Anti-canine distemper virus monoclonal antibody, plasmid vector and preparation method

Abstract

This invention belongs to the field of bioengineering technology. It provides a recombinant protein whose amino acid sequence is composed of two dominant antigenic epitopes of the canine distemper virus H protein tandemly. The amino acid sequence is converted into a corresponding nucleotide sequence using E. coli-preferred codons. This nucleotide sequence is then chemically synthesized and a recombinant expression vector is constructed, thereby increasing the expression level of the recombinant protein in E. coli. This invention also involves immunizing mice with this recombinant protein, sorting B lymphocytes that specifically bind to the recombinant protein using flow cytometry, amplifying the heavy and light chain variable regions of B lymphocyte antibodies using single-cell PCR, constructing a complete mouse IgG antibody sequence recombinant expression vector from the obtained sequences, expressing monoclonal antibodies via transient transfection of HEK293F cells, purifying the monoclonal antibodies, and labeling them with colloidal gold particles. The optimal monoclonal antibody pairing combination is determined through orthogonal experiments, which is of great significance for the early diagnosis and prevention of canine distemper virus.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology. Specifically, this invention relates to a novel monoclonal antibody against the recombinant H protein of canine distemper virus. The method involves sorting B lymphocytes that specifically bind to the recombinant protein using flow cytometry, amplifying the heavy and light chain variable region sequences of the B lymphocyte antibody using single-cell PCR, constructing a complete mouse IgG antibody sequence expression vector to express the canine distemper virus H protein monoclonal antibody, and applying it to the early diagnosis of canine distemper virus. Background Technology

[0002] Canine distemper is a disease caused by canine distemper virus (CDV), resulting in immunosuppression and systemic infection. Infected dogs experience respiratory, digestive, and central nervous system symptoms. It has a wide range of infecting animals, including pandas, dogs, minks, foxes, wolves, terrestrial carnivores, and aquatic mammals such as whales and seals. Canine distemper virus belongs to the Paramyxoviridae family, Measlesvirus genus, and is an enveloped, non-segmented, negative-sense RNA virus. The viral genome is approximately 15690 bp in length. The viral particles are mostly spherical, with the nucleoprotein within the protein envelope measuring 600–800 nanometers in length and 18 nanometers in diameter. As early as 1995, it was reported that the persistent infection caused by both measles virus and canine distemper virus in hosts is related to viral replication, assembly, and budding release, with the F and H proteins playing particularly important roles. PAGE electrophoresis analysis showed that the F protein is approximately 63 kDa. Besides assembling into the viral envelope, the F and H proteins also mediate viral replication by invading cells through cellular receptors. Clinical symptoms of canine distemper depend on viral virulence, environment, host age, and immune status. Over 50% of infections are subclinical. Common symptoms include lethargy, fever, upper respiratory tract infection, bilateral ocular and nasal discharge that transitions from serous to mucolytic, watery cough, and difficulty breathing. Infected dogs may develop keratoconjunctivitis. Reports indicate that dogs that survive the disease may develop persistent anosmia. Dogs of all ages are susceptible to canine distemper virus, especially puppies aged 3-6 months. Due to the lack of maternal antibodies, early symptoms are mild, including anorexia, decreased appetite, vomiting, and diarrhea with watery stools. Tenesmus and intussusception may follow, leading to severe dehydration and emaciation from fluid loss. Therefore, dogs with systemic symptoms often die suddenly.

[0003] Domestic and international methods for etiological examination of canine distemper virus infection mainly include agarose gel electrophoresis, PCR, molecular probe technology, and other gene detection technologies. These technologies can not only qualitatively detect canine distemper virus but also quantitatively analyze it in samples. However, RNA extraction is difficult to achieve successfully and is cumbersome and time-consuming. Although PCR has high specificity, high sensitivity, and accurate quantification, it is complex to operate and requires specially trained technicians and specialized equipment. Immunoassay techniques, represented by ELISA double-antibody sandwich method, monoclonal antibody technology, and solid-phase immunoassay, are particularly outstanding in the rapid diagnosis of canine distemper virus. They not only simplify the detection steps but also improve the specificity and sensitivity of canine distemper virus detection.

[0004] Therefore, the preparation of monoclonal antibodies against canine distemper virus has become the main method for specific detection and diagnosis of canine distemper virus. The conventional method for preparing monoclonal antibodies against canine distemper virus involves preparing ascites fluid from Balb / c mice using monoclonal cell lines containing canine distemper virus protein, and then purifying the monoclonal antibody using Protein A affinity chromatography. However, due to the uncertain yield of ascites fluid from a single mouse and large individual variability, the obtained monoclonal antibody exhibits significant batch-to-batch variability, resulting in poor detection accuracy. Summary of the Invention

[0005] Design objective: To address the shortcomings of traditional monoclonal antibody preparation methods, this study designed and synthesized recombinant canine distemper virus H protein and screened for lymphocyte B cell antibody sequences that specifically bind to recombinant protein H using flow cytometry and single-cell PCR. Monoclonal antibodies were then prepared by transient transexpression of these sequences, significantly reducing the preparation time compared to traditional methods. Furthermore, the resulting monoclonal antibodies exhibited high stability and uniformity, greatly minimizing batch-to-batch variability.

[0006] Design scheme: To achieve the above design objectives, this application: (1) Using canine distemper virus H protein as the target antigen, two specific dominant antigenic epitopes of the antigen were analyzed and selected. The sequence alignment results showed that the selected antigenic epitopes had no obvious homology with other protein sequences. (2) In order to promote the stimulation of the immune system of Balb / c mice by the selected dominant antigenic epitopes and enhance the immune effect, the two selected dominant antigenic epitopes were tandemly linked and a His tag was added to the carbon terminus of the sequence to form a recombinant protein amino acid sequence. (3) Using the E. coli preferred codon, the recombinant protein amino acid sequence was converted into the corresponding nucleotide sequence to facilitate the efficient expression of the recombinant protein in E. coli. (4) The nucleotide sequence obtained in the previous step was chemically synthesized and ligated by enzyme digestion. The synthesized nucleotide fragment was inserted into the prokaryotic expression vector pET-28a(+) to construct a recombinant protein expression vector. (5) The recombinant protein expression vector was transformed into E. coli ER2566 competent cells, and kanamycin resistance selection medium was added to screen for recombinant protein expression strains. (6) After large-scale culture of the recombinant protein expression strain, the bacteria were lysed by sonication and centrifuged at low temperature. The supernatant was taken and eluted through a nickel agarose affinity chromatography column to obtain the purified recombinant protein. (7) After multiple immunizations of Balb / c mice with the recombinant protein H, the spleens were taken. The B lymphocyte suspension was sorted by BD FACS flow cytometry and collected in a 96-well PCR plate containing appropriate amounts of cell lysis buffer, RNase inhibitors and PCR reaction reagents. A mixture of forward primers was designed for different leader sequences of the variable regions of the antibody heavy and light chains. The reverse primers were specifically complementary to the constant regions of the antibody. The mRNA was reverse transcribed into cDNA. The corresponding antibody nucleotide sequence was cloned by RT-PCR. The purified antibody nucleotide sequence was analyzed by gel electrophoresis and sequenced to finally obtain the antibody nucleotide sequence that can bind to the recombinant protein. (8) The heavy and light chain variable region sequences were used to construct a complete mouse IgG expression vector and expressed monoclonal antibodies in HEK293 cells. The monoclonal antibodies were purified by Protein A affinity chromatography and labeled with colloidal gold particles. (9) Using the colloidal gold immunochromatographic screening platform, it was found that the combination of 7F3 monoclonal antibody coating and 2B5 colloidal gold-labeled monoclonal antibody is the best combination for detecting canine distemper virus.

[0007] Specific implementation plan: Although the following embodiments provide a relatively detailed textual description of the design concept of the present invention, these textual descriptions are merely simple textual descriptions of the design concept of the present invention, and not limitations on the design concept of the present invention. Any combination, addition or modification that does not exceed the design concept of the present invention falls within the protection scope of the present invention.

[0008] Example 1: Selection of dominant antigenic epitopes for canine distemper virus H protein

[0009] Using protein H as the target antigen, the hydrophilicity and antigenicity of its antigenic epitope sequences were analyzed using the biological software DNAssist 2.0, and dominant antigenic epitopes A and B were selected. Sequence comparison results showed that the selected dominant antigenic epitope sequences A and B had high specificity and no significant homology with other protein sequences.

[0010] Example 2: Tandem of dominant antigenic epitopes of H protein

[0011] To enhance the stimulation of the mouse immune system by the selected antigenic epitopes and facilitate subsequent experiments, the A and B dominant antigenic epitope sequences of the H protein were linked by a flexible fragment (four consecutive glycine residues) and repeated four times. A His tag was added to the C-terminus of the sequence to obtain the amino acid sequence of the recombinant protein.

[0012] Example 3: Optimization of the nucleotide sequence encoding recombinant proteins

[0013] To improve the expression level of recombinant protein in *E. coli*, while keeping the amino acid sequence of the recombinant protein unchanged, the amino acid sequence encoding the recombinant protein was converted into the corresponding nucleotide sequence according to the codons preferred by *E. coli*. Nucleotide sequences corresponding to the restriction enzyme sites BamHI and EcoRI were then added upstream and downstream of the transverse codons, respectively. This process was performed by General Biosystems Anhui Co., Ltd. The synthesized target gene was cloned into the pMD19-T vector (Takara Bio Engineering Dalian Co., Ltd.).

[0014] Example 4: Construction of recombinant protein expression vector

[0015] The pMD19-T vector and pET-28a(+) vector (Novagen, Germany) containing the target gene were double-digested at 37°C using restriction endonucleases BamHI and EcoRI (Takara Bio Engineering Dalian Co., Ltd.).

[0016] After 12 hours, the enzyme digestion products were electrophoresed on 1% agarose gels, and the target gene and pET-28a(+) vector (Asigen Biotech Hangzhou Co., Ltd.) were recovered separately. Using T4 ligase (Takara Bio Engineering Dalian Co., Ltd.), the recovered target gene and pET-28a(+) vector were ligated at a specific ratio at 4℃ for 12 hours. The ligation product was then transformed into DH5α competent cells (Hangzhou Xianzhi Biotechnology Co., Ltd.) and plated on LB agar plates containing kanamycin resistance (50 μg / mL). After incubation at 37℃ for 12 hours, single clones were picked from the plates and transferred to LB liquid medium containing kanamycin resistance (50 μg / mL). After incubation at 37℃ for 12 hours, plasmids were extracted using a plasmid purification kit (Asigen Biotech Hangzhou Co., Ltd.). The correct recombinant expression vector was obtained after double digestion with BamHI and EcoRI.

[0017] Example 5: Construction of recombinant H antigen expression strain

[0018] The constructed recombinant expression vector was transformed into E. coli ER2566 competent cells and plated on LB agar plates containing kanamycin resistance (50 μg / mL) and cultured overnight at 37°C. The next day, single clones from the plates were picked and cultured in LB liquid medium containing kanamycin resistance (50 μg / mL) at 37°C for 8 hours. One mL of the medium was stored, and the remaining medium was induced with IPTG (isopropyl thio-β-D-galactopyranoside) (final concentration 1.0 mmol / L) for 4 hours to prepare protein electrophoresis samples. 12% polyacrylamide gel electrophoresis results showed successful recombinant protein expression, yielding the recombinant protein expression strain.

[0019] Example 6: Purification of recombinant canine distemper virus protein

[0020] The recombinant protein expression strain was inoculated into LB liquid medium, and kanamycin was added to a final concentration of 50 μg / mL. After incubation at 37°C in a shaker for 8 hours, the strain was diluted 1:100 with LB liquid medium containing 50 μg / mL kanamycin and aliquoted into bacterial culture flasks. The flasks were then incubated at 37°C in a shaker until OD600 = 0.8. IPTG (isopropyl thio-β-D-galactopyranoside) was added to a final concentration of 1.0 mmol / L, and induction was continued for 4 hours. After centrifugation to collect the bacterial cells, the cells were lysed by low-temperature sonication. The supernatant was then passed through a nickel-agarose affinity chromatography column, and the purified recombinant protein was obtained after washing and elution.

[0021] Example 7: Preparation of monoclonal antibody against recombinant protein of canine distemper virus

[0022] Female Balb / c mice aged 4-6 weeks were used for the primary immunization. Each mouse received a subcutaneous injection of 100 μg of recombinant protein emulsified with Freund's complete adjuvant at multiple sites, totaling 400 μl per mouse. A booster immunization was performed 20 days later by emulsifying 80 μg of recombinant protein with Freund's incomplete adjuvant, totaling 400 μl per mouse, and injecting it subcutaneously at multiple sites. A third booster immunization was performed 15 days later, using the same method as the second booster. Twenty days later, a booster injection of 120 μg of recombinant protein was administered intraperitoneally. Mice were sacrificed 72 hours later, and the spleen was removed and minced. Trypsin was added to digest the spleen tissue, separating it into single cells. Different fluorescently labeled antibodies were used to stain different lymphocytes. Simultaneously, fluorescently labeled probes prepared from the recombinant protein were added to stain target B lymphocytes. Flow cytometry fluorescence sorting (FACS) was used to isolate single B cells expressing specific antibodies. mRNA was extracted from single B cells and cDNA was synthesized by RT-PCR. Using the cDNA as a template, the nucleic acid sequences encoding the light and heavy chains of the antibody were amplified using universal degenerate primers for mouse single-chain antibody SCFV. The encoding sequences were digested with enzymes and inserted into the pcDNA3.1(+) vector to construct recombinant plasmids expressing the specific antibody light and heavy chains. The light chain plasmid and heavy chain plasmid of the same antibody were mixed at a 1:1 mass ratio and transfected into HEK293F cells for the expression and assembly of the monoclonal antibody light and heavy chains. The cell culture medium was collected and the monoclonal antibody was purified by affinity polymerization with Protein A. The purity was detected by silver staining after SDS-PAGE electrophoresis. Monoclonal ELISA screening was performed the next day, and the screening steps are as follows:

[0023] Coating: Dilute the canine distemper virus H recombinant protein with coating buffer to a final concentration of 1 μg / mL, add 100 μL / well to the microplate (Shenzhen Jincanhua Industrial Co., Ltd.), incubate overnight at 4°C, and then wash once with washing buffer using a DEM-3 plate washer (Sun Yat-sen University Da An Gene Co., Ltd.).

[0024] Blocking: Add 200 μL of blocking solution to each well, block at 37°C for 2 hours, and wash once with washing solution using a plate washer;

[0025] Sample loading: Add overnight bacterial culture supernatant and control serum, 100 μL / well, incubate at 37°C for 1 h, and wash 3 times with washing buffer using a plate washer;

[0026] Add enzyme-labeled antibody: Add 100 μL / well of freshly diluted HRP-labeled secondary antibody (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.), incubate at 37°C for 30 minutes, and then wash 4 times with washing buffer using a plate washer;

[0027] Add colorimetric reagents: Add 50 μL of colorimetric reagent A and 50 μL of colorimetric reagent B to each well, and develop the color at 37°C in the dark for 10 minutes.

[0028] To terminate the reaction, add 2M H2SO4 at a rate of 50 μL / well;

[0029] Results Interpretation: OD values ​​were read at 450 nm using a microplate reader after zeroing the blank wells. Immune mouse serum was used as a positive control. Results showed that four positive clones had high OD values, and sequencing yielded four sequences: 4A1, 2B5, 3E9, and 7F3.

[0030] The relevant solution formulations are as follows:

[0031] Coating solution: Na2CO3 1.5g, NaHCO3 2.9g, add ddH2O to make up to 1000mL (pH 9.6).

[0032] Blocking solution: Na2HPO4·12H2O 2.68g, NaH2PO4·2H2O 0.39g, NaCl 8.5g, 20g bovine serum albumin, add ddH2O to bring the volume to 1000mL (pH 7.4).

[0033] Washing solution: Na2HPO4·12H2O 2.68g, NaH2PO4·2H2O 0.39g, NaCl 8.5g, Tween-20 0.5mL, add ddH2O to make up to 1000mL (pH 7.4).

[0034] Colorimetric solution A: Dissolve 200 mg TMB in 100 mL of anhydrous ethanol, and add ddH2O to bring the volume to 1000 mL.

[0035] Colorimetric solution B: 2.1g citric acid, 71g Na2HPO4·12H2O, add ddH2O to make up to 1000mL.

[0036] When using: 1 mL of colorimetric solution A + 1 mL of colorimetric solution B + 0.4 μL of 30% H2O2

[0037] Stop solution: 2M H2SO4, 21.7mL concentrated H2SO4, add ddH2O to make up to 1000mL.

[0038] Example 8: Preparation of colloidal gold pads

[0039] Add 5 ml of 0.01% colloidal gold solution to 10 μL of 0.2 mol / L potassium carbonate solution, mix thoroughly, then add 50 μg of monoclonal antibody, mix well, and let stand at room temperature for 2 hours. Then add 100 μL of 10% BSA (bovine serum albumin) solution for blocking. After blocking for 2 hours, centrifuge (10000 rpm / min, 20 min), discard the supernatant, and dissolve the precipitate thoroughly in 500 μL of reconstitution solution. The dissolved gold solution is then uniformly sprayed onto a 6 mm wide glass fiber at a rate of 6 μl / cm using a gold spraying apparatus (Shanghai Jinbiao Biotechnology Co., Ltd.), and then dried in an electric heating drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.) at 37℃ for 1 hour.

[0040] The relevant solution formulations are as follows:

[0041] 0.01% colloidal gold solution: 1 ml of 1% chloroauric acid solution, 1.4 ml of 1% citric acid solution, add ultrapure water, heat to dissolve and react, and then bring the volume to 100 ml.

[0042] 1% chloroauric acid solution: Dissolve 1g of AuCL3.HCl.4H2O powder in ultrapure water and bring the volume to 100ml.

[0043] 1% Citric Acid Solution: Dissolve 1g of citric acid crystals in ultrapure water and bring the volume to 100ml.

[0044] 0.2 mol / L potassium carbonate solution: Dissolve 27.64 g of potassium carbonate in ultrapure water and bring the volume to 1000 ml. Reconstituted solution: Dissolve 6.057 g of Tris base in 800 ml of ultrapure water, adjust the pH to 8.0 with an appropriate amount of HCl, and bring the volume to 1000 ml with ultrapure water.

[0045] Example 9: Preparation of nitrocellulose membrane (NC membrane)

[0046] Canine distemper virus monoclonal antibodies (4A1, 2B5, 3E9, 7F3) were diluted with coating buffer (final concentration 1 mg / ml) and uniformly coated onto nitrocellulose membranes (Sartorius) at a rate of 1 μl / cm using a gold-spraying membrane scribing apparatus (Shanghai Jinbiao Biotechnology Co., Ltd.); this is the T line. Goat anti-mouse solution (final concentration 1 mg / ml) was uniformly coated onto the nitrocellulose membrane at a rate of 1 μl / cm using the same apparatus (Shanghai Jinbiao Biotechnology Co., Ltd.); this is the C line. After coating, the nitrocellulose membranes were dried in an electrically heated drying oven (Shanghai Yiheng Scientific Instruments Co., Ltd.) at 37°C for 12 hours.

[0047] Example 10: Preparation of Colloidal Gold Immunoassay Card

[0048] Assemble the test strips: On the PVC base plate, overlap and paste the following in sequence: (1) Spray canine distemper virus monoclonal antibody (4A1, 2B5, 3E9, 7F3) as the detection area and goat anti-mouse IgG as the quality control area of ​​the NC membrane; (2) Spray a gold pad with colloidal gold-labeled anti-canine distemper virus monoclonal antibody (4A1, 2B5, 3E9, 7F3); (3) The sample pad is a glass fiber membrane treated with 2% Tween-20; (4) Absorbent paper, after assembly, cut to a width of 4mm, attach the reagent card shell and press it tightly to obtain the colloidal gold immunochromatographic test card.

[0049] Example 11: Screening of paired monoclonal antibodies

[0050] Canine distemper virus positive and negative samples were added to the assay diluent at a certain ratio and mixed well. 80 μL of the sample was loaded into each well and left at room temperature for 15 min. The T and C line signals on the NC membrane were read by a colloidal gold chromatography reader (Hangzhou Weizan Technology Co., Ltd.) and the measured value T / (T+C) was calculated. See Table 1 and Table 2 for details.

[0051]

[0052]

[0053]

[0054] The table above shows that the 7F3 monoclonal antibody coating paired with the 2B5 monoclonal antibody-labeled colloidal gold is the optimal antibody pairing for detecting canine distemper virus.

Claims

1. A recombinant H protein-specific monoclonal antibody 2B5 against canine distemper virus, comprising a light chain and a heavy chain, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO.1; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

2.

2. A recombinant H protein-specific monoclonal antibody 7F3 against canine distemper virus, comprising a light chain and a heavy chain, characterized in that: The amino acid sequence of the light chain variable region is shown in SEQ ID NO.3; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

4.

3. A gene encoding a specific monoclonal antibody 2B5 against canine distemper virus recombinant H protein as described in claim 1, characterized in that: The nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO.5; The nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO.

6.

4. A gene encoding a specific monoclonal antibody 7F3 against recombinant H protein of canine distemper virus as described in claim 2, characterized in that: The nucleotide sequence encoding the variable region of the light chain is shown in SEQ ID NO.7; The nucleotide sequence encoding the variable region of the heavy chain is shown in SEQ ID NO.

8.

5. A plasmid vector, characterized by: The plasmid vector contains nucleic acid molecules as shown in the nucleotide sequences of SEQ ID NO.5 and SEQ ID NO.

6.

6. A plasmid vector, characterized by: The plasmid vector contains nucleic acid molecules as shown in the nucleotide sequences of SEQ ID NO.7 and SEQ ID NO.

8.

7. A method for preparing a recombinant H protein-specific monoclonal antibody against canine distemper virus as described in claim 1 or 2, comprising: (a) The light chain variable region nucleotide sequence and the heavy chain variable region nucleotide sequence were bridged with the light chain constant region and the heavy chain constant region nucleotide sequence of mouse IgG1 by PCR, respectively, and then digested with enzymes and ligated into plasmid vectors to construct eukaryotic cell expression vectors. (b) The eukaryotic cell expression vector from step (a) was transfected into HEK293F cells to express a recombinant H protein-specific monoclonal antibody against canine distemper virus. (c) Purify monoclonal antibodies and label them with colloidal gold particles respectively, and determine the optimal monoclonal antibody pairing combination through orthogonal experiments; The nucleotide sequence of the light chain variable region is shown in SEQ ID NO.5, and the nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO.6; Alternatively, the nucleotide sequence of the light chain variable region is as shown in SEQ ID NO.7, and the nucleotide sequence of the heavy chain variable region is as shown in SEQ ID NO.8.

Images