CN117801099A - Recombinant monoclonal antibody of anti-bluetongue virus VP7 protein - Google Patents

Abstract

The invention belongs to the field of biotechnology, and specifically relates to a recombinant monoclonal antibody against bluetongue virus VP7 protein. The present invention obtains a monoclonal antibody against bluetongue virus VP7 protein, obtains the light and heavy chain variable region sequence of the monoclonal antibody, and constructs a variable region containing the antibody by homologous recombination with a mouse antibody expression vector. The mouse recombinant expression plasmid in the region successfully expressed the mouse recombinant monoclonal antibody against the bluetongue virus VP7 protein after transfection. Through Western blotting, indirect ELISA, cell immunofluorescence and other experiments, it was identified that the recombinant monoclonal antibody of this strain can specifically react with the VP7 protein of bluetongue virus and can react with the natural VP7 protein in bluetongue virus-infected cells. The expression of this recombinant monoclonal antibody avoids the shortcoming of unstable antibody expression during hybridoma cell antibody expression, and lays the foundation for the use of this monoclonal antibody to establish a bluetongue disease pathogen and antibody detection method.

Description

A recombinant monoclonal antibody against bluetongue virus VP7 protein

Technical field

The invention belongs to the field of biotechnology, and specifically relates to a recombinant monoclonal antibody against bluetongue virus VP7 protein.

Background technique

Bluetongue disease is a disease caused by bluetongue virus (BTV) in ruminants such as sheep, cattle, and deer, and is mainly spread by blood-sucking midges of the genus Culicoides. Sheep are the most susceptible to the disease, and its clinical symptoms mainly include depression, elevated body temperature, facial edema and congestion, and oral and nasal mucosa ulcers or congestion. The World Organization for Animal Health (WOAH) lists it as a notifiable disease. BTV is a double-stranded RNA virus belonging to the genus Orbovirus of the family Reoviridae. Its genome is composed of 10 RNA fragments of different sizes, encoding a total of 7 structural proteins and 4 non-structural proteins. There are many serotypes of BTV, and there is no or very low cross-reaction between different serotypes. Early diagnosis and prevention are important means to control the epidemic of this disease. Structural protein VP7 is highly conserved among different serotypes, with an amino acid homology of more than 94%. It is the main antigenic protein for establishing a specific diagnostic method for bluetongue serogroups.

As BTV continues to mutate, at least 29 serotypes have been discovered. Conventional virus detection methods mainly rely on the specific binding of antibodies to pathogens. However, the expression of monoclonal antibodies obtained by conventional hybridoma cell technology is unstable.

Contents of the invention

In view of the above technical problems, the present invention provides a recombinant monoclonal antibody against BTV VP7 protein. The recombinant monoclonal antibody BTV VP7 protein has good binding ability, can react specifically with the BTV VP7 protein, avoids the shortcomings of unstable antibody expression in hybridoma technology, and is a new method for detecting and diagnosing bluetongue pathogens and antibodies. Establishment provides an important foundation.

Specifically include the following:

In a first aspect, the present invention provides a recombinant monoclonal antibody against BTV VP7 protein. The amino acid sequence of the heavy chain variable region of the recombinant monoclonal antibody is shown in SEQ ID NO. 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. The sequence is shown as SEQ IDNO.3.

In a second aspect, the present invention provides a nucleic acid, characterized in that the nucleic acid encodes the light chain variable region and the heavy chain variable region of the recombinant monoclonal antibody described in the first aspect.

Preferably, the gene sequence encoding the heavy chain variable region of the recombinant monoclonal antibody is shown in SEQ ID NO.2, and the gene sequence encoding the light chain variable region of the recombinant monoclonal antibody is shown in SEQ ID NO.4 .

In a third aspect, the present invention provides a method for preparing the recombinant monoclonal antibody described in the first aspect, which method includes the following steps:

(1) Construct the murine recombinant monoclonal expression plasmid containing the light chain and heavy chain variable region sequences described in the first aspect;

(2) Use serum-free medium to dilute the light chain and heavy chain murine recombinant expression plasmids respectively. According to the mass ratio of light chain to heavy chain plasmids of 2:1, transfect 293F suspension cells for expression and obtain murine recombinant plasmids. Cloning antibodies.

Preferably, the step (1) is: obtaining the light chain and heavy chain variable region sequences encoding the recombinant monoclonal antibody of claim 1 through PCR amplification, and comparing them with the mouse sequence containing the light chain and heavy chain constant regions respectively. The mouse-derived recombinant expression plasmid is constructed by homologous recombination.

In a fourth aspect, the present invention provides an immunoconjugate, which includes:

(i) The recombinant monoclonal antibody described in the first aspect above;

(ii) and a conjugated moiety selected from the group consisting of a detectable label, a drug, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or a VLP, or a combination thereof.

In a fifth aspect, the present invention provides the use of the recombinant monoclonal antibody described in the first aspect in preparing reagents, test strips, or kits for detecting bluetongue disease.

In a sixth aspect, the present invention provides the use of the recombinant monoclonal antibody described in the first aspect in the in vitro detection of bluetongue for the purpose of non-disease diagnosis.

In a seventh aspect, the present invention provides a BTV ELISA detection kit according to the first aspect, characterized in that the kit comprises the recombinant monoclonal antibody according to the first aspect.

Preferably, the kit further comprises an ELISA plate, a blocking solution, a diluent, an enzyme-labeled secondary antibody, a washing solution, a color developer, and a stop solution.

The beneficial effects of the present invention are: ① The present invention obtains a mouse-derived recombinant monoclonal antibody against BTV VP7 protein through genetic engineering technology; ② The recombinant monoclonal antibody has good binding ability to VP7 protein and can bind to BTVVP7 protein Specific reactions occur; ③ the invention can quickly and easily obtain recombinant monoclonal antibodies; ④ and the recombinant monoclonal antibodies can specifically bind to the BTV VP7 protein in infected cells and can be used for the detection and diagnosis of BTV.

Description of drawings

FIG1 is the RT-PCR amplification results of the light chain and heavy chain of the antibody 2A7 of the present invention;

Figure 2 The PCR amplification results of the light chain and heavy chain variable regions of the antibody 2A7 according to the present invention;

Figure 3 shows the immunoblotting results of the expression of the light chain and heavy chain of the recombinant monoclonal antibody 2A7 prepared by the present invention;

FIG4 is an immunoblot result showing the binding of the recombinant monoclonal antibody 2A7 prepared by the present invention to the BTV VP7 protein;

Figure 5 shows the cellular immunofluorescence results of the combination of recombinant monoclonal antibody 2A7 prepared by the present invention and BTV;

Figure 6 shows the immunoblotting results of the specific binding of the recombinant monoclonal antibody 2A7 prepared by the present invention to the BTV VP7 protein.

Detailed ways

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 also easily available to those skilled in the art.

Example 1 Preparation of recombinant monoclonal antibody 2A7

1. Obtaining monoclonal antibody 2A7

BALB/c mice were immunized with BTV VP7 recombinant protein, and their spleen cells were fused with SP2/0 cells to obtain hybridoma cells. A hybridoma cell line 2A7 secreting monoclonal antibodies against BTV VP7 protein was screened and obtained.

1.1 Obtaining monoclonal antibody light chain and heavy chain genes

The total RNA of hybridoma cells secreting monoclonal antibody 2A7 was extracted by Trizol lysis method, and then reverse transcription was performed using a reverse transcription kit to synthesize antibody cDNA. The obtained cDNA was used as a template and PCR reaction was performed under the conditions of 98℃, 5min pre-denaturation; 94℃, 30s denaturation; 54℃, 30s annealing; 72℃, 2min extension; after 30 cycles, 72℃, 6min, and then the results were identified by agarose gel electrophoresis.

The results are shown in Figure 1. The amplified sequence has an obvious band around 700 bp, which is in line with the expected results and indicates the successful amplification of the monoclonal antibody 2A7 light and heavy chain sequence of the present invention.

The amplified products were connected to the pUC19 vector to form pUC19-VK and pUC19-VH plasmids and sequenced to obtain the antibody light chain and heavy chain variable region sequences. After sequencing, the coding DNA sequence of the heavy chain variable region of the monoclonal antibody is as follows: CAGGTGCAGCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCC TGTCCATCACATGCACCGTCTCAGGGTTCTCATTAACTAGCTATGGTGTACACTGGGTTCGCCAGCCTCCAGGAAAGGGTCTGGAGTGGCTGGTAGTGATATGGCGTGATGGAAGCACAACCTATAATTCAGCTCTCAAATCCAGACTGAACATCAGCAAGGACAACT CCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTCCAAACTGATGACACAGCCATTTACTACTGTGCCAGAAAGGGGGGGGATAATTACGGGGGAGGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (shown in SEQ ID NO. 2);

The coding DNA sequence of the monoclonal antibody light chain variable region is as follows: GACATTGTGCTGACACAGTC TCCTGCTTCCTTAGTTGTATCTCTGGGGCAGAGGGCCACCTTCTCATGCAGGGCCAGCCGAAGTGTCAGTACATCTAGCTATAGTTATATGCACTGGTACCAACAGAAACCAGGACGGCCACCCAAACTCCTCATCAGGTATGCATCCAGCCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACT TCACCCTCAACATCCATCCTGTGGATGAGGAGGATACTGCAACATATTACTGTCAGCACAGTTGGAGGATTTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGG (shown in SEQ ID NO. 4);

According to the codon coding rules, it can be seen that the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as follows: QVQ LKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLVVIWRDGSTTYNSALKS RLNISKDNSKSQVFLKMNSLQTDDTAIYYCARKGGDNYGEAMDYWGQGTSVTVSS (shown in SEQ ID NO. 1);

The amino acid sequence of the monoclonal antibody light chain variable region is as follows: DIVLTQSPASLVVSLGQRATFSCRASRSVSTSSYSYMHWYQQKPGRPPKLLIRYASSLESGVPARFSGSGSGTDFTLNIHPVDEEDTATYYCQHSWRIWTFGGGTKLEIKR (shown in SEQ ID NO. 3).

2. Preparation of recombinant monoclonal antibody 2A7

2.1 Amplification of recombinant monoclonal antibody variable region sequences

(1) Primer design:

The antibody light and heavy chain variable region amplification primers were designed using the principle of homologous recombination. Heavy chain primer: F-primer: CTAGTAGC AACTGCAACCGGTCAGGTGCAGCTGAAGGAGTCA (shown in SEQ ID NO.5); R-primer: CC CTTGGTGGTGGCGCTCGAGGCTGAGGAGACGGTGACTGA (shown in SEQ ID NO.6); Light chain primer: F-primer: CCCTTGGTGGTGGCGCTCGAGGCTGAGGAGACGGTGACTGA (shown in SEQ ID NO.7), R-primer: TTGGTGCAGCATCCGTACGCCGTTTGATTTCCAGCTTGGTGC (shown in SEQ ID NO.8).

(2)PCR amplification

The light chain and heavy chain cloning plasmids pUC19-VK and pUC19-VH were used as templates, respectively. A 50 μL reaction system was prepared with MaxDNA polymerase, light and heavy chain variable region primers and deionized water. Amplification was performed according to the program of 98°C for 10 s, 57°C for 30 s, 72°C for 15 s, 35 cycles, and 72°C for 10 s. The amplification results were then identified by agarose gel electrophoresis.

The results are shown in FIG2 . The size of the light chain variable region is about 350 bp, and the size of the heavy chain variable region is about 500 bp, indicating that the variable region sequence of the recombinant monoclonal antibody 2A7 of the present invention was successfully amplified.

2.2 Construction of recombinant monoclonal antibody light chain and heavy chain expression plasmids

Use T4 DNA polymerase, expression vectors pcDNA-MK and pcDNA-MH containing mouse light chain and heavy chain constant region sequences to prepare a 10 μL connection system, connect for 4 minutes at 25°C, and incubate on ice for 10 minutes to obtain the light chain of the monoclonal antibody. and heavy chain murine recombinant expression plasmids.

2.3 Expression of recombinant monoclonal antibody 2A7

When the density of 293F suspension cells reaches 5×10 ⁶ /mL, use the transfection reagent PEI and expression plasmid according to the mass ratio of 3:1, and the light chain and heavy chain recombinant expression plasmid according to the mass ratio of 2:1, in the absence of serum. The culture medium was diluted and transfected into 293F suspension cells. After 8 to 10 days, the cell supernatant was collected for immunoblotting detection.

The results are shown in Figure 3. The light chain is approximately 25kDa and the heavy chain is approximately 55kDa, indicating the successful expression of the recombinant monoclonal antibody 2A7 of the present invention.

2.4 Identification of recombinant monoclonal antibody 2A7

(1) Immunoblotting detection of recombinant monoclonal antibody 2A7

The purified BTV VP7 recombinant protein was prepared for Western Blot. The primary antibody was used with the prepared recombinant monoclonal antibody 2A7, incubated at 4°C overnight, washed three times with PBST, 10 min each time, and the secondary antibody was HRP-labeled goat anti-mouse IgG antibody. (1:10000), incubate at room temperature for 1 hour, wash with PBST three times, and finally develop color for observation.

The results are shown in Figure 4. There is an obvious band at 40 kDa, indicating that the recombinant monoclonal antibody 2A7 prepared in the present invention can bind to the BTV VP7 recombinant protein.

(2) Indirect ELISA detection of recombinant monoclonal antibody 2A7

Coating: Dilute the expressed BTV VP7 protein to 0.5 μg/mL with CBS buffer (0.05M carbonate-bicarbonate buffer pH 9.6), add 100 μL/well to the enzyme plate, and coat overnight at 4°C. ;Wash the plate 5 times with PBST buffer;

Blocking: Block the enzyme plate with PBST buffer containing 5% skim milk powder, 200 μL/well, and incubate at 37°C for 1 hour; wash the plate 5 times with PBST buffer;

Detection: Add the above recombinant monoclonal antibody 2A7 to the enzyme plate at 100 μL/well, incubate at 37°C for 1 hour, and wash the plate 5 times with PBST buffer;

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

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

Judgment: Calculate the absorbance difference between the sample and the negative control (OD ₄₅₀ sample - OD ₄₅₀ negative control). If the difference is greater than zero, it indicates that there is a signal different from the negative control in the sample and is considered positive.

The results of indirect ELISA are shown in Table 1 below. The absorbance differences of the recombinant monoclonal antibody 2A7 were all greater than zero, and were determined to be positive samples, indicating that the recombinant monoclonal antibody 2A7 prepared by the present invention can bind to the BTV VP7 protein.

Table 1 Indirect ELISA results of recombinant monoclonal antibody 2A7

2A7 negative control Absorbance difference OD ₄₅₀ 0.267 0.045 0.222 OD ₄₅₀ 0.195 0.043 0.152

(3) Immunofluorescence detection of recombinant monoclonal antibody 2A7

BSR cells were spread on climbing slides one day in advance, then infected with BTV and cultured at 37°C for 24 hours. The cell culture supernatant was discarded, fixed with 4% paraformaldehyde for 15 minutes, washed three times with PBS, and then washed with 0.5% Triton X-100. After 10 min of action, wash 3 times with PBS, block with 3% BSA for 1 h, aspirate and discard, add recombinant monoclonal antibody 2A7, incubate at 4°C overnight, and then use goat anti-mouse IgG antibody (Alexa 568), incubate at room temperature in the dark for 1 hour, wash 3 times with PBS, add Hoechst 33342 to stain the cell nuclei for 7 minutes, wash 3 times with PBS, and observe and collect images under a fluorescence microscope after sealing.

The test results are shown in Figure 5. There is red fluorescence in the cytoplasm of BSR infected with BTV, while the negative control has no fluorescence, indicating that the recombinant monoclonal antibody 2A7 prepared in the present invention can bind to BTV in infected cells.

(4) Specificity detection of recombinant monoclonal antibody 2A7

BTV-infected BSR cells (golden hamster kidney cells) and KC cells (drosophila cells) were collected and prepared for Western Blot. The primary antibody was incubated with recombinant monoclonal antibody 2A7 at 4°C overnight, and washed three times with PBST, 10 min each time. The secondary antibody was HRP-labeled goat anti-mouse IgG antibody (1:10000), incubated at room temperature for 1 hour, washed three times with PBST, 10 minutes each time, and finally developed for color observation.

The results are shown in Figure 6. There are obvious bands at 40kDa in the wells of BSR cells and KC cells, indicating that the recombinant monoclonal antibody 2A7 prepared in the present invention can specifically bind to the BTV VP7 protein in BSR cells and KC cells. It does not specifically bind to other BTV proteins.

Claims (10)

1. The recombinant monoclonal antibody of the anti-bluetongue virus VP7 protein is characterized in that the amino acid sequence of a heavy chain variable region of the recombinant monoclonal antibody is shown as SEQ ID NO.1, and the amino acid sequence of a light chain variable region of the recombinant monoclonal antibody is shown as SEQ ID NO. 3.

2. A nucleic acid encoding the light and heavy chain variable regions of the recombinant monoclonal antibody of claim 1.

3. The nucleic acid of claim 2, wherein the nucleic acid sequence encoding the heavy chain variable region of the recombinant monoclonal antibody is shown in SEQ ID No.2 and the nucleic acid sequence encoding the light chain variable region of the recombinant monoclonal antibody is shown in SEQ ID No. 4.

4. The method of producing a recombinant monoclonal antibody according to claim 1, wherein the method comprises the steps of:

(1) Constructing a recombinant expression plasmid for expressing the recombinant monoclonal antibody light chain and heavy chain of claim 1;

(2) The murine recombinant expression plasmid containing the variable region sequences of the light chain and the heavy chain is diluted by a serum-free culture medium respectively, and the mass ratio of the light chain to the heavy chain is 2:1, adding 293F suspension cells, and culturing to obtain the murine recombinant monoclonal antibody.

5. The method of claim 4, wherein the step (1) is: the light chain and heavy chain variable region sequences of the recombinant monoclonal antibody of claim 1 are obtained through RT-PCR amplification, and the light chain and heavy chain murine recombinant expression plasmids are respectively constructed in a homologous recombination mode with murine recombinant expression vectors with light chain and heavy chain constant region sequences.

6. An immunoconjugate, the immunoconjugate comprising:

(i) The recombinant monoclonal antibody of claim 1;

(ii) And a coupling moiety selected from the group consisting of: a detectable label, a drug, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.

7. The use of the recombinant monoclonal antibody according to claim 1 in the preparation of a reagent, a test strip, or a kit for the detection of bluetongue.

8. Use of the recombinant monoclonal antibody of claim 1 in an in vitro assay for bluetongue for the purpose of non-disease diagnosis.

9. A bluetongue virus ELISA detection kit, comprising the recombinant monoclonal antibody of claim 1.

10. The ELISA detection kit of claim 9, further comprising an ELISA plate, a blocking solution, a diluent, an ELISA secondary antibody, a wash solution, a color reagent, and a stop solution.