Murine recombinant monoclonal antibody for specifically recognizing African horse pestivirus VP7 protein

Abstract

The invention belongs to the technical field of immunology and in vitro diagnosis, and particularly relates to a mouse recombinant monoclonal antibody for specifically recognizing African horse pestivirus VP7 protein. According to the invention, a hybridoma cell strain 2C12 for African horse pestivirus VP7 protein is obtained through screening by using a hybridoma cell technology, total RNA is extracted and is subjected to reverse transcription to form cDNA, the cDNA is used as a template and is amplified by using a nested PCR method to obtain light chain and heavy chain variable region sequences of mouse IgG antibody molecules, the light chain and heavy chain variable region sequences are constructed to a plasmid vector containing a mouse source constant region, and the mouse IgG antibody is obtained. And co-transfecting 293F suspension cells for expression and purification to obtain the murine recombinant monoclonal antibody for specifically recognizing the African horse pestivirus VP7 protein. The recombinant antibody can specifically recognize the recombinant VP7 protein of the African horse plague virus, but does not recognize the recombinant VP7 protein of bluetongue virus and epidemic hemorrhagic fever virus, so that support is provided for further establishing a specific African horse plague diagnosis technology.

Description

Technical Field

[0001] The invention belongs to the technical field of immunology and in vitro diagnosis, and specifically relates to a mouse-derived recombinant monoclonal antibody that specifically recognizes the VP7 protein of African horse sickness virus. Background Art

[0002] African horse sickness (AHS) is a non-infectious disease caused by the African horse sickness virus (AHSV) infecting equine animals. Infected animals will show clinical symptoms such as fever, subcutaneous edema and organ hemorrhage. AHSV is mainly transmitted between hosts by the blood-sucking insect Culicoides. The mortality rate caused by African horse sickness is related to the species of equine animals infected and the strain or serotype of the virus. Among equine animals, horses are most susceptible to infection, with a mortality rate of 50-95%, followed by mules, with a mortality rate of about 50%. In African regions, donkeys are highly resistant to African horse sickness virus and only experience subclinical infection. However, in European and Asian countries, donkeys are moderately susceptible, with a mortality rate of 10%. Zebras are significantly resistant and have no clinical symptoms except for an increase in body temperature.

[0003] AHSV is a member of the genus Orbivirus in the family Reoviridae. The genus Orbivirus also includes bluetongue virus and epidemic hemorrhagic fever virus, which have similar morphological and biochemical characteristics. Its genome consists of ten double-stranded RNA (dsRNA) segments encoding seven structural proteins (VP1-VP7) and at least four nonstructural proteins (NS1-NS4). VP7 is the major antigenic protein in African horse sickness virus, which is conserved among the nine serotypes and is also a serogroup-specific antigen used to establish diagnostic methods for AHSV. Summary of the invention

[0004] In response to the above technical problems, the present invention provides a mouse-derived recombinant monoclonal antibody that specifically recognizes the VP7 protein of African horse sickness virus. The recombinant monoclonal antibody can specifically recognize the recombinant VP7 protein of African horse sickness virus expressed in Escherichia coli and BHK21 cells, but does not recognize the recombinant VP7 protein of bluetongue virus and epidemic hemorrhagic fever virus.

[0005] Specifically include the following:

[0006] In a first aspect, the present invention provides a murine recombinant monoclonal antibody that specifically recognizes the VP7 protein of African horse sickness virus, wherein the recombinant monoclonal antibody comprises an antibody heavy chain and an antibody light chain;

[0007] The variable region CDR of the antibody heavy chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3;

[0008] The variable region CDR of the antibody light chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.6, CDR2 with an amino acid sequence as shown in SEQ ID NO.7, and CDR3 with an amino acid sequence as shown in SEQ ID NO.8.

[0009] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown as SEQ ID NO.4, and the amino acid sequence of the variable region of the antibody light chain is shown as SEQ ID NO.9.

[0010] In a second aspect, the present invention provides a nucleic acid encoding the antibody heavy chain and antibody light chain of the recombinant monoclonal antibody described in the first aspect.

[0011] Preferably, the gene sequence encoding the variable region of the antibody heavy chain is shown as SEQ ID NO.5, and the gene sequence encoding the variable region of the antibody light chain is shown as SEQ ID NO.10.

[0012] In a third aspect, the present invention provides a use of the recombinant monoclonal antibody described in the first aspect in the in vitro detection of African horse sickness virus for non-disease diagnosis purposes.

[0013] In a fourth aspect, the present invention provides a use of the recombinant monoclonal antibody described in the first aspect in the preparation of a reagent, a test strip, or a kit for detecting African horse sickness virus.

[0014] In a fifth aspect, the present invention provides an African horse sickness virus detection kit, the kit comprising the recombinant monoclonal antibody described in the first aspect above.

[0015] Preferably, the kit further comprises an ELISA plate, an African horse sickness virus antigen, a blocking solution, a diluent, an enzyme-labeled secondary antibody, a washing solution, a color developer, and a stop solution.

[0016] Preferably, the African horse sickness virus antigen is selected from the recombinant VP7 protein of African horse sickness virus.

[0017] In a sixth aspect, the present invention provides a method for preparing the recombinant monoclonal antibody according to the first aspect, the method comprising the following steps:

[0018] (1) amplifying the light chain variable region sequence and the heavy chain variable region sequence encoding the recombinant monoclonal antibody described in the first aspect, and constructing them into plasmid vectors containing mouse constant regions, respectively, to obtain the light chain expression plasmid and the heavy chain expression plasmid of the recombinant antibody;

[0019] (2) The light chain expression plasmid and the heavy chain expression plasmid were transfected into 293F suspension cells at a ratio of 1:1, and the recombinant monoclonal antibody was obtained by culture and purification.

[0020] The invention has the following beneficial effects: ① the invention uses hybridoma cell technology to screen and obtain a hybridoma cell line 2C12 targeting the VP7 protein of the African horse sickness virus, the hybridoma cell line 2C12 is lysed with Trizol, total RNA is extracted, and the heavy chain and light chain variable region sequences encoding the antibody are amplified by a nested PCR method; the heavy chain and light chain variable region sequences are constructed into a plasmid vector containing a mouse IgG constant region, and the mouse recombinant monoclonal antibody 2C12 targeting the VP7 protein of the African horse sickness virus is successfully expressed; ② the mouse recombinant cloned antibody can recognize the recombinant VP7 protein expressed by Escherichia coli and specifically react with the recombinant VP7 protein expressed in BHK21 cells, and can be used for the detection and diagnosis of the African horse sickness virus; ③ the recombinant monoclonal antibody can specifically recognize the recombinant VP7 protein of the African horse sickness virus, but does not recognize the recombinant VP7 protein of the blue tongue virus and the epidemic hemorrhagic fever virus of the same genus, and has good specificity. The mouse recombinant antibody can be used to establish an African horse sickness virus competitive ELISA antibody detection method, which is suitable for the specific detection and diagnosis of the African horse sickness virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 PCR amplification results of the heavy and light chain variable regions of the recombinant monoclonal antibody 2C12;

[0022] Figure 2 SDS-PAGE verification results of the purified recombinant monoclonal antibody 2C12;

[0023] Figure 3 Specificity identification diagram of recombinant monoclonal antibody 2C12;

[0024] Figure 4 Immunofluorescence image of recombinant monoclonal antibody 2C12 and recombinant VP7 protein of African horse sickness virus. DETAILED DESCRIPTION

[0025] The embodiments of the present invention are described in detail below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. In addition, if not explicitly stated, all reagents used in the following embodiments are commercially available, or can be synthesized according to text or known methods, and the reaction conditions not listed are easily available to those skilled in the art.

[0026] Example 1 Preparation of mouse recombinant monoclonal antibody 2C12 that specifically recognizes VP7 protein of African horse sickness virus

[0027] The hybridoma cell line 2C12 targeting the VP7 protein of African horse sickness virus was screened by hybridoma cell technology; the hybridoma cell line 2C12 cells were lysed with Trizol and the total RNA was extracted, and then the cDNA was obtained using a reverse transcription kit. The obtained cDNA was used as a template to amplify the heavy chain and light chain variable region sequences using the nested PCR method, and verified by agarose gel electrophoresis. The PCR amplification results of the light chain and heavy chain of the recombinant monoclonal antibody 2C12 are shown in Figure 2. Figure 1 shown.

[0028] After the amplified product was recovered from the gel, it was connected to the pMD20-T vector using a TA cloning kit and then sent for sequencing. The sequencing results were analyzed using the International Immunogenetics Information System (IMGT) to obtain the heavy chain and light chain variable region sequences of the recombinant monoclonal antibody 2C12. The details are as follows:

[0029] The amino acid sequence of the heavy chain variable region of the recombinant monoclonal antibody 2C12 is as follows: EVRLQESGPSLVKPSQTLSLTCSVSGDSFTSGFWHWIRKFPGLKLEHMGYITYSGSTYYNPSLKRRISITRDTSKNQYYLHLNSVTTGDTATYYCARLDFDYWGQGTTLTVSS (shown in SEQ ID NO.4);

[0030] The gene sequence encoding the heavy chain variable region of the recombinant monoclonal antibody 2C12: GAGGTGCGGCTTCAGGAGTCAG GACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACCTGTTCTGTCTCTGGCGACTCCTTCACCAGTGGTTTCTGGCACTGGATCCGGAAATTCCCAGGACTTAAGCTTGAGCACATGGGATACATTACCTACAGTGGTTCCACTTACTACAATCCATCTCTCAAACGTCGAATCTCCATCACTCGAGACACATCCAAGAACCAGTACTACCTACACTTGAATTCTGTGACTACTGGGGACACAGCCACATATTACTGTGCTAGACTGGACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (shown in SEQ ID NO.5);

[0031] Wherein, CDR1: GDSFTSGF (shown in SEQ ID NO.1);

[0032] CDR2:ITYSGST (shown in SEQ ID NO.2);

[0033] CDR3: ARLDFDY (shown in SEQ ID NO. 3);

[0034] The amino acid sequence of the light chain variable region of the recombinant monoclonal antibody 2C12 is as follows: DIVLTQAAFSNPVTLGTSASISCRSSKNLLHSDGITYLYWYLQRPGQSPQLLIYRVSNLASGVPNRFSGSESGTDFTLRISRVEAEDVGIYYCAQLLELPYTFGGGTKLEIKR (shown in SEQ ID NO.9);

[0035] The gene sequence encoding the light chain variable region of the recombinant monoclonal antibody 2C12 is as follows: GATATTGTGCTGACGCAGGCTGC CTTCTCCAATCCAGTCACTCTTGGAACATCAGCTTCCATCTCCTGCAGGTCTAGTAAGAATCTCCTACATAGTGATGGCATCACTTATTTGTATTGGTATCTGCAGAGGCCAGGCCAGTCTCCTCAACTCCTGATATATCGGGTGTCCAATCTGGCCTCAGGAGTCCCAAACAGGTTCAGTGGCAGTGAGTCAGGAACTGATTTCACACTGAGAATCAGCAGAGTGGAGGCTGAGGATGTGGGTATTTATTACTGTGCTCAACTGCTAGAGCTCCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGG (shown in SEQ ID NO.10);

[0036] Among them, CDR1: KNLLHSDGITY (shown in SEQ ID NO.6);

[0037] CDR2: RVS (shown in SEQ ID NO. 7);

[0038] CDR3: AQLLELPYT (shown in SEQ ID NO. 8).

[0039] The light chain variable region sequence and the heavy chain variable region sequence encoding the recombinant monoclonal antibody 2C12 were respectively constructed into plasmid vectors containing mouse constant regions to form a light chain recombinant vector PTT5-LV-2C12 and a heavy chain recombinant vector PTT5-HV-2C12.

[0040] One day before transfection, adjust the cell density of 293F cells to 1×10 6 cells / mL, and cultured until the next day when the cell density reached 2×10 6 cells / mL. Mix the recombinant vector PTT5-LV-2C12 and PTT5-HV-2C12 in a ratio of 1:1, and then mix with the transfection reagent in a ratio of 1:3 and incubate at room temperature for 10-15 minutes. Then slowly add it to the 293F suspension cells and culture them at 37°C, 8% CO2 and 125rpm for 7 days.

[0041] The cell supernatant after 7 days of culture was collected, filtered and added to the Protein A column pre-equilibrated with Binding / Wash buffer (0.15M NaCl, 20mM Na2HPO4, pH 7.0), the flow rate was controlled at 2mL / min, and then 10 column volumes of Binding / Wash buffer were used to wash. The antibody was then eluted with 4 column volumes of Elution buffer (0.1Mglycin e, pH 3.0), and 1 / 10 volume of Neutralization buffer (1M Tris-HCl, pH 8.5) was added to the collected antibody. The antibody was drawn into a 10kDa ultrafiltration tube for concentration, and finally the concentrated mouse recombinant monoclonal antibody 2C12 was obtained. The purified recombinant monoclonal antibody 2C12 was analyzed for purity and molecular weight using SDS-PAGE.

[0042] SDS-PAGE verification results are as follows Figure 2 As shown, it was demonstrated that the mouse recombinant antibody 2C12 was successfully expressed and purified.

[0043] Example 2 Reactivity of mouse recombinant monoclonal antibody 2C12 with African horse sickness virus VP7 protein

[0044] The function of mouse recombinant monoclonal antibody 2C12 was verified by Western blotting (WB), indirect ELISA and immunofluorescence analysis (IFA).

[0045] Immunoblotting detection of mouse recombinant monoclonal antibody 2C12: The samples were prepared with the expressed recombinant VP7 protein of African horse sickness virus, recombinant VP7 protein of blue tongue virus (BTV) and recombinant VP7 protein of epidemic hemorrhagic fever virus (EHDV) for immunoblotting. The primary antibody was the mouse recombinant monoclonal antibody 2C12 (1:1000) prepared in Example 1. The samples were incubated at room temperature for 1 hour, washed 3 times with PBST for 10 minutes each time, and HRP-labeled goat anti-mouse IgG (1:10000) was used as the secondary antibody. The samples were incubated at room temperature for 1 hour, washed 3 times with PBST, and finally observed with color development.

[0046] WB results are as follows Figure 3 As shown, it is shown that the mouse recombinant monoclonal antibody 2C12 prepared in the present application can specifically recognize the recombinant VP7 protein of African horse sickness virus, but does not recognize the recombinant VP7 protein of bluetongue virus and epidemic hemorrhagic fever virus of the same genus.

[0047] Indirect ELISA detection of mouse recombinant monoclonal antibody 2C12: Dilute the AHSV recombinant VP7 protein to 0.5 μg / mL, 100 μL / well with CBS buffer (0.05M carbonate-bicarbonate buffer, pH 9.6) and coat it on the ELISA plate at 4°C overnight. Wash the plate 5 times with PBST buffer. Block the ELISA plate with PBST buffer containing 5% skimmed milk powder, 200 μL / well, and incubate at 37°C for 1h. Wash the plate 5 times with PBST buffer. Dilute the mouse recombinant monoclonal antibody 2C12 prepared in Example 1 at 1:1000, 100 μL / well, add it to the ELISA plate, set up a negative control at the same time, incubate at 37°C for 1h, and wash the plate 5 times with PBST buffer. Add 1:40000 dilution (0.01M PBS pH7.2) of HRP-labeled goat anti-mouse IgG to the ELISA plate, 100 μL / well, incubate at 37°C for 1 hour. Wash the plate 5 times with PBST buffer. Develop with TMB colorimetric solution in the dark, 100 μL / well, incubate at 37°C for 10 minutes; add 100 μL / well of stop solution, and read the OD 450 The value of .

[0048] The indirect ELISA results are shown in Table 1, indicating that the mouse recombinant monoclonal antibody 2C12 described in the present application can react with the recombinant VP7 protein of African horse sickness virus.

[0049] Table 1 Indirect ELISA results of mouse recombinant monoclonal antibody 2C12

[0050] Recombinant monoclonal antibody 2C12 Negative control <![CDATA[OD 450 ]]> 3.5933 / 3.4442 0.0613 / 0.0641

[0051] IFA detection of mouse recombinant monoclonal antibody 2C12: The mouse recombinant monoclonal antibody 2C12 prepared in Example 1 was used to perform immunofluorescence analysis on BHK21 cells transfected with the recombinant plasmid pcDNA3.1-AHSVVP7 expressing the recombinant VP7 protein of AHSV. The recombinant plasmid pcDNA3.1-AHSVVP7 was obtained by cloning the gene expressing the recombinant VP7 protein of African horse sickness virus into the vector pcDNA3.1 / myc-His(-)B. Specifically, BHK21 cells were plated, and the recombinant plasmid pcDNA3.1-AHSVVP7 was transfected into BHK21 cells using Lipofectamine 3000, cultured at 37°C for 24 hours, the cell culture supernatant was discarded, fixed with 4% paraformaldehyde for 10 minutes, washed 3 times with PBS, and then treated with 0.5% Triton X-100 for 10 minutes, washed 3 times with PBS, blocked with 3% BSA for 1 hour, and the old solution was discarded, and the recombinant monoclonal antibody 2C12 (1:1000) prepared in Example 1 was added, and mouse negative serum was used as a negative control. Incubated overnight at 4°C, the cell nucleus was stained with Hoechst 33342 for 10 minutes, washed 3 times with PBS, and goat anti-mouse IgG (Alexa Fluor 500) was added. 488) were incubated at room temperature in the dark for 1 h, and the slides were sealed and observed and images were collected.

[0052] IFA results are as follows Figure 4 As shown, it is demonstrated that the murine recombinant monoclonal antibody 2C12 described in the present application is able to recognize the recombinant VP7 protein of African horse sickness virus expressed in BHK21 cells.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A mouse recombinant monoclonal antibody that specifically recognizes the VP7 protein of African horse sickness virus, characterized in that: The recombinant monoclonal antibody comprises an antibody heavy chain and an antibody light chain; The variable region CDR of the antibody heavy chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.1, CDR2 with an amino acid sequence as shown in SEQ ID NO.2, and CDR3 with an amino acid sequence as shown in SEQ ID NO.3; The variable region CDR of the antibody light chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.6, CDR2 with an amino acid sequence as shown in SEQ ID NO.7, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

8.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the variable region of the antibody heavy chain is shown in SEQ ID NO.4, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.

9.

3. A nucleic acid, characterized in that The nucleic acid encodes the antibody heavy chain and antibody light chain of the recombinant monoclonal antibody according to claim 1 or 2.

4. The nucleic acid according to claim 3, characterized in that The gene sequence encoding the variable region of the antibody heavy chain is shown in SEQ ID NO.5, and the gene sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO.

10.

5. Use of the recombinant monoclonal antibody as claimed in claim 1 or 2 in in vitro detection of African horse sickness virus for non-disease diagnosis purposes.

6. Use of the recombinant monoclonal antibody as claimed in claim 1 or 2 in the preparation of a reagent, test strip or kit for detecting African horse sickness virus.

7. An African horse sickness virus detection kit, characterized in that: The kit comprises the recombinant monoclonal antibody according to claim 1 or 2.

8. The detection kit according to claim 7, characterized in that The kit also includes an ELISA plate, an African horse sickness virus antigen, a blocking solution, a diluent, an enzyme-labeled secondary antibody, a washing solution, a color developer, and a stop solution.

9. The detection kit according to claim 8, characterized in that The African horse sickness virus antigen is selected from the recombinant VP7 protein of African horse sickness virus.

10. The method for preparing a recombinant monoclonal antibody according to claim 1 or 2, characterized in that: The method comprises the following steps: (1) amplifying the light chain variable region sequence and the heavy chain variable region sequence encoding the recombinant monoclonal antibody of claim 1 or 2, and constructing them into plasmid vectors containing mouse constant regions, respectively, to obtain the light chain expression plasmid and the heavy chain expression plasmid of the recombinant antibody; (2) The light chain expression plasmid and the heavy chain expression plasmid were transfected into 293F suspension cells at a ratio of 1:1, and the recombinant monoclonal antibody was obtained by culture and purification.

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