An antibody for specifically treating mouth sore of baby goat and application thereof

Abstract

The application belongs to the technical field of antibodies, and particularly relates to an antibody for specifically treating orf of milk goat kids and application thereof. Virus liquid of a diseased goat is collected from a lesion site, and ORFV virus strains are obtained through subculture and amplification of goat skin fibroblasts; milk goats are immunized with a weak vaccine to prepare high-titer immune serum; flow sorting technology is used to separate ORFV-specific B cells from PBMC (peripheral blood mononuclear cells); antibody genes are amplified through nested PCR, expression vectors are constructed, and 293T cells are transfected to express monoclonal antibodies. The monoclonal antibody No. 2 obtained in the application has high affinity and specificity, the heavy chain CDR1-3 region sequence is SEQ ID NO: 1-3, and the light chain CDR1-3 region sequence is SEQ ID NO: 4-6. The antibody can be used for diagnosis, treatment and prevention of orf of goats, and has the characteristics of high efficiency, specificity and stability, and provides a new biological preparation for prevention and control of orf of goats.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antibodies, and particularly relates to an antibody for specifically treating mouth sore of goat kids and application thereof. BACKGROUND

[0002] Orf disease, also known as sheep contact-transmitted pustular dermatitis, sheep contact-transmitted pustular dermatitis, sheep contact-transmitted pustular dermatitis, and sheep contact-transmitted pustular dermatitis, is a contagious disease of sheep caused by the sheep contact-transmitted pustular dermatitis virus (ORFV). The disease mainly harms the oral cavity (lip, tongue mucosa), lung, breast, and skin near the hoof of the affected sheep, and is widely prevalent in countries where sheep and goats are raised. The clinical feature of the disease is that the infected sheep develop oral pustules, which can expand and become proliferative warty lesions. Sometimes, extensive necrosis occurs in the oral cavity, and it can spread down to the esophagus. Although adult sheep flocks can also be infected, lactating kids are most commonly infected with the disease. Clinically, the disease is most severe in newborn kids of dairy goats, cashmere goats, or introduced meat sheep. The incidence rate varies, and the incidence rate of the first occurrence in a large-scale farm is as high as 100% in about one week. In newborn kids, the incubation period of the disease is 3-7 days, and once the disease occurs, the lesion quickly evolves into malignant mouth sore, often appearing cauliflower-like pustule signs on the oral mucosa, secondary bacterial infection causing extensive necrosis of the oral mucosa, affecting the milk allowance and feeding of the kids, and increasing the mortality rate of the kids or causing developmental retardation leading to stunted kids. The direct mortality rate of the disease is low, but the secondary harm caused by the disease is serious, especially for large-scale dairy goat breeding. The disease is one of the key diseases to be prevented and controlled.

[0003] At present, a commercialized ORFV attenuated vaccine has been put on the market, but active immunization of kids does not play a due role in preventing early-stage kids from getting the disease. Mainly, newborn kids generally get the disease within 7 days after birth, and the herpes lesion often occurs before the protective effect of active immunization of newborn kids is produced. Therefore, artificial indirect passive immunization prevention is mainly adopted for the prevention and control of the disease, that is, the late-pregnant ewes are immunized, and the kids obtain specific antibodies and immune factors through feeding of colostrum to prevent the disease. However, the recommended immunization method of the vaccine is oral mucosa scratch inoculation, which is inconvenient to operate, especially because the antibody maintenance time is short, the immunization needs to be performed 6-8 weeks before delivery in the late pregnancy, so that the effective maternal antibody, immune cell, and immune factor can be transmitted to the kids through colostrum. However, the immunization at this time has a certain risk of stress abortion, and the abortion problem of the first-time pregnant ewe is more prominent, which affects and restricts the application and immunization coverage of the vaccine. The kids born by some ewes without oral sore vaccine immunization or with uncertain immunization effect are infected with the disease to different degrees due to the lack of colostrum transmission of effective maternal antibody, resulting in failure of specific passive immunization.

[0004] So far, due to the lack of specific monoclonal antibodies or serum antibodies specific to the aphthovirus in the market, the treatment of the sick lambs is based on the specific treatment of applying antiviral and antibiotic drugs and the symptomatic treatment of reducing inflammation, relieving clinical manifestations, and preventing food refusal malnutrition by artificially feeding goat milk through the esophagus. There are actual problems of poor etiological control and low treatment effect, and it is necessary to develop and develop specific monoclonal antibodies or serum antibodies for etiological prevention and treatment products to make up for and solve the clinical problems of limited coverage of vaccine immunization prevention leading to multiple outbreaks of goat lambs and the severe malignant oral herpes lesions of the disease, which makes the conventional drug treatment effect low. SUMMARY

[0005] The present application provides an antibody for specifically treating oral herpes of goat lambs, wherein the heavy chain variable region of the antibody comprises CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the light chain variable region comprises CDR1, CDR2 and CDR3 sequences of SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0006] In some embodiments, the antibody can specifically bind to the B2L protein of ORFV.

[0007] The present application also provides a nucleic acid molecule encoding the above-mentioned antibody.

[0008] The present application also provides an expression vector comprising the above-mentioned nucleic acid molecule.

[0009] The present application also provides a host cell comprising the above-mentioned expression vector.

[0010] The present application also provides a method for preparing an ORFV-specific monoclonal antibody, comprising the following steps:

[0011] (1) Collecting virus liquid from the lesion site of the sick goat, and amplifying ORFV virus by subculture in goat skin fibroblasts cells (GSF, purchased from the Cell Bank of Kunming Institute of Zoology, Chinese Academy of Sciences);

[0012] (2) Immunizing a goat with ORFV attenuated vaccine to prepare high-titer immune serum;

[0013] (3) Separating PBMC (peripheral blood mononuclear cells) from the peripheral blood of the immunized goat;

[0014] (4) Separating ORFV-specific B cells from PBMC by flow sorting technology;

[0015] (5) amplifying the antibody gene by nested PCR;

[0016] (6) constructing an expression vector and transfecting a host cell to express the monoclonal antibody.

[0017] In some embodiments, the method for preparing the virus liquid in step (1) is as follows: extracting the blister fluid from the papules of the diseased sheep, adding sterile normal saline, adding penicillin and streptomycin at a final concentration of 2000 IU / mL, centrifuging at 3000 rpm for 10 minutes, filtering the supernatant with a 0.22 μm filter membrane, and then storing the filtered supernatant.

[0018] In some embodiments, the immunization method in step (2) is as follows: the first immunization dose is 200 μL of the attenuated vaccine, and the multi-point intradermal injection method is used to immunize the neck and back; the booster immunization is performed every 2 weeks after the first immunization, the immunization dose is halved, the immunization site is the tail, and a total of 6 booster immunizations are performed.

[0019] The application also provides a kit comprising the above-mentioned antibody.

[0020] The application finally provides the use of the above-mentioned antibody in the preparation of a medicine for preventing or treating orf.

[0021] Compared with the prior art, the application has at least the following beneficial effects:

[0022] (1) High specificity: the monoclonal antibody No. 2 prepared by the application has high specificity for ORFV, can accurately recognize and bind the B2L protein of the virus, and has excellent antigen binding activity as shown by the titer determination result.

[0023] (2) Optimized preparation method: the application establishes a complete preparation process for the ORFV-specific monoclonal antibody, which is optimized at each link from virus isolation, immunization scheme design to B cell sorting and antibody expression. In particular, the multi-point intradermal injection and tail booster immunization scheme significantly improves the immunization effect, and the effective neutralization titer of the serum reaches more than 1:5000.

[0024] (3) Diagnostic value: the diagnostic kit based on the monoclonal antibody can be used for rapid and accurate diagnosis of orf, solving the problems of long time consumption and insufficient specificity of the traditional diagnostic method. The PCR detection result shows that the method can specifically recognize the ORFV virus.

[0025] (4) Therapeutic potential: the monoclonal antibody can be directly used for the treatment of orf, providing a safe and effective biological agent for clinical use, and avoiding the problem of drug resistance in traditional antibiotic treatment.

[0026] (5) Good stability: The complete sequence of the antibody variable region is obtained by sequencing, which can be large-scale recombinant expressed, ensuring the consistency and stability of the antibody between batches. It provides a reliable foundation for the industrial production of the antibody.

[0027] (6) Wide application: The antibody can be used not only for diagnosis and treatment, but also for vaccine quality evaluation, virology research and other fields, with broad application prospects.

[0028] (7) Cost-effective: Compared with polyclonal antibodies, the production process of monoclonal antibodies is more stable, the cost is lower, and it is suitable for large-scale production and application. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Figure 1 is a picture of the lesion of the goat lamb with malignant mouth sore; wherein A is the lip; B is the tongue; and C is the lung.

[0030] Figure 2 Figure 2 is a picture of GSFs culture; wherein A is a normal GSFs culture monolayer; and B is a cell lesion of ORFV infected GSFs.

[0031] Figure 3 Figure 3 is a picture of PCR reaction of B2L gene primer after GSF culture amplification of the collected lamb mouth sore material and then expansion of ORFV; M lane is DL2000, 1 lane is negative control, 2 lane is positive control, and 3-9 lanes are isolated sheep mouth sore virus.

[0032] Figure 4 Figure 4 is a sorting picture of IgG+ B cells; wherein A is a PBMC cell gate picture set by using forward scattering light and side scattering light; B is a single cell gate picture set; C is a live cell gate picture set; D is a B cell gate picture set; and E is a cell gate picture set expressing anti-ORFV specific IgG.

[0033] Figure 5 Figure 5 is a picture of titer determination of the ORFV monoclonal antibody. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and advantages to be solved by the present application more clear, the following will be described in detail in combination with the drawings and specific embodiments.

[0035] Example 1, etiology diagnosis of lamb mouth sore

[0036] 1.1 Amplification of pathogen of the material: collect the virus liquid of the lesion site of the diseased sheep, and the specific site is, for example Figure 1The treatment method is as follows: 2 mL of vesicle fluid is extracted from the papules of the affected sheep of the sheep contagious pustular dermatitis using a sterile disposable syringe, 4 mL of sterile normal saline is added, and penicillin and streptomycin are added at a final concentration of 2000 IU / mL. Centrifugation is performed at 3000 rpm for 10 minutes, and the supernatant is carefully aspirated. The supernatant is filtered through a 0.22 μm filter membrane, and then it is divided into 1 mL per branch and stored at -20°C. The goat skin fibroblasts cell (GSF, purchased from the Kunming Institute of Zoology Cell Bank of the Chinese Academy of Sciences) is subcultured and expanded. The GSF is inoculated in a 6-well plate and cultured until the cell monolayer is fully covered, and then the sample treatment liquid is inoculated in the 6-well plate with GSF to blind passage 1-3 generations. After the cell monolayer of GSF appears cell plaques (CPE), the virus is collected and stored for detection. The comparison between virus-infected cells and normal cells is shown in FIG. 1. Figure 2 .

[0037] 1.2 Pathogen amplification and PCR detection: The ORFV-B2L gene primers (upstream: 5'-CGCGGATCCATGTGGCCGTTCTCCTCCATC-3' (SEQ ID NO: 33); downstream: 5'-AGCCTCGAGTTAATTTATTGGCTTGCAGAAC-3' (SEQ ID NO: 34); the amplification fragment size is 1137 bp) are used to amplify the virus to be detected, and the electrophoresis gel imaging is detected. The PCR result is shown in FIG. 2, and a positive band appears near 1137 bp, indicating that the pathogen amplification of the sample is the sheep contagious pustular dermatitis virus (ORFV). Combined with the pathogenic characteristics of cell subculture, it is inferred that the sheep contagious pustular dermatitis virus (ORFV) infection is the main pathogenic factor of the lamb mouth sore. Figure 3 .

[0038] Example 2, Preparation of Sheep Mouth Sore Virus Specific Immune Serum

[0039] 2.1 Immunization of sheep: The attenuated vaccine of the sheep contagious pustular dermatitis virus is used for immunization of the fattening male lambs of the dairy goats. The first immunization dose is 2 portions of the attenuated vaccine of the sheep contagious pustular dermatitis virus, and the volume is 1000 μL. The multi-point intradermal injection method is used for immunization on the neck and back. The booster immunization is performed every 2 weeks after the first immunization, and the immunization dose is halved. The immunization site is the tail. The booster immunization is performed for 6 times.

[0040] 2.2 Serum titer determination: blood collection was performed 7 days after the last booster immunization. The vacuum blood collection tube was placed at 45 degrees, and the blood was allowed to stand at room temperature for 45 minutes. The blood was centrifuged at 4000 rpm for 10 minutes at room temperature, and the supernatant was separated. The serum antibody titer level was determined according to the TCID50 neutralization test method. The GSF cells were recovered and cultured in vitro to the logarithmic growth phase in a T25 cell culture bottle, and the cell culture medium was DMEM + 10% FBS. After trypsin digestion of the cells, the cell density was adjusted to 3*10 5 cells / mL, and 100 μL of the cell suspension was added to each well of a 96-well plate. The serum to be tested was diluted by 10 times in gradient with the cell culture medium, and 50 μL was added to each well of the cell culture plate. The undiluted serum to be tested was recorded as 10 -0 , the 10 times diluted serum was recorded as 10 -1 , and so on. Then, 50 μL of the virus treatment solution described in 2.1 was added to each well, and 8 replicate wells were prepared for each serum gradient. After incubation at 37°C, the proportion of pathological cells was calculated after 3 days, as shown in Table 1.

[0041] Table 1, serum titer determination

[0042]

[0043] lgTCID50=L-D(s-0.5), L is the logarithm of the highest dilution; D is the difference between the logarithms of the dilutions; S is the total ratio of positive wells. lgTCID50=-3.875, which means that the serum can be diluted by 103.875 times to neutralize the same amount of virus. When the effective neutralization titer of the serum reaches 1:5000 or more, PBMC (peripheral blood mononuclear cells) can be separated from the blood.

[0044] 2.3 Preparation of PBMC: fresh anticoagulant whole blood from immunized sheep with serum titer meeting the requirements was diluted with 1* dilution and washing solution at a ratio of 1:1, and then gently mixed and reserved. An appropriate amount of mononuclear cell separation medium was added to a sterile centrifuge tube, and the diluted blood sample was gently placed on the surface of the separation medium at a ratio of "separation medium:diluted whole blood=1:2", and the interface between the two liquids was kept clear. Centrifugation was performed at 800g, with an increase of 9 and a decrease of 0, at room temperature for 20 min. After centrifugation, the liquid surface in the tube was from top to bottom in the order of diluted plasma layer, PBMC layer, separation medium layer and red blood cell layer. The plasma layer was carefully discarded, and the PBMC layer was transferred to a 15 mL centrifuge tube. The centrifuge tube was added with 10 mL of 1* dilution and washing solution to resuspend the cells, and centrifuged at 250g for 10 min at room temperature. The supernatant was discarded. This step was repeated 1-2 times. The obtained cell suspension could be used for flow staining and sorting.

[0045] 2.4 Flow sorting of antigen-specific B cells: Resuspend PBMC in 15 mL centrifuge tube with 2 mL DPBS, add 6 mL erythrocyte lysate, incubate at room temperature for 5-10 min. 300g, 4°C centrifuge for 5 min, discard supernatant, resuspend cells with 5 mL neutralization solution, which is DPBS+2% FBS. 300g, 4°C centrifuge for 5 min, discard supernatant, resuspend cells with neutralization solution to 5*10 6 6 cells / mL, aliquot in 1.5 mL EP tube with 100 μL per tube. Cell markers used for sorting are 7AAD, CD19, IgG, B2L with fluorescent labels. FACS sorting results are shown in Figure 4 Figure 4 A in which FSC is forward scatter light and SSC is side scatter light, according to cell volume and structure complexity to circle PBMC gate. Figure 4 B in which FSC-A / FSC-W is used to remove adherent cells, circle single cell population. Figure 4 C in which 7ADD is used to label cells, circle 7ADD-negative live cells. Figure 4 D in which anti-goat CD19-FITC antibody is used to label cells, circle CD19-positive cell population. Figure 4 E in which Q2 population that is double positive for anti-goat IgG-PC5.5 and anti-B2L-APC is the B cells expressing ORFV-specific antibodies.

[0046] 2.5 Amplification and cloning of antibody genes: After FACS separation of antigen-specific B cells, sort them into 96-well plates containing lysis buffer, lyse B cells and release intracellular RNA. Use RevertAid RT reverse transcription kit to synthesize cDNA and use its product as template for nested PCR. First round of nested PCR amplifies partial heavy chain, light chain lambda and kappa, primer sequences are shown in Table 2. First round of nested PCR primer amplifies target region and nearby sequences, second round of nested PCR primer binding site is inside first round product, specifically amplifies target fragment.

[0047] Table 2, first round of nested PCR primer sequences

[0048]

[0049] Second round of nested PCR amplifies heavy chain, light chain lambda and kappa variable region, primer sequences are shown in Table 3.

[0050] Table 3, second round of nested PCR primer sequences

[0051]

[0052] ​2.6 Expression, screening and identification of monoclonal antibodies: the amplified antibody gene fragments are detected by agarose gel electrophoresis, the fragments with correct size are selected for gel recovery, and after recovery, the homologous recombinase is used to connect to the vector fragments (the vector is pFuse, which contains CMV promoter, sv40 polyA sequence, signal peptide sequence, sheep Fc, Amp resistance gene), the reaction condition is 50°C, 30min. 10ul of the ligation product is added to 100ul of E. coli competent cells, incubated on ice for 30min, 42°C for 90s, incubated on ice for 3min, 1ml of LB medium is added, and incubated at 37°C for 1h, the shaking speed is set at 220rpm. The competent cells are plated on solid plates containing antibiotics and incubated at 37°C overnight. Single colonies are picked and cultured in liquid medium containing antibiotics. The Omega plasmid extraction kit is used to extract the plasmid, and the paired heavy chain and light chain plasmids are filtered through a 0.22um filter membrane, then the Freestyle293 cells are transfected. Then the cell number and cell mortality rate are continuously monitored, when the mortality rate is 30-35%, the cells are separated and the cell culture medium is collected, centrifuged at 12000rpm, 4°C for 15min, the supernatant is collected and used for ELISA detection of antibody titer. ORFV is diluted 1:40 with coating solution, 100ul per well is added for antigen coating, the prepared monoclonal antibody is used as the primary antibody, and the HRP labeled rabbit anti-sheep IgG (H+L) is used as the secondary antibody, Figure 5 The horizontal coordinate is the concentration of the added antibody, and the vertical coordinate is the absorbance. The experimental results show that the EC50 value of No. 1 monoclonal antibody is 1.263uM, the EC50 value of No. 2 is 8.444nM, the EC50 value of No. 3 is NA, the EC50 value of No. 4 is 14.94nM, and the EC50 value of No. 5 is 13.56nM. The candidate monoclonal antibodies 2, 4 and 5 have better antigen binding activity and can be used as alternatives for subsequent preparation. The No. 2 with the highest expression and the most stable is selected for subsequent variable region analysis.

[0053] 2.7 Sequence analysis of monoclonal antibody variable region: the plasmid sequence corresponding to the No. 2 monoclonal antibody with the highest titer is sent to Anshengda Life Science Technology Co., Ltd. for sequencing, and the sequencing results are shown in Table 4. After sequencing, a large amount of plasmid can be prepared, and PEI method is used to transfect Freestyle293 cells and prepare antibodies.

[0054] Table 4, amino acid sequence of monoclonal antibody

[0055]

[0056] The above is the preferred embodiment of the present application. It should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered within the scope of protection of the present application.

Claims

1. An antibody for specific binding of B2L protein of ORFV of milk goat kid mouth sore, characterized in that: The heavy chain of the antibody is shown as SEQ ID NO: 8, and the light chain is shown as SEQ ID NO:

7.

2. A nucleic acid molecule encoding the antibody of claim 1.

3. An expression vector comprising the nucleic acid molecule of claim 2.

4. A host cell comprising the expression vector of claim 3.

5. A kit characterized in that: The kit comprises the antibody of claim 1.

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