Recombinant monoclonal antibody of murine origin specifically recognizing VP7 protein of african horse sickness virus

Abstract

The application belongs to the field of immunology and in vitro diagnosis technology, and particularly relates to a mouse-derived recombinant monoclonal antibody specifically recognizing African horse sickness virus VP7 protein. The hybridoma cell strain 2C12 for the African horse sickness virus VP7 protein is screened by using the hybridoma cell technology, total RNA is extracted and reversely transcribed into cDNA, and the mouse IgG antibody molecule light chain and heavy chain variable region sequences are amplified by using the nest PCR method with the cDNA as a template. The light chain and heavy chain variable region sequences are constructed into a plasmid vector containing a mouse-derived constant region, and are co-transfected into 293F suspension cells for expression and purification, so that the mouse-derived recombinant monoclonal antibody specifically recognizing the African horse sickness virus VP7 protein is obtained. The recombinant antibody can specifically recognize the recombinant VP7 protein of the African horse sickness virus, but cannot recognize the recombinant VP7 proteins of the blue tongue virus and the epidemic hemorrhagic fever virus, which provides support for further establishing specific African horse sickness diagnosis technology.

Description

Technical Field

[0001] The present invention belongs to the technical fields of immunology and in vitro diagnosis, and particularly 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 of equine species caused by the African horse sickness virus (AHSV). Infected animals develop clinical symptoms such as fever, subcutaneous edema, and organ hemorrhage. AHSV is mainly transmitted between hosts by the blood-sucking insect Culicoides midge. The mortality rate caused by AHS is related to the species of equine species infected and the strain or serotype of the virus. Among equine species, horses are most susceptible to infection, with a mortality rate of 50-95%, followed by mules, with a mortality rate of approximately 50%. In Africa, donkeys are highly resistant to AHSV 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, apart from an elevated body temperature, do not show clinical symptoms.

[0003] AHSV is a member of the genus Orbivirus within the family Reoviridae. This genus also includes bluetongue virus and hemorrhagic fever virus, which share 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, conserved among nine serotypes. It is also a serogroup-specific antigen used in the development of 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 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 preparing 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, which comprises 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 coding the recombinant monoclonal antibody in the first aspect to construct into a plasmid vector containing a murine constant region, respectively, to obtain a light chain expression plasmid and a heavy chain expression plasmid of the recombinant antibody;

[0019] (2) transfecting the light chain expression plasmid and the heavy chain expression plasmid into 293F suspension cells at 1:1, culturing and purifying to obtain the recombinant monoclonal antibody.

[0020] The beneficial effects of the present application are: ① the hybridoma cell strain 2C12 against the African horse sickness virus VP7 protein is screened by the hybridoma cell technology, total RNA is extracted after the hybridoma cell strain 2C12 is lysed by Trizol, the heavy chain and light chain variable region sequences coding the antibody are amplified by the nest PCR method; and the heavy chain and light chain variable region sequences are constructed into a plasmid vector containing a murine IgG constant region, and the murine recombinant monoclonal antibody 2C12 against the African horse sickness virus VP7 protein is successfully expressed; ② the murine recombinant monoclonal antibody can recognize the recombinant VP7 protein expressed by E. 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 not the recombinant VP7 protein of the same genus bluetongue virus and epidemic hemorrhagic fever virus, has good specificity, and the murine recombinant antibody can be used to establish an African horse sickness virus competitive ELISA antibody detection method, and is suitable for the specific detection and diagnosis of the African horse sickness virus. BRIEF DESCRIPTION OF DRAWINGS

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

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

[0025] The following examples of the present invention are described in detail. It should be noted that the following examples are illustrative and are intended only to explain the present invention and are not to be construed as limiting the present invention. In addition, unless otherwise expressly stated, all reagents used in the following examples are commercially available or can be synthesized according to the literature or known methods. Reaction conditions not listed are readily available to those skilled in the art.

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

[0027] Hybridoma cell line 2C12 targeting the VP7 protein of African horse sickness virus was screened using hybridoma cell technology; the hybridoma cell line 2C12 cells were lysed with Trizol and total RNA was extracted. Then, a reverse transcription kit was used to obtain its cDNA. 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] The amplified product was recovered from the gel and ligated 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 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 is as follows: GAGGTGCGGCTTCAGGAGTCAG GACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACCTGTTCTGTCTCTGGCGACTCCTTCACCAGTGGTTTCTGGCACTGGATCCGGAAATTCCCAGGACTTAAGCTTGAGCACATGGGATACATTACCTACAGTGGTTCCACTTACTACAATCCATCTCTCAAACGTCGAATCTCCATCACTCGAGACACATCCAAGAACCAGTACTACCTACACTTGAATTCTGTGACTACTGGGGACACAGCCACATATTACTGTGCTAGACTGGACTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (shown in SEQ ID NO. 5);

[0031] Among them, 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 heavy chain variable region sequence encoding the recombinant monoclonal antibody 2C12 were respectively constructed into plasmid vectors containing mouse constant regions to form the light chain recombinant vector PTT5-LV-2C12 and the heavy chain recombinant vector PTT5-HV-2C12.

[0040] One day before transfection, the 293F cells were adjusted to a cell density of 1×10 6 cells / mL, and cultured until the next day when the cell density reached 2×10 6 Begin transfection at 100 cells / mL. Mix the recombinant vectors PTT5-LV-2C12 and PTT5-HV-2C12 at a 1:1 ratio, then mix with the transfection reagent at a 1:3 ratio and incubate at room temperature for 10-15 minutes. Then, slowly add the recombinant vectors to the 293F suspension cells and culture at 37°C, 8% CO2, and 125 rpm for 7 days.

[0041] The cell supernatant after 7 days of culture was collected, filtered, and added to a 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 impurities. 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 shown that the mouse recombinant antibody 2C12 was successfully expressed and purified.

[0043] Example 2 Reactivity of murine 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: Immunoblotting was performed using samples expressed with recombinant VP7 protein of African horse sickness virus, recombinant VP7 protein of bluetongue virus (BTV), and recombinant VP7 protein of epidemic hemorrhagic fever virus (EHDV). The primary antibody was the mouse recombinant monoclonal antibody 2C12 (1:1000) prepared in Example 1. The cells were incubated at room temperature for 1 hour, washed three times with PBST, and each time for 10 minutes. The secondary antibody was HRP-labeled goat anti-mouse IgG (1:10000). The cells were incubated at room temperature for 1 hour, washed three times with PBST, and finally developed for observation.

[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 hemorrhagic fever virus of the same genus.

[0047] Indirect ELISA detection of mouse recombinant monoclonal antibody 2C12: The AHSV recombinant VP7 protein was diluted to 0.5 μg / mL and 100 μL / well with CBS buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6) and coated 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 1 hour. Wash the plate 5 times with PBST buffer. The mouse recombinant monoclonal antibody 2C12 prepared in Example 1 was diluted 1:1000, 100 μL / well, and added to the ELISA plate. At the same time, a negative control was set up, incubated at 37°C for 1 hour, and washed 5 times with PBST buffer. Add HRP-labeled goat anti-mouse IgG at a dilution of 1:40,000 (0.01 M PBS pH 7.2) to the ELISA plate at 100 μL / well and incubate at 37°C for 1 hour. Wash the plate five times with PBST buffer. Develop with TMB colorimetric solution in the dark at 100 μL / well and incubate at 37°C for 10 minutes. Add 100 μL / well of stop solution and read the OD value. 450 The value of .

[0048] The indirect ELISA results are shown in Table 1, indicating that the murine 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: Immunofluorescence analysis was performed on BHK21 cells transfected with the recombinant plasmid pcDNA3.1-AHSVVP7 expressing the recombinant VP7 protein of AHSV using the mouse recombinant monoclonal antibody 2C12 prepared in Example 1. 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. The cells were cultured at 37°C for 24 hours, and the cell culture supernatant was discarded. The cells were fixed with 4% paraformaldehyde for 10 minutes, washed 3 times with PBS, and then treated with 0.5% Triton X-100 for 10 minutes. After washing 3 times with PBS, the cells were blocked with 3% BSA for 1 hour. After the old solution was discarded, the recombinant monoclonal antibody 2C12 (1:1000) prepared in Example 1 was added, and mouse negative serum was used as a negative control. The cells were incubated overnight at 4°C, and then the cell nuclei were stained with Hoechst 33342 for 10 minutes. The cells were washed 3 times with PBS, and goat anti-mouse IgG (Alexa Fluor 500) was added. 488) Incubate at room temperature for 1 h in the dark, observe and collect images after mounting.

[0052] The IFA results, as shown in Figure 4 indicate that the murine recombinant monoclonal antibody 2C12 described in the present application can recognize the recombinant VP7 protein of African horse sickness virus expressed in BHK21 cells.

[0053] The above only is the preferred embodiment of the present application, it should be pointed out that, for those skilled in the art, without departing from the principles of the present application, can also make a number of improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

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, wherein 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, wherein 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 according to claim 1 or 2 in the preparation of a reagent, test strip, or kit for detecting African horse sickness virus.

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

7. The detection kit according to claim 6, wherein The kit also includes an enzyme-labeled plate, 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.

8. The detection kit according to claim 7, wherein The African horse sickness virus antigen is selected from the recombinant VP7 protein of African horse sickness virus.

9. The method for preparing a recombinant monoclonal antibody according to claim 1 or 2, wherein: 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 according to claim 1 or 2, and constructing them into plasmid vectors containing mouse constant regions, respectively, to obtain the light chain expression plasmid and heavy chain expression plasmid of the recombinant monoclonal 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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