0004] Although scFv has unique biological properties and has important application value in the prevention and control of important viral diseases and in the field of scientific research. However, there are technical difficulties in the effective preparation of single-chain antibodies of the African swine fever virus CD2v protein, and the corresponding single-chain antibodies cannot be effectively obtained by directly using conventional methods. In view of this, it is necessary to provide a single-chain antibody, gene, primer, device, drug and preparation method of the African swine fever virus CD2v protein to solve or at least alleviate the above-mentioned technical defects of how to effectively prepare the single-chain antibody of the African swine fever virus CD2v protein. Summary of the invention

[0005] The main purpose of the present invention is to provide a single-chain antibody, gene, primer, device, drug and preparation method of African swine fever virus CD2v protein to solve the above-mentioned technical problem of how to effectively prepare the single-chain antibody of African swine fever virus CD2v protein.

[0006] To achieve the above objectives, the present invention provides a gene encoding a single-chain antibody of the CD2v protein of African swine fever virus, wherein the gene has a nucleotide sequence as shown in SEQ ID NO.1.

[0007] The present invention also provides a single-chain antibody against the African swine fever virus CD2v protein, wherein the single-chain antibody is expressed using any of the genes described above.

[0008] The present invention also provides a single-chain antibody against the African swine fever virus CD2v protein, wherein the single-chain antibody has an amino acid sequence as shown in SEQ ID NO.2.

[0009] The present invention also provides a method for preparing a single-chain antibody of the African swine fever virus CD2v protein, comprising the steps of:

[0010] S1, constructing a recombinant plasmid using any of the genes described above, and expressing it through an E. coli expression system;

[0011] S2, obtaining inclusion body precipitates from the expressed bacteria; incubating the inclusion body precipitates with inclusion body lysis buffer, and collecting the supernatant; the inclusion body lysis buffer contains urea;

[0012] S3, dialyzing and renaturing the supernatant to obtain a single-chain antibody against the African swine fever virus CD2v protein.

[0013] Furthermore, the E. coli expression system includes the E. coli strain BL21 or the E. coli strain Shuffle T7.

[0014] Furthermore, the temperature expressed in step S1 is 22-40°C.

[0015] Furthermore, the concentration of urea in the inclusion body lysis buffer is 6-10 mol / L.

[0016] The present invention also provides an African swine fever virus detection device, which includes any of the single-chain antibodies described above.

[0017] The present invention also provides a drug against African swine fever virus, wherein the drug comprises any of the single-chain antibodies described above.

[0018] The present invention also provides a primer for preparing a single-chain antibody of the African swine fever virus CD2v protein as described above, wherein the primer includes VH-F1, VH-R1, VLκ-F1, and VLκ-R1; or, the primer includes VH-F1 and VLκ-R1;

[0019] The nucleotide sequence of the VH-F1 is shown in SEQ ID NO.3, the nucleotide sequence of the VH-R1 is shown in SEQ ID NO.4, the nucleotide sequence of the VLκ-F1 is shown in SEQ ID NO.5, and the nucleotide sequence of the VLκ-R1 is shown in SEQ ID NO.6.

[0020] The beneficial effects of the present invention include at least:

[0021] The present invention prepares a single-chain antibody of the CD2v protein of the African swine fever virus. The present invention uses the CD2v positive monoclonal antibody hybridoma cell 4B8 as a template, successfully amplifies the heavy chain variable region and light chain variable region genes through degenerate primers, and connects them through GS linker to form a scFv gene sequence. Studies have found that 4B8-scFv is expressed in the form of inclusion bodies in Escherichia coli BL21 (DE3), and a high-purity antibody molecule is obtained through urea denaturation and dialysis renaturation. The antibody has good CD2v protein reactivity, providing a new antibody molecule for ASF immunological prevention and control research.

[0022] Specifically, in order to obtain a single-chain antibody (scFv) specific for the CD2v protein of African swine fever virus (ASFV), the present invention uses CD2v positive monoclonal antibody hybridoma cells as templates, obtains antibody heavy chain variable region (VH) and light chain variable region (VL) genes by PCR amplification, and uses Overlap-PCR to introduce a short peptide (Gly4Ser) 2 between VH and VL to form scFv. It is inserted into the prokaryotic expression vector pET28a (+), and transformed into Escherichia coli BL21 (DE3), induced to express by 1mM IPTG at 25°C, and the refolded scFv protein is evaluated for its reaction activity by ELISA and IFA. The results showed that the monoclonal antibody hybridoma cell line 4B8 with good secretion blocking effect was obtained through blocking ELISA screening of CD2v protein, and the heavy chain variable region, light chain variable region and 4B8-scFv gene connected by GS linker of monoclonal antibody 4B8 were successfully amplified; SDS-PAGE results showed that 4B8-scFv was expressed in E. coli BL21 (DE3) in the form of inclusion bodies, and urea denaturation and dialysis renaturation were used to obtain 4B8-scFv with high purity (relative molecular weight of about 34kDa); indirect ELISA and IFA results showed that 4B8-scFv had good reactivity with CD2v protein. The preparation of CD2v scFv provides a new antibody molecule for in-depth research on the biological function of CD2v protein and ASF diagnosis and prevention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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 some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the contents shown in these drawings without paying creative work.

[0024] Figure 1 This is a graph for evaluating the immunogenicity of the ASFV recombinant CD2v protein in Example 1;

[0025] Figure 2 This is an indirect ELSIA detection diagram of ASFV anti-CD2v monoclonal antibody in Example 2;

[0026] Figure 3 This is a blocking ELSIA assay diagram of the ASFV anti-CD2v monoclonal antibody in Example 2;

[0027] Figure 4This is a gene amplification map of the heavy chain variable region and light chain variable region of the 4B8 monoclonal antibody in Example 3; wherein A is the heavy chain variable region, and B is the light chain variable region;

[0028] Figure 5 This is the gene amplification diagram of scFv in Example 3;

[0029] Figure 6 This is a double enzyme digestion verification diagram of pET28a-CD2v 4B8-scFv in Example 3;

[0030] Figure 7 Schematic diagram of the plasmid construction of pET28a-CD2v 4B8-scFv in Example 3;

[0031] Figure 8 This is an analysis diagram of the antibody expression of ASFV CD2v 4B8-scFv at 37°C in Example 3;

[0032] Fig. 9 This is a denaturation purification analysis diagram of ASFV CD2v 4B8-scFv after expression at 37°C in Example 3;

[0033] Fig.10 This is an analysis diagram of the antibody expression of ASFV CD2v 4B8-scFv at 25°C in Example 3;

[0034] Fig.11 This is a denaturation purification analysis diagram of ASFV CD2v 4B8-scFv after expression at 25°C in Example 3;

[0035] Fig.12 This is an SDS-PAGE analysis chart (ultrafiltration) of the ASFV CD2v 4B8-scFv antibody inclusion body renaturation in Example 3;

[0036] Fig.13 This is an SDS-PAGE analysis diagram (dialysis) of the renaturation of ASFV CD2v 4B8-scFv antibody inclusion bodies in Example 3;

[0037] Fig.14 This is a Western-blot verification diagram of the ASFV CD2v 4B8-scFv antibody inclusion body after renaturation in Example 3;

[0038] Fig.15 This is a graph showing the ELISA test results of the anti-CD2v scFv activity in Example 3;

[0039] Fig.16 This is the indirect immunofluorescence image of 4B8-scFv and CD2v protein in Example 3;

[0040] Fig.17 This is a schematic diagram of the simulation of the homologous structure of the anti-CD2v single-chain antibody in Example 3.

[0041] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with the implementation methods and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] Furthermore, the technical solutions between the various embodiments of the present invention may be combined with each other, but this must be based on the fact that a person of ordinary skill in the art can implement the technology. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention. In the present invention, when conducting comparative tests, except for the conditions that need to be compared, other conditions remain the same.

[0044] In the present invention, the CD2v protein is the African swine fever virus CD2v extracellular domain protein; the competent state BL21 (DE3) and Shuffle T7 were purchased from Beijing Jinsha Biotechnology Co., Ltd.; the Trozl gel recovery purification kit and the plasmid extraction kit were purchased from Omega Bio-Tek; the restriction endonuclease was purchased from Thermo Fisher, USA, the mouse anti-His tag antibody, the horseradish peroxidase (HRP)-labeled goat anti-pig IgG antibody and the horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody were all purchased from Invitrogen, and the 3,3',5,5'-tetramethylbenzidine (TMB) color developing solution was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0045] The present invention provides a gene encoding a single-chain antibody of the CD2v protein of the African swine fever virus, wherein the gene has a nucleotide sequence as shown in SEQ ID NO.1.

[0046] The present invention also provides a single-chain antibody against the African swine fever virus CD2v protein, wherein the single-chain antibody is obtained by expressing any of the genes described above.

[0047] In the present invention, the single-chain antibody of African swine fever virus CD2v protein has an amino acid sequence as shown in SEQ ID NO.2, and its structural feature is that the heavy chain variable region (amino acid sequence is EV KLQESGGGLVQPKGSLKLSCATSGFTFNTNAMNWVRQAPGKGLEWVARIRSKSYNYATY YADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVREGIWGQGTSVTVSSAKTTPPSVY) of the monoclonal antibody (produced by 4B8 hybridoma cells) in the present invention is connected by a (Gly4Ser)2 flexible linker.

[0048] In the present invention, the nucleotide sequence shown in SEQ ID NO.1 is:

[0049] GAGGTAAAGCTGCAGGAGTCTGGTGGAGGATTGGTGCAGCCTAAAGGGTCATTGAAACTCTCATGTGCAACCTCTGGATTCACCTTCAATACCAATGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAAAGTTATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCACAAAGCATTCTCTATCTGCAAATGAACAACTTGAAAACTGAGGACACAGCCATGTATTACTGTGTGAGAGAGGGCATTTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATTCAGGCGGAGGTGGCTCTGGAGGTGGCGGCTCGGATATTGTGCTGACACAGACTACACCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGACCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTAAAAAACAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTTCTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATAGTTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAAATCAAATAA。

[0050] The amino acid sequence shown in SEQ ID NO.2 is as follows:

[0051] EVKLQESGGGLVQPKGSLKLSCATSGFTFNTNAMNWVRQAPGKGLEWVARIRSKSYNYATYYADSVKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVREGIWGQGTSVTVSSAKTTPPSVYS GGGGSGGGGSDIVLTQTTPSLTVTAGEKVTMTCKSSQSLLNSKKQKNYLTWYQQKPGQPPKLLIFWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIK.

[0052] The present invention also provides a method for preparing a single-chain antibody of the African swine fever virus CD2v protein, comprising the steps of:

[0053] S1. Use any of the genes described above to construct a recombinant plasmid and express it through an E. coli expression system.

[0054] The E. coli expression system includes the E. coli strain BL21 or the E. coli strain Shuffle T7; preferably, the E. coli expression system includes the E. coli strain BL21, that is, BL21 (DE3) competent cells; specifically, the recombinant plasmid pET28a-CD2v 4B8-scFv can be transformed into BL21 (DE3) competent cells.

[0055] In the present invention, the expression temperature in step S1 can be 22-40° C., preferably 22-28° C., so as to reduce the expression of irrelevant proteins.

[0056] In the present invention, the expression process may include: inducing expression through isopropyl thiogalactoside, specifically, after the OD600 of the culture system reaches 0.6-0.8, isopropyl thiogalactoside is added to the culture system, and then the culture is continued for 16-20 hours to induce expression.

[0057] S2, obtaining inclusion body precipitate from the expressed bacteria; the process may include: lysing the expressed bacteria and collecting the cell lysis precipitate by centrifugation; then, incubating the cell lysis precipitate with a non-ionic surfactant and collecting the inclusion body precipitate by centrifugation; the incubation may be performed on ice, and the incubation time may be 8-12 minutes; the non-ionic surfactant may include TritonX-100.

[0058] The inclusion body lysis buffer is used to incubate the inclusion body precipitate, and the supernatant is collected by centrifugation; the incubation can be performed on ice, and the incubation time can be 18-22 minutes; the inclusion body lysis buffer contains urea, and the concentration of the urea in the inclusion body lysis buffer is 6-10 mol / L.

[0059] In the present invention, the process of incubating the inclusion body precipitate with an inclusion body lysis buffer comprises: incubating the inclusion body precipitate with an inclusion body lysis buffer for multiple times, during each incubation, the urea concentration in the inclusion body lysis buffer is increased successively, and the supernatant obtained by centrifugation after the last incubation is used for dialysis renaturation; in the present invention, during the last incubation, the urea concentration in the inclusion body lysis buffer is between 6-10 mol / L; in Example 3 of the present invention, a total of 4 incubations are performed, and the urea concentrations in the inclusion body lysis buffer are 2 mol / L, 4 mol / L, 6 mol / L, and 8 mol / L, respectively.

[0060] In the present invention, the inclusion body lysis buffer is prepared as follows: 4.728 g of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCL), 0.336 g of disodium ethylenediaminetetraacetate, 120 / 240 / 360 / 480 g of urea at a final concentration of 2, 4, 6, 8 M, 0.3 g of reduced glutathione (GSH), 0.123 g of oxidized glutathione (GSSG), about 500 mL of ultrapure water is added thereto, the mixture is fully dissolved by a magnetic stirrer, and then the volume is fixed to about 980 mL, the pH is adjusted to 8.0 by concentrated hydrochloric acid and sodium hydroxide, and the mixture is stored in a refrigerator at 4°C for later use.

[0061] S3, dialyzing and renaturing the supernatant to obtain a single-chain antibody against the African swine fever virus CD2v protein.

[0062] The present invention also provides an African swine fever virus detection device, which includes any of the single-chain antibodies described above.

[0063] The present invention also provides a drug for African swine fever virus, which comprises any of the single-chain antibodies described above.

[0064] The present invention also provides a primer for preparing a single-chain antibody of the African swine fever virus CD2v protein as described above, wherein the primer includes VH-F1, VH-R1, VLκ-F1, and VLκ-R1; or, the primer includes VH-F1 and VLκ-R1.

[0065] The nucleotide sequence of the VH-F1 is shown in SEQ ID NO.3, the nucleotide sequence of the VH-R1 is shown in SEQ ID NO.4, the nucleotide sequence of the VLκ-F1 is shown in SEQ ID NO.5, and the nucleotide sequence of the VLκ-R1 is shown in SEQ ID NO.6.

[0066] The present invention successfully amplified the heavy chain variable region (VH) and light chain variable region (VL) of the mouse monoclonal antibody 4B8 by degenerate primers, and successfully obtained the 4B8-scFv gene fragment by Overlap-PCR, and constructed it into the prokaryotic expression vector pET28a+ vector, and successfully expressed CD2V scFv by the Escherichia coli expression system. This is the first report of ASFV CD2v scFv targeting, which provides a new tool for in-depth research on the biological function of ASFV CD2v and the development of diagnostic methods.

[0067] The present invention uses two competent strains in the 4B8-scFv expression process, one is the commonly used exogenous gene expression strain BL21 (DE3) and the other is Shuffle T7, which has been verified by multiple research reports to improve the solubility of single-chain antibodies. The strain has a better oxidation environment and can promote the formation of disulfide bonds through disulfide isomerase (DsbC) and the like to help scFv fold correctly in the strain. Therefore, the present invention transforms the pET28a-CD2v 4B8-scFv plasmid into BL21 (DE3) and Shuffle T7 competent cells respectively and induces expression. Solubility analysis shows that in the two expression strains, most of the 4B8-scFv protein is expressed in the form of inclusion bodies and exists in the cell lysis precipitate, which is consistent with the reported results of the early prokaryotic expression system expressing scFv. In addition, although the existing research used the P17 tag with a size of 33aa in the tail of T7 phage to add to the scFv sequence and found that the tag can promote the solubility of scFv, the present invention attempted to add the P17 tag to 4B8-scFv, but did not achieve the expected effect. Therefore, this method was not used to prepare the single-chain antibody of African swine fever virus CD2v protein.

[0068] The following are specific examples of the present invention:

[0069] Example 1: Immunological evaluation

[0070] 1. Animal testing

[0071] Five healthy susceptible piglets aged 4-5 weeks were selected and inoculated with 1 mL of recombinant CD2v protein (40 μg) mixed with adjuvant, 500 μL each in the left and right necks. A negative control group was set up, and blood was collected every 7 days after the first immunization; a second immunization was performed at an interval of 14 days with the same dose and method; and CD2v indirect ELISA was used to detect changes in antibody levels in serum.

[0072] 2. Indirect ELISA

[0073] Animal experiment ELISA: The recombinant CD2v protein was coated on the ELISA plate at a concentration of 4 μg / mL, 100 μL / well, overnight at 4°C; on the next day, 200 μL / well of BSA blocking solution was added, and the plate was incubated at 24°C for 3 h; the serum was separated from the blood collected in the animal experiment, and the serum was diluted to 25 times with diluent, 100 μL / well, and incubated at 24°C for 60 min; HRP-labeled sheep anti-pig IgG was diluted 1:7000, 100 μL / well, and incubated at 24°C for 30 min; TMB 50 μL / well was added, incubated in the dark for 5 min, and then 2 mol / L sulfuric acid stop solution was added to terminate the reaction, and the OD450 reading was read by an ELISA instrument.

[0074] The results of the immunogenicity evaluation of CD2v protein are as follows:

[0075] See also Figure 1 As shown, CD2v-specific antibodies in immunized pigs were detected by indirect ELISA. The results showed that CD2v-specific IgG antibodies could be detected one week after the first CD2v immunization of pigs, and the antibody level continued to increase. The antibody level increased further after the second immunization, reaching the highest value 14 days after the second immunization and continued to maintain until the 7th week. The results show that the recombinant CD2v protein has good immunogenicity. The immune serum (specific IgG antibody) of the CD2v protein will be used to evaluate the blocking effect of subsequent monoclonal antibodies.

[0076] Figure 1 In the table, P1-P5 are the numbers of the pigs in the immunization group, and N1-N5 are the numbers of the pigs in the blank control group.

[0077] Example 2: Evaluation of monoclonal antibodies

[0078] Mice were immunized with recombinant CD2v protein, spleen lymphocytes were collected and fused with myeloma cells SP2 / 0, and then 5 CD2v-positive hybridoma cells were obtained by indirect ELISA screening, namely 4B8, 5A4, 6C6, 6E11, and 9G2.

[0079] Monoclonal antibody indirect ELISA: The recombinant CD2v protein was coated on the ELISA plate at a concentration of 4 μg / mL, 100 μL / well, at 4°C overnight; on the next day, 200 μL / well of BSA blocking solution was added, and the plate was incubated at 24°C for 3 h; the prepared monoclonal antibody was diluted to different concentrations, 100 μL / well, and the plate was incubated at 24°C for 30 min; HRP-labeled goat anti-mouse IgG was diluted 1:7000, 100 μL / well, and the plate was incubated at 24°C for 30 min; TMB 50 μL / well was added, incubated in the dark for 5 min, and then 2 mol / L sulfuric acid stop solution was added to terminate the reaction, and the OD450 reading was read using an ELISA instrument.

[0080] Monoclonal antibody blocking ELISA: The recombinant CD2v protein was coated on the ELISA plate at a concentration of 1 μg / mL, 100 μL / well, and incubated at 4°C overnight; on the next day, 200 μL / well of BSA blocking solution was added, and the plate was incubated at 24°C for 3 h; the porcine CD2v immune positive serum in Example 1 was diluted 10 times, 100 μL / well, and incubated at 24°C for 60 min; the prepared monoclonal antibody was diluted 1:10000, 100 μL / well, and incubated at 24°C for 30 min; HRP-labeled goat anti-mouse IgG was diluted 1:7000, 100 μL / well, and incubated at 24°C for 30 min; TMB 50 μL / well was added, incubated in the dark for 5 min, and then 2 mol / L sulfuric acid stop solution was added to terminate the reaction, and the OD450 reading was read by an ELISA instrument.

[0081] The results of the CD2v monoclonal antibody assay are as follows:

[0082] The recombinant CD2v protein was used as the coating antigen, and the reactivity of 5 monoclonal antibodies was measured by indirect ELISA, and their ELISA blocking efficiency was evaluated. Figure 2-3 As shown in the figure, the monoclonal antibodies produced by the five hybridoma cells all have good reactivity with CD2v protein; the blocking ELISA results of CD2v immune positive serum show that monoclonal antibody 4B8 has the best blocking effect. The monoclonal antibodies produced by 4B8, 5A4, 6C6, 6E11, and 9G2 are all positive monoclonal antibodies, and only 4B8 monoclonal antibody has a good blocking effect.

[0083] Figure 2 Among them, 4B8, 5A4, 6C6, 6E11, and 9G2 are monoclonal antibodies produced by five hybridoma cell lines, respectively.

[0084] Figure 3 Among them, 4B8, 5A4, 6C6, 6E11, and 9G2 are monoclonal antibodies produced by 5 hybridoma cell lines respectively; Positive is a blocking test using porcine CD2v immune positive serum, and Negative is a test using porcine negative serum.

[0085] Example 3: Preparation of single-chain antibodies

[0086] 1. Antibody heavy chain variable region (VH) and light chain variable region (VL) gene amplification

[0087] After extracting the total RNA of 4B8 hybridoma cells by Trizol, cDNA was obtained by reverse transcription using it as a template. The amplification primers used with cDNA as a template are shown in Table 1. The PCR reaction conditions are 95℃ pre-denaturation for 3min; 94℃ denaturation for 25s, 58℃ for 25s, 72℃ extension for 10s, 35 cycles; 72℃ for 5min.

[0088] Table 1 Antibody heavy chain and light chain amplification primers

[0089]

[0090]

[0091] 2. 4B8-scFv (VH-linker-VL) gene amplification

[0092] The amplified VH and VL genes were used as templates, and the VH-F1 and VLκ-R1 in Table 1 were used as primers to amplify the scFv gene sequence. The PCR reaction conditions were 95°C pre-denaturation for 3 min; 94°C denaturation for 25 s, 58°C for 25 s, 72°C extension for 15 s, 35 cycles; 72°C for 5 min. After the amplification, agarose gel electrophoresis was used to detect whether there was a band at the target size position.

[0093] The results of CD2v 4B8-scFv sequence amplification are as follows:

[0094] Total RNA of 4B8 hybridoma cells was extracted and reverse transcribed into cDNA, which was used as a template to successfully amplify the genes of the antibody heavy chain variable region (VH, 399 bp) and light chain variable region (VL, 369 bp). For details, see Figure 4 As shown. The scFv gene sequence (VH-linker-VL) was successfully obtained by Overlap-PCR. For specific amplification results, see Figure 5 As shown; in the present invention, the nucleotide sequence of the scFv gene is shown in SEQ ID NO.1.

[0095] Figure 4-5 In the figure, M is DL-1000 DNA Marker; 1 corresponds to VH gene; 2 corresponds to VL gene; 3-5 correspond to scFv gene.

[0096] 3. Construction of pET28a-CD2v 4B8-scFv plasmid

[0097] The scFv gene and pET28a(+) vector were double-digested with BamH I and Xho I restriction endonucleases at 37°C for 1 h, purified by gel recovery, ligated overnight at 4°C, transformed into DH5α competent cells by heat shock method, extracted plasmid after shaking culture, enzyme digestion verification and sequencing to obtain pET28a-CD2v 4B8-scFv.

[0098] The results of the plasmid construction are as follows:

[0099] The scFv and pET28a vectors were double-digested by BamH I and Xho I restriction endonucleases, respectively, and the recombinant plasmid pET28a-CD2v 4B8-scFv was constructed by ligation reaction. The double-digestion verification electrophoresis results showed that the expected vector fragment (5329 bp) and the target band (744 bp) were obtained (see Figure 6 The recombinant plasmid is 6079 bp in size (including a 6 bp restriction site at the 3' end). VL and VH are connected by a flexible linker to form scFv, and a His tag is added to the 3' end of VH. For details, see Figure 7 shown.

[0100] Figure 6 In the figure, M is DL-5000 DNA Marker; 1 is the double restriction digestion product of the recombinant plasmid pET28a-CD2v 4B8-scFv.

[0101] 4. Expression of 4B8-scFv and denaturation of inclusion body proteins

[0102] The recombinant plasmid pET28a-CD2v 4B8-scFv was transformed into BL21 (DE3) competent cells and Shuffle T7 competent cells, respectively, and cultured in LB medium supplemented with kanamycin at 37°C for 12 h until the medium became turbid. Then, the culture medium was added to TB medium supplemented with kanamycin at a volume ratio of 1:50, and cultured in a constant temperature shaker at 37°C, 220 rpm for 2 h. After OD600 reached 0.8, isopropylthiogalactoside (IPTG) was added to induce expression for 18 h. The final concentration of IPTG in the culture system was 1 mmol / L. The induced expression was carried out at 25°C and 37°C, respectively. After the expression was completed, the cells were harvested by centrifugation at 4°C, 5,000 rpm, for 15 min. The supernatant was discarded and the cells were collected. The cell pellet was resuspended in PBS and ultrasonically disrupted on ice for 20 min (corresponding to Figure 8 and Fig.10 The supernatant and precipitate were collected and analyzed by SDS-PAGE.

[0103] TritonX-100 was added to the collected cell lysis precipitate, the lysate was incubated on ice for 10 min, and then centrifuged at 20,000 rpm for 20 min to precipitate inclusion bodies, the supernatant was removed, and the precipitate was incubated in inclusion body lysis buffer containing 2M, 4M, 6M, and 8M urea concentrations in sequence (each time incubated on ice for 20 min), 20 mL of inclusion body lysis buffer was used for each incubation, and centrifugation was performed after each incubation (centrifuge conditions 10°C, 20,000 rpm centrifugation for 20 min) to continue to precipitate inclusion bodies, and soluble supernatants were collected. The soluble supernatants were labeled as 2S, 4S, 6S, and 8S, respectively, and analyzed by SDS-PAGE; wherein 2S, 4S, 6S, and 8S correspond to the supernatants obtained after incubation with inclusion body lysis buffer containing 2M, 4M, 6M, and 8M urea concentrations, respectively (corresponding to the precipitated proteins treated with urea solutions of different concentrations).

[0104] The test results are as follows:

[0105] The recombinant plasmid pET28a-CD2v 4B8-scFv was transformed into E. coli strains BL21 (DE3) and ShuffleT7 to induce expression. The results showed that at 37°C ( Figure 8 ) and 25℃( Fig.10 ) conditions, the recombinant scFv protein (inclusion body) with a size of about 34 kDa was successfully induced to express, and during the expression process, the expression level in BL21(DE3) was higher than that in Shuffle T7.

[0106] In order to obtain soluble scFv antibodies, the results of SDS-PAGE electrophoresis after urea denaturation and purification showed that scFv gradually dissolved in urea solution with the increase of urea concentration, and at 25°C, the irrelevant proteins in the 8 mol / L urea-soluble protein group were significantly lower than those expressed at 37°C. For details, see Fig. 9 and Fig.11 shown.

[0107] Figure 8 and Fig.10 In the figure, M is the prestained protein molecular weight standard (26616); T is the total protein (liquid before centrifugation after induced lysis); S is the supernatant after induced lysis; P is the precipitate after induced lysis.

[0108] Fig. 9 and Fig.11 In the figure, M is the prestained protein molecular weight standard (26616); 2M-8M are proteins precipitated by treating with urea solutions of different concentrations, corresponding to 2S, 4S, 6S, and 8S, respectively.

[0109] 5. Ultrafiltration and dialysis refolding of inclusion body proteins

[0110] Ultrafiltration refolding: Select a 50ml ultrafiltration tube of appropriate specifications according to the predicted size of the target protein, add 8M urea protein solution to the ultrafiltration tube, centrifuge at 10℃, 4,000rpm for 10min, until all the urea protein is ultrafiltered. When about 200μL solution remains in the ultrafiltration tube, add PBS prepared in advance, centrifuge at 10℃, 4,000rpm for 10min, repeat twice, and then replace the protein solution with PBS.

[0111] Dialysis refolding: Gradually add ultrapure water to the 8M urea protein solution to dilute the urea concentration until the urea concentration is 2 M. Add 5-10 mL of the diluted urea protein solution to the pre-treated dialysis bag, use PBS as the dialysis fluid, and dialyze at 4°C for 48 hours with constant stirring, and replace PBS every 24 hours.

[0112] 6. Western Blot

[0113] The harvested recombinant protein was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane, blocked with 5% skim milk at room temperature for 3 h, washed with PBST (0.05%) 5 times, 5 min each time, incubated at room temperature for 1 h with mouse anti-His tag antibody (1:3000 dilution) and 1 h with PBST (0.05%), washed 5 times, 5 min each time with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody (1:2000 dilution) as a secondary antibody, incubated at room temperature for 1 h, washed 5 times, 5 min each time with PBST (0.05%), and detected with a DAB commercial kit.

[0114] The recombinant scFv protein induced and expressed at 25°C was renatured (ultrafiltration renaturation or dialysis renaturation), see Figure 12-14 As shown in FIG. 1 , the SDS-PAGE electrophoresis results showed that ultrafiltration failed to obtain the target protein of the correct size and required purity, while dialysis renaturation obtained a recombinant scFv protein of the correct size and high purity (see Fig.13 2 parts); Western-blot identification results showed that the Shuffle T7 competent expression protein treatment group after ultrafiltration did not see the target band of the expected size, while the BL21 (DE3) competent state obtained a small amount of protein with low purity. The results showed that the protein dissolved in urea was dialyzed and renatured, and a scFv protein with high concentration and good purity was successfully obtained. The scFv used in the subsequent experiments of this example was derived from the protein dialyzed product after 8Mol urea treatment of BL21 (DE3) competent expression (induced expression under 25°C).

[0115] Fig.12In the figure, M is the prestained protein molecular weight standard (26616); 1 is the protein ultrafiltration product after Shuffle T7 competent expression and 8Mol / L urea treatment; 2 is the protein ultrafiltration product after BL21 (DE3) competent expression and 8Mol / L urea treatment; 3 is the protein after Shuffle T7 competent expression and 8Mol / L urea treatment; 4 is the protein after BL21 (DE3) competent expression and 8Mol / L urea treatment.

[0116] Fig.13 In the figure, M is the prestained protein molecular weight standard (26616); 1 is the protein dialyzate after Shuffle T7 competent expression treated with 8Mol / L urea; 2 is the protein dialyzate after BL21 (DE3) competent expression treated with 8Mol / L urea.

[0117] Fig.14 In the figure, M is the prestained protein molecular weight standard (26616); 1 is the protein ultrafiltration product after Shuffle T7 competent expression and 8Mol / L urea treatment; 2 is the protein ultrafiltration product after BL21(DE3) competent expression and 8Mol / L urea treatment; 3 is the protein dialyzed product after BL21(DE3) competent expression and 8Mol / L urea treatment.

[0118] 7. scFv indirect ELISA validation

[0119] The recombinant CD2v protein was coated on the ELISA plate at a concentration of 6 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL, 100 μL / well, and incubated at 4°C overnight; the next day, 200 μL / well of BSA blocking solution was added, and the plate was incubated at 24°C for 3 h; the scFv was diluted to 1 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL with diluent, and then 100 μL / well was incubated at 24°C for 60 min; HRP-labeled goat anti-mouse IgG was diluted 1:7000, and then 100 μL / well was incubated at 24°C for 30 min; TMB 50 μL / well was added, incubated in the dark for 5 min, and then 2 mol / L sulfuric acid stop solution was added to terminate the reaction, and the OD450 reading was read by an ELISA reader.

[0120] To verify the reactivity of 4B8-scFv with CD2v, see Fig.15 As shown, the indirect ELISA test results showed that compared with the negative control (0 μg), scFv at a concentration of 1 μg / mL showed a good binding effect with CD2v and was dose-dependent, indicating that scFv has good CD2v protein reactivity.

[0121] Fig.15, 6, 4, 2, and 1 in the horizontal axis represent the CD2V protein coating concentrations, respectively; at the same CD2V protein coating concentration, along the positive extension direction of the horizontal axis, the columns represent scFv concentrations of 20 μg / mL, 10 μg / mL, 5 μg / mL, 1 μg / mL and NC (negative control), respectively.

[0122] 8. Indirect Immunofluorescence

[0123] The pcDNA3.1-CD2v plasmid (which contains the African swine fever virus CD2v extracellular domain target gene) was transfected into 293T cells, with the pcDNA 3.1 vector as a negative control. After fixation with 4% paraformaldehyde for 20 minutes, the cells were washed four times with PBST (0.05%), blocked with 3% BSA at room temperature for 1 hour, and washed four times with PBST (0.05%). 4B8-scFv diluted with 1% BSA (1:1000) was added to the negative and positive controls, incubated at 37°C for 45 minutes, washed four times with PBST (0.05%), Alexa Fluor 488 (1:2000) was added, incubated at 37°C in the dark for 30 minutes, washed four times with PBST (0.05%), and DAPI was added to stain the nucleus for 10 minutes before observation and filming.

[0124] See also Fig.16 As shown, 4B8-scFv was fluorescently stained with Alexa Fluor 488, and the results showed that 4B8-scFv had binding activity with CD2v expressed by 293T cells.

[0125] Fig.16 In the figure, Alexa Fluor 488 is an anti-mouse fluorescent secondary antibody that shows bright green; DAPI is a DNA fluorescent dye that shows blue; Merge is a mixture of the Alexa Fluor 488 image and the DAPI image; CD2V- refers to cells transfected with an empty vector that do not express CD2v; CD2V+ refers to cells transfected with a CD2v vector that express CD2v.

[0126] 9. 4B8-scFv antibody gene sequence analysis and homologous structure simulation

[0127] See also Fig.17As shown, after sequence alignment analysis on the IMGT website, the amplified VH and VL have a high degree of homology (>90%) with the known mouse immunoglobulin sequence, which conforms to the gene structure of the variable region of mouse IgG antibody. Its VH and VL genes are connected by a flexible linker (G4S) 2, thereby further promoting its structural stability. The results of SWISS-MODEL homology simulation show that the similarity between 4B8-scFv and its corresponding template sequence is 68.05%, and the homology ≥30% meets the homology modeling requirements. The quality assessment results of UCLA-DOELAB SAVES show that the ERRAT score of the model is higher than 85 points, and the VERIFY3D score is greater than the standard value of 80%. It shows that the structure has good stability and meets the quality requirements of homologous structure simulation. In the present invention, the amino acid sequence of 4B8-scFv is shown in SEQ ID NO.2.

[0128] The above technical solutions of the present invention are only preferred embodiments of the present invention, and the patent scope of the present invention is not limited thereto. All equivalent structural changes made by using the contents of the present invention specification and drawings under the technical concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A gene encoding a single-chain antibody against the CD2v protein of African swine fever virus, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. A single-chain antibody against the CD2v protein of African swine fever virus, characterized in that: The single-chain antibody is expressed using the gene according to claim 1.

3. A single-chain antibody against African swine fever virus CD2v protein, characterized in that: The amino acid sequence of the single-chain antibody is shown in SEQ ID NO.

2.

4. A method for preparing a single-chain antibody against the CD2v protein of African swine fever virus, characterized in that: Includes steps: S1, constructing a recombinant plasmid using the gene as claimed in claim 1, and expressing it through an Escherichia coli expression system; S2, obtaining inclusion body precipitates from the expressed bacteria; incubating the inclusion body precipitates with inclusion body lysis buffer, and collecting the supernatant; the inclusion body lysis buffer contains urea; S3, dialyzing and renaturing the supernatant to obtain a single-chain antibody against the African swine fever virus CD2v protein.

5. The preparation method according to claim 4, characterized in that: The E. coli expression system includes the E. coli strain BL21 or the E. coli strain Shuffle T7.

6. The preparation method according to claim 4, characterized in that: The temperature expressed in step S1 is 22-40°C.

7. The preparation method according to claim 4, characterized in that: The concentration of urea in the inclusion body lysis buffer is 6-10 mol / L.

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