A mouse genetic engineering antibody for recognizing bluetongue virus VP7 protein and its preparation method

Abstract

The invention uses hybridoma cell technology to screen and obtain a hybridoma cell line 2B2 that recognizes the VP7 protein of the bluetongue virus. The hybridoma cell line 2B2 is lysed by Trizol to extract total cell RNA, and amplified by a nested PCR method after reverse transcription to obtain heavy chain and light chain variable region sequences of antibody molecules; the heavy chain and light chain variable region sequences are constructed into an expression vector containing a mouse IgG constant region, and a genetically engineered antibody that recognizes the VP7 protein of the bluetongue virus is successfully expressed; the genetically engineered antibody can react with the recombinant VP7 protein of the bluetongue virus, and can recognize the natural VP7 protein in the BHK-21 cells and KC cells infected with the bluetongue virus; the genetically engineered antibody can be used to establish detection methods such as bluetongue virus competitive ELISA, sandwich ELISA and colloidal gold test strips, thereby providing a technical basis for effectively preventing and controlling bluetongue disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to a mouse genetic engineering antibody for identifying the VP7 protein of bluetongue virus and a preparation method thereof. Background Art

[0002] Bluetongue is a contagious viral disease that is primarily vector-borne, affecting wild and domestic ruminants such as sheep, goats, cattle, buffalo, deer, etc. Bluetongue virus is primarily transmitted between susceptible vertebrate hosts by Culicoides, and the global distribution of the disease depends largely on the distribution of the vectors. Bluetongue virus (BTV) infection is not obvious in the vast majority of animals, but can cause death in a subset of infected sheep, deer, and wild ruminants. The clinical signs of bluetongue mainly include fever, congestion, facial edema and hemorrhage, mucosal erosions, coronitis and lamellar inflammation, pleural and pericardial hemorrhages.

[0003] Bluetongue virus is a representative member of the genus Orbivirus in the family Reoviridae. BTV consists of three protein layers. The outer capsid layer contains two proteins, VP2 and VP5. VP2 is the major neutralizing antigen and determinant of serotype specificity. Removal of the outer VP2 / VP5 layer leaves a double-layered icosahedral core particle, including an outer layer composed entirely of VP7 capsids and a complete inner capsid composed of VP3, which encapsulates 10 dsRNA genome segments and minor structural proteins (VP1, VP4 and VP6). VP7 is the major determinant of serogroup specificity and is used to establish a competitive enzyme-linked immunosorbent assay to detect anti-BTV antibodies. Enzyme-linked immunosorbent assay (ELISA) and virus neutralization (VN) are the most commonly used serological tests, and the competitive ELISA based on monoclonal antibodies has the advantages of high sensitivity, strong specificity, rapidity and reliability.

[0004] The present invention screens and obtains a hybridoma cell line 2B2 that recognizes the VP7 protein of the bluetongue virus, and amplifies the heavy chain and light chain variable region sequences of the antibody by a nested PCR method; and constructs the heavy chain and light chain variable region sequences into an expression vector containing a mouse IgG constant region, and successfully expresses and obtains a genetically engineered antibody that recognizes the VP7 protein of the bluetongue virus; the genetically engineered antibody can recognize the VP7 protein in BTV-infected BHK-21 cells and KC cells, and reacts with the recombinant VP7 protein of the bluetongue virus. Summary of the invention

[0005] In response to the above technical problems, the present invention provides a genetically engineered antibody for recognizing the VP7 protein of bluetongue virus. The genetically engineered antibody has the characteristics of high titer, strong affinity and good specificity, and can recognize the natural VP7 protein in BTV-infected BHK-21 cells and KC cells, and react with the recombinant VP7 protein of bluetongue virus.

[0006] Specifically include the following:

[0007] In a first aspect, the present invention provides a genetically engineered antibody for recognizing the VP7 protein of a bluetongue virus, wherein the genetically engineered antibody comprises an antibody heavy chain and an antibody light chain;

[0008] The variable region CDR of the antibody heavy chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.5, CDR2 with an amino acid sequence as shown in SEQ ID NO.6, and CDR3 with an amino acid sequence as shown in SEQ ID NO.7;

[0009] The variable region CDR of the antibody light chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.8, CDR2 with an amino acid sequence as shown in SEQ ID NO.9, and CDR3 with an amino acid sequence as shown in SEQ ID NO.10.

[0010] Preferably, the amino acid sequence of the variable region of the antibody heavy chain is shown as SEQ ID NO.1, and the amino acid sequence of the variable region of the antibody light chain is shown as SEQ ID NO.3.

[0011] In a second aspect, the present invention provides a nucleic acid encoding the antibody heavy chain and antibody light chain of the genetically engineered antibody described in the first aspect above.

[0012] Preferably, the gene sequence encoding the variable region of the antibody heavy chain is shown as SEQ ID NO.2, and the gene sequence encoding the variable region of the antibody light chain is shown as SEQ ID NO.4.

[0013] In a third aspect, the present invention provides the use of the genetically engineered antibody described in the first aspect in the in vitro detection of bluetongue virus for non-disease diagnosis purposes.

[0014] In a fourth aspect, the present invention provides the use of the genetically engineered antibody described in the first aspect in the preparation of a reagent, a test strip, or a kit for detecting bluetongue virus.

[0015] In a fifth aspect, the present invention provides a bluetongue virus ELISA detection kit, the kit comprising the genetically engineered antibody described in the first aspect above.

[0016] Preferably, the kit comprises an ELISA plate, a bluetongue virus antigen, a blocking solution, a diluent, the genetically engineered antibody described in the first aspect, an enzyme-labeled secondary antibody, a washing solution, a color developer, and a stop solution.

[0017] Preferably, the blocking solution is PBST buffer containing 5% skimmed milk powder.

[0018] Preferably, the diluent is 0.01M PBS pH 7.2.

[0019] Preferably, the washing solution is PBST buffer.

[0020] Preferably, the enzyme-labeled secondary antibody is a goat anti-mouse secondary antibody labeled with HRP.

[0021] Preferably, the bluetongue virus antigen is selected from the bluetongue virus VP7 recombinant protein.

[0022] In a sixth aspect, the present invention provides a method for preparing the genetically engineered antibody according to the first aspect, the method comprising the following steps:

[0023] (1) amplifying the light chain variable region sequence and the heavy chain variable region sequence encoding the genetically engineered antibody described in the first aspect, and constructing them into vectors containing mouse constant regions, respectively, to obtain the light chain expression plasmid and the heavy chain expression plasmid of the genetically engineered antibody;

[0024] (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 genetically engineered antibody was obtained by culture and purification.

[0025] The beneficial effects of the present invention are as follows: ① the present invention screens a hybridoma cell line 2B2 for recognizing the VP7 protein of the bluetongue virus by using hybridoma cell technology, and then lyses the hybridoma cell line 2B2 with Trizol to extract total RNA, and amplifies the heavy chain and light chain variable region sequences encoding the antibody by using a nested PCR method after reverse transcription; and constructs the heavy chain and light chain variable region sequences into a vector containing a mouse constant region, and successfully expresses and obtains a genetically engineered antibody for recognizing the VP7 protein of the bluetongue virus; ② the genetically engineered antibody can recognize the natural VP7 protein in BHK-21 cells and KC cells infected with BTV, and reacts with the recombinant VP7 protein of the bluetongue virus, and can be used for the detection and diagnosis of the bluetongue virus; ③ the genetically engineered antibody can be used to establish detection methods such as competitive ELISA, sandwich ELISA, and colloidal gold test strips for the bluetongue virus. Compared with the existing antibody detection method, the method has better sensitivity and specificity, and is suitable for the detection and diagnosis of the bluetongue virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 PCR amplification results of the light and heavy chains of the genetically engineered antibody 2B2;

[0027] Figure 2 SDS-PAGE verification results of genetically engineered antibody 2B2;

[0028] Figure 3 WB results of genetically engineered antibody 2B2 and bluetongue virus recombinant VP7 protein

[0029] Figure 4 WB results of genetically engineered antibody 2B2 and BHK-21 cells and KC cells infected with bluetongue virus;

[0030] Figure 5 Results of immunofluorescence test between genetically engineered antibody 2B2 and bluetongue virus VP7 protein. DETAILED DESCRIPTION

[0031] 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.

[0032] Example 1 Preparation of genetically engineered antibody 2B2 against bluetongue virus VP7 protein

[0033] Hybridoma cell line 2B2 targeting the VP7 protein of bluetongue virus was screened by hybridoma cell technology; the hybridoma cell line 2B2 cells were lysed with Trizol to extract total RNA, and then its cDNA was obtained using a reverse transcription kit. The obtained cDNA template was used to amplify the light chain and heavy 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 hybridoma cell line 2B2 are shown in the figure. Figure 1 shown.

[0034] The amplified product was recovered from the gel and then double-digested, then constructed into the pUC19 vector and sent for sequencing. The sequencing results were analyzed using IMGT to obtain the light chain and heavy chain variable region sequences of the hybridoma cell line 2B2. The details are as follows:

[0035] The amino acid sequence of the heavy chain variable region of the hybridoma cell line 2B2 is as follows: EVQLQQSGPELGKPGASVKMSCKASGYT FTDFVMHWMKQKPGQGLEWIGYIYPYNDGGKYNAKFKGKATLTSDKSSSSVYMELSSLTSEDSAVYYCARGGPAFAMDYWGQGTSLTVSS (shown in SEQ ID NO. 1);

[0036] The gene sequence encoding the heavy chain variable region of the hybridoma cell line 2B2: GAGGTCCAGCTGCAGCAGTCTGGAC CGGAGCTGGGAAAGCCTGGGGCTTCAGTGAAGATGTCCTGTAAGGCCTCTGGATACACATTCACTGACTTCGTTATGCACTGGATGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTTATCCTTACAATGATGGTGGTAAGTACAATGCGAAATTCAAGGGCAAGGCCACACTGACTTCAGACAAATCCTCCAGTTCAGTCTACATGGAGCTCAGCAGCCTGACCTCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGGGGGCCCCGCCTTTGCTATGGACTACTGGGGTCAAGGAACCTCACTCACCGTCTCCTCA (shown in SEQ ID NO.2);

[0037] Wherein, CDR1: GYTFTDFV (shown in SEQ ID NO.5);

[0038] CDR2: IYPYNDGG (shown in SEQ ID NO. 6);

[0039] CDR3: ARGGPAFAMDY (shown in SEQ ID NO. 7);

[0040] The amino acid sequence of the light chain variable region of the hybridoma cell line 2B2 is as follows: NIVMTQSPKSMSMSVGERVTLSCKASENV VTYVSWYQQKPEQSPKLLIYGASNRYIGVPDRFTGSGSATDFTLTISNVQAEDLADYHCGQ NYNYPTFGAGTKLELKR (shown in SEQ ID NO. 3);

[0041] The gene sequence encoding the light chain variable region of the hybridoma cell line 2B2: AACATTGTAATGACCCAATCTCCCAA ATCCATGTCCATGTCAGTAGGAGAGGGTCACCTTGAGCTGCAAGGCCAGTGAGAATGTGGTTACTTATGTATCCTGGTATCAACAGAAACCAGAACAGTCTCCTAAACTGCTAATATACGGGGCATCCAACCGGTACATTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAATGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAAAATTACAACTATCCTACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGG (shown in SE Q ID NO.4);

[0042] Among them, CDR1: ENVVTY (shown in SEQ ID NO.8);

[0043] CDR2: GAS (shown in SEQ ID NO. 9);

[0044] CDR3: GQNYNYPT (shown in SEQ ID NO. 10).

[0045] The light chain variable region sequence and heavy chain variable region sequence encoding the hybridoma cell line 2B2 were respectively constructed onto vectors containing mouse constant regions to form a light chain recombinant vector PTT5-LV-2B2 and a heavy chain recombinant vector PTT5-HV-2B2.

[0046] One day before transfection, cells were plated at 1×10 6 The cells were inoculated at a density of cells / mL and counted on the second day of culture. When the cell viability was >95% and the viable cell density was ≥2×10 6 cells / mL, directly used for transfection. The light chain recombinant vector PTT5-LV-2B2 and the heavy chain recombinant vector PTT5-HV-2B2 were mixed at a ratio of 1:1, and then mixed with the transfection reagent PEI at a ratio of 1:3 and incubated at room temperature for 10-15 minutes, and then slowly added to the 293F suspension cells and cultured at 37°C, 8% CO2, and 125rpm. After culturing for 18h-22h, feed was added, and the culture was continued for 7 days to end the culture.

[0047] Collect the cell culture supernatant, collect the cell culture supernatant into a 50ml centrifuge tube, centrifuge at 1000rpm for 10min; filter the supernatant and add it to a Protein A column pre-equilibrated with Binding / Wash Buffer (0.15M NaCl, 20mM Na2HPO4, pH 7.0), and use 10 column volumes of Binding / Wash Buffer for washing; use 4 column volumes of Elution Buffer (0.1M glycine, pH 3.0) to elute the antibody; collect the antibody, and add 1 / 10 volume of Neutralization Buffer (1M Tris-HCl, pH 8.5) to the collected antibody; absorb the antibody into a 10kDa ultrafiltration tube for concentration, so that the final volume reaches about 1 to 2ml, and obtain the concentrated genetic engineering antibody 2B2. The purified antibody is analyzed for its purity and molecular weight using SDS-PAGE.

[0048] SDS-PAGE verification results are as follows Figure 2 As shown, it was demonstrated that the genetically engineered antibody 2B2 was successfully expressed and purified.

[0049] Example 2 Reactivity of genetically engineered antibody 2B2 with bluetongue virus VP7 protein

[0050] The function of genetically engineered antibody 2B2 was verified by WB, ELISA and immunofluorescence assays.

[0051] 1. Immunoblotting detection of genetically engineered antibody 2B2

[0052] The recombinant VP7 protein of bluetongue virus and BHK-21 cells and KC cells infected with BTV were prepared for WB. The genetically engineered antibody 2B2 (1:1000) prepared in Example 1 was used as the primary antibody. The cells were incubated at room temperature for 1 hour and washed 3 times with PBST for 10 minutes each time. The secondary antibody was HRP-labeled goat anti-mouse IgG (1:10000). The cells were incubated at room temperature for 1 hour and washed 3 times with PBST for color development and observation.

[0053] The results are as follows Figure 3 and Figure 4 As shown, it is shown that the genetically engineered antibody 2B2 prepared in the present application can not only recognize the recombinant VP7 protein of bluetongue virus, but also recognize the VP7 protein in BTV-infected BHK-21 cells and KC cells.

[0054] 2. Indirect ELISA detection of genetically engineered antibody 2B2

[0055] Coating: Dilute BTV VP7 recombinant protein to 0.5 μg / mL with CBS buffer (0.05 M carbonate-bicarbonate buffer, pH 9.6), and coat it in the ELISA plate at 100 μL / well at 4°C overnight. Wash the plate 5 times with PBST buffer.

[0056] Blocking: Block the ELISA plate with PBST buffer containing 5% skim milk powder, 200 μL / well, incubate at 37°C for 1 hour. Wash the plate 5 times with PBST buffer.

[0057] Detection: The genetically engineered antibody 2B2 prepared in Example 1 was diluted 1:1000, 100 μL / well, and added to the ELISA plate. A negative control was set up and incubated at 37° C. for 1 h. The plate was washed 5 times with PBST buffer.

[0058] Add enzyme-labeled secondary antibody: Add 1:40000 dilution (diluent is 0.01M PBS pH7.2) of HRP-labeled goat anti-mouse secondary antibody to the ELISA plate, 100 μL / well, incubate at 37°C for 1 hour. Wash the plate 5 times with PBST buffer.

[0059] Color development: Use TMB color development solution in the dark, 100 μL / well, incubate at 37°C for 10 min; add stop solution 100 μL / well, read OD 450 The value of .

[0060] The indirect ELISA results are shown in Table 1, indicating that the genetically engineered antibody 2B2 described in the present application can react with the recombinant VP7 protein of the bluetongue virus.

[0061] Table 1 Indirect ELISA results of genetically engineered antibody 2B2

[0062] Genetically engineered antibody 2B2 Negative control <![CDATA[OD 450 Value]]> 3.7710 / 3.7389 0.1171 / 0.1258

[0063] 3. Immunofluorescence detection of genetically engineered antibody 2B2

[0064] Immunofluorescence analysis was performed on BHK-21 cells infected with BTV using the genetically engineered antibody 2B2 prepared in Example 1. BHK-21 cells were plated and then infected with BTV. The cells were cultured at 37°C for 24 hours, 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 genetically engineered antibody 2B2 (1:1000) prepared in Example 1 was added, and mouse negative serum was set 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) were incubated at room temperature in the dark for 1 h, and the slides were sealed and observed and images were collected.

[0065] Immunofluorescence test results Figure 5 As shown, it is shown that the genetically engineered antibody 2B2 described in the present application can recognize BHK-21 cells infected with bluetongue virus.

[0066] 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 genetically engineered antibody that recognizes the VP7 protein of bluetongue virus, characterized in that: The genetically engineered 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.5, CDR2 with an amino acid sequence as shown in SEQ ID NO.6, and CDR3 with an amino acid sequence as shown in SEQ ID NO.7; The variable region CDR of the antibody light chain includes CDR1 with an amino acid sequence as shown in SEQ ID NO.8, CDR2 with an amino acid sequence as shown in SEQ ID NO.9, and CDR3 with an amino acid sequence as shown in SEQ ID NO.

10.

2. The genetically engineered 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.1, and the amino acid sequence of the variable region of the antibody light chain is shown in SEQ ID NO.

3.

3. A nucleic acid, characterized in that The nucleic acid encodes the antibody heavy chain and antibody light chain of the genetically engineered 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.2, and the gene sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO.

4.

5. Use of the genetically engineered antibody according to claim 1 or 2 in in vitro detection of bluetongue virus for non-disease diagnosis purposes.

6. Use of the genetically engineered antibody according to claim 1 or 2 in the preparation of a reagent for detecting bluetongue virus.

7. A bluetongue virus ELISA detection kit, characterized in that: The kit comprises the genetically engineered antibody according to claim 1 or 2.

8. The ELISA test kit according to claim 7, characterized in that, The kit comprises an ELISA plate, a blue tongue virus antigen, a blocking solution, a diluent, the genetic engineering antibody according to claim 1 or 2, an enzyme-labeled secondary antibody, a washing solution, a color developer, and a stop solution.

9. The ELISA test kit according to claim 7, characterized in that, The bluetongue virus antigen is selected from the bluetongue virus VP7 protein.

10. The method for preparing a genetically engineered 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 genetically engineered antibody of claim 1 or 2, and constructing them into vectors containing mouse constant regions, respectively, to obtain the light chain expression plasmid and the heavy chain expression plasmid of the genetically engineered 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 genetically engineered antibody was obtained by culture and purification.

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