CN105754952B - Monoclonal antibody of anti-capripoxvirus K3L protein C terminal and application thereof - Google Patents

Abstract

The invention discloses a hybridoma cell strain capable of secreting a monoclonal antibody against a capripoxvirus K3L protein C terminal, a monoclonal antibody secreted against capripoxvirus K3L holoprotein, and applications of the hybridoma cell strain and the antibody.A hybridoma cell strain K3L 35 capable of secreting a monoclonal antibody against a capripoxvirus K3L protein C terminal is preserved in China center for type culture Collection with the preservation number of CCTCC NO: C2015223. the K3L 35 hybridoma cell strain can secrete a monoclonal antibody against a K3L protein C terminal.the K3L 35 hybridoma cell strain and the secreted monoclonal antibody can be applied to preparation of reagents for diagnosing or detecting capripoxvirus infection, and can also be applied to preparation of reagents for tests.

Description

Monoclonal antibody of anti-capripoxvirus K3L protein C terminal and application thereof

Technical Field

The invention relates to a monoclonal antibody and application thereof, in particular to a hybridoma cell strain capable of secreting a C-terminal monoclonal antibody against capripoxvirus K3L protein, a C-terminal monoclonal antibody against capripoxvirus K3L protein secreted by the hybridoma cell strain, and application of the hybridoma cell strain and the antibody.

Background

Capripox is a highly contagious and devastating viral disease in sheep plagues, which is legally reported by the world animal health Organization (OIE) and is characterized by fever, conjunctivitis, rhinitis, skin and mucosal acne, dyspnea and death. Capripox is mainly prevalent in some areas of africa, middle east, india, nepal turkey, and is prevalent in the Qinghai, Gansu, Hunan, inner Mongolia, etc. in China. The Sheep Pox Virus (SPV) belongs to poxviridae, chordopoxvirinae subfamily and goatpox virus, is mainly transmitted by aerosol, close contact or insect bite, sheep is the main host of the sheep pox virus, and can be infected in all age groups, but the death rate of cases mainly occurs in lambs, is up to 100 percent, causes great economic loss to the sheep industry, and is considered as a potential biological weapon in economy. Therefore, diagnosis and control of sheep pox have been the focus of research.

The Chinese patent 2011103869424 discloses a capripoxvirus virus, including goat pox virus, sheep pox virus and cow lumpy skin disease virus chip detection device. The patent can simultaneously identify goat pox virus, sheep pox virus and cow lumpy skin disease virus of sheep pox virus by designing a specific probe. The invention aims to establish a method for detecting goat pox virus, sheep pox virus and cow lumpy skin disease virus of capripox virus by using a microarray chip which has the advantages of high sensitivity, strong specificity, time and labor saving and easy observation of results.

Chinese patent application 2013105940610 and 201310594930X respectively disclose colloidal gold, a gold-labeled antibody and a preparation method thereof for a capripoxvirus colloidal gold test reagent strip. The colloidal gold adopts the optimum labeling amount of the capripoxvirus P32 monoclonal antibody, and the prepared gold-labeled antibody compound is scanned in a visible light range to generate the maximum absorption peak.

The methods are established aiming at nucleic acid or structural protein in the middle and later periods of virus replication, so that the nucleic acid or structural protein can only detect whether the virus exists after the virus infects a host for 12 hours or more, and the application of the C-terminal monoclonal antibody of the anti-K3L protein prepared by the invention in the detection of early infection of the capripoxvirus can detect the expression of K3L at the earliest 2 hours of virus infected cells and reaches the maximum 24 hours.

Disclosure of Invention

The invention provides a hybridoma cell strain capable of secreting a C-terminal monoclonal antibody against capripoxvirus K3L protein, a monoclonal antibody secreted by the hybridoma cell strain against capripoxvirus K3L protein, and applications of the hybridoma cell strain and the monoclonal antibody.

The hybridoma cell strain K3L 35 capable of secreting the C-terminal monoclonal antibody of the anti-capripoxvirus K3L protein is preserved in the China center for type culture Collection of Wuhan university in Wuhan, 2015 12, 17 days, and the preservation number is CCTCC NO: C2015223.

The K3L 35 hybridoma cell strain can secrete the K3L protein C-terminal monoclonal antibody.

The K3L 35 hybridoma cell strain and the secreted monoclonal antibody can be applied to the preparation of a reagent for diagnosing or detecting the capripoxvirus infection and can also be applied to the preparation of a reagent for testing.

The preparation method of the monoclonal antibody for resisting the C terminal of the K3L protein comprises the following steps:

the invention relates to a method for obtaining soluble protein of K3L by using goat pox virus, which comprises the steps of synthesizing a K3L gene complete sequence after performing codon preference analysis and codon optimization modification according to an SPV K3L gene sequence, connecting the complete sequence with a pUC57 vector, transforming a connection product into DH5 α competent cells, extracting plasmids and sequencing, carrying out accurate sequencing recombinant plasmid pUC 57-K3L, subcloning a K3L gene into a prokaryotic expression vector pET11d, carrying out recombinant plasmid pET11 d-K3L, transforming the recombinant plasmid into B L (DE 6345) plysS competent cells, carrying out induction expression on recombinant bacteria by using G as an inducer to obtain the soluble protein of K3L, collecting supernatant after mass expression, carrying out collection and treatment of the protein, finally obtaining the immune protein of a mouse Balb, carrying out selective fusion of collected mouse lymphocyte and spleen cell with the spleen cell, carrying out selective fusion of collected mouse lymphocyte 890 with the spleen cell, and carrying out selective fusion of the spleen cell obtained hybridoma obtained by collecting IPT 11 and spleen cell.

K3L as one of Hrf of poxviruses, the function of immune regulatory gene has been the research hotspot in the field, L angland in 2002 is found that K3L gene-deleted virus can replicate on Vero but not on BHK cells, and E3L gene-deleted virus can replicate on BHK cells but not on Hela cells, after constructing K3L and E3L gene VACV-deleted strains, thereby proving that K3L and E3L are VACV host range factors (L angland et al, 2002) related research finds that K3L comprises two functional regions, an N-terminal Z-DNA binding region, homologous to RNA editing enzymes (ADAR), related to pathogenicity of the virus, K3L N-terminal deleted virus of VACV can replicate effectively on nasal mucosa but cannot transfer from nasal cavity to lung or brain, greatly reducing the virulence of the virus to the host (Kim al, 2009), C3L gene has binding activity of eIF2 which inhibits the binding of the eIF2, PKF 962, and IFN a gene which can inhibit the phosphorylation of the homologous protein of the PKF 3.

The C-terminal monoclonal antibody of the anti-K3L protein prepared by the invention can be applied to infection and immunization research of the capripox virus and can also be applied to preparation of a capripox virus detection reagent or a kit, in the process of prevention and control of infectious diseases, if early detection is carried out on a certain specific molecule of a pathogen, great convenience can be brought to formulation of later-stage prevention and control measures, and a benefit is brought to further transmission of the disease reduction.

Drawings

FIG. 1 is a graph of codon mass distribution GC content of SPV K3L before codon optimization.

FIG. 2 is a graph of codon mass distribution GC content of SPV K3L after codon optimization.

FIG. 3 is a graph showing the mass distribution of SPV K3L at a position of the codon used before codon optimization.

FIG. 4 is a graph showing the mass distribution of codons used by SPV K3L after codon optimization at the same position as in FIG. 3.

FIG. 5 is a graph of the codon GC content of SPV K3L before optimization.

FIG. 6 is a graph showing the codon GC content of SPV K3L after optimization.

FIG. 7 is an electrophoretogram of recombinant pUC 57-K3L vector after PCR amplification with cloning primers.

FIG. 8 shows the electrophoretograms of the double restriction enzymes of FIG. 5pUC 57-K3L and pET11 d-K3L, wherein FIG. A shows 5pUC 57-K3L, and FIG. B shows pET11 d-K3L.

FIG. 9 shows the sequencing results of pET11 d-K3L.

FIG. 10 is an SDS-PAGE analysis of SPV K3L protein expression.

FIG. 11 is an electrophoretogram of K3L recombinant protein after purification.

FIG. 12 is a drawing showing the Western Blot analysis of the purified K3L recombinant protein.

FIG. 13 shows the results of detection of K3L C-terminal Monoclonal Antibody Isotyping Kit.

FIG. 14 is a mass spectrum of C-terminal synthetic peptide of K3L.

FIG. 15 is a mass spectrum of N-terminal synthetic peptide of K3L.

FIG. 16 is an electrophoretogram identifying a monoclonal antibody by Western Blot.

FIG. 17 is a graph showing the expression level of K3L detected at different time points after cells were inoculated with capripoxvirus.

FIG. 18 is a photograph of immunofluorescence analysis showing the localization of K3L protein of capripoxvirus detected in cells.

FIG. 19 is a photograph of immunofluorescence analysis of DAPI after staining of nuclei.

FIG. 20 is a photograph of an immunofluorescence assay of FIGS. 18 and 19 superimposed.

Detailed Description

The invention is illustrated below with reference to examples. The following is merely illustrative of relevant details of the invention and should not be taken as limiting the scope of the invention.

In the following examples:

1. strains and cells

The strain used in the present invention is the goalpox strain, and the Vero cells and SP2/0 cells used in the present invention are both preserved by the applicant (Lanzhou veterinary research institute, national academy of agricultural sciences).

2. Primary reagent

pUC57 cloning vector, Top10 competent cells, plasmid extraction kit, Xba I and BamH I restriction enzymes, T4 DNA ligase were purchased from Dalibao bioengineering Co., Ltd, DMEM medium, RPMI-1640 cell medium and fetal bovine serum were purchased from Gibco, gel recovery kit was purchased from AXYGEN, pET11d expression vector, B L (DE 3) plysS host bacteria, His binding resin and nitrocellulose membrane were purchased from Novagen, agar powder, peptone and yeast powder were purchased from OXOID, Ampicillin (AMP), IPTG, horseradish peroxidase (HRP) -labeled rabbit anti-goat IgG and Weffa complete adjuvant and Freund incomplete adjuvant were purchased from Sigma, horseradish peroxidase (HRP) -labeled goat anti-mouse IgG was purchased from Biowrold, Prestaining protein marker, Signal (FMS), Thermo substrate, FI Biophys Biophyte Biote Biolite, and Pebae Biophyte Biolite, Prestaining protein marker Biolite, Prep Biote Biolite, Prep Biolite, Prep Biote Biolite, Prep Biote Biolite.

3. Main instrument

The system comprises a noon-honest ZWY-240 constant-temperature culture oscillator, an Eppendorf 5417R high-speed freezing centrifuge, an Eppendorf5810R high-speed freezing centrifuge, a Mastercycle Eppendorf PCR instrument, a fine DNP-9052 electric heating constant-temperature incubator, a Liangping JA2003 electronic balance, a Heal Force BIOsafe 12 biological safety cabinet and a Berle semi-dry rotary film instrument.

First, SPV K3L complete gene codon optimization synthesis, cloning and identification

According to the sequence of the SPV K3L gene, performing codon preference analysis and codon optimization to synthesize a complete sequence, performing 1% agarose gel electrophoresis analysis on a synthesized product, cutting a target strip with the size of about 300 bp under an ultraviolet lamp, purifying a target gene by using a DNA gel recovery kit of the company AXYGEN, connecting a sheep pox virus K3L gene PCR product with a pUC57 vector overnight at 4 ℃, performing standing culture overnight at 37 ℃, picking a single clone colony by using a sterilized gun tip and placing the single clone in a L B culture medium containing AMP resistance, performing shaking culture overnight at 37 ℃, taking a 2ml overnight culture, extracting a recombinant plasmid according to a photo-ligation biological plasmid extraction kit specification, performing enzyme digestion identification, and performing restriction enzyme digestion identification on a pUC 3L-SPV L mu 6861 plasmid sequencing, a Xb 638, a 6865-42, and adding a recombinant plasmid DNA for DNA fragment identification after performing ultraviolet gel electrophoresis analysis on a pUC L DNA fragment, wherein the PCR product is used for identifying that the size of a pUC 3614 bp DNA fragment is correct, and the size of a DNA fragment is used for performing DNA fragment electrophoresis analysis and adding a DNA fragment for identifying a correct expression of a DNA fragment of a pUC L DNA PCR product through an Inv L DNA gel electrophoresis analysis.

The sequence of the SPV K3L gene before codon optimization is SEQ ID NO 1;

the SPV K3L gene sequence after codon optimization is SEQ ID NO 2;

the sequence of the protein for encoding SPV K3L is SEQ ID NO 3.

After codon optimization, the codon grade is improved, the quality is higher, the GC content is obviously reduced, corresponding results are shown in figures 1 to 6, the proportion of high-quality codons of SPV after codon optimization is greatly improved in comparison with that before optimization (figure 1) in figure 2, the quality of all nucleotide sequence sites of SPV K3L after codon optimization is improved in comparison with that before optimization (figure 3) in figure 4, and the GC content of SPV K3L after codon optimization is reduced in comparison with that before optimization (figure 5), so that the soluble expression of K3L in escherichia coli is facilitated.

Second, construction of prokaryotic expression vector of SPV K3L gene

The method comprises the steps of carrying out BamH I and Xba I double enzyme digestion on a pET11 prokaryotic expression vector and a pUC-SPV K recombinant plasmid with correct identification under a water bath condition at 37 ℃, adding ddH2 to 20 mu 5 into a reaction system which is pET 11/pUC-SPV K10 mu 0, BamH I1 mu 2, Xho I1 mu 3 and 2K buffer 2 mu 4, and showing fragments with expected sizes through electrophoresis (see figure 8A), recovering the vector and a target fragment according to a gel recovery kit specification, connecting the vector and the target fragment under a water bath condition at 16 ℃, wherein the reaction system is SPV K6 gene recovery fragment 15 mu 7, the pET11 vector recovery fragment 3 mu 8, 10 1T ligase buffer 2.5 mu 9, adding ddH2 to 25 mu 0 into T ligase 1 mu, transforming the connection product to B (DE) plysS 221 cells, carrying out sequencing on a PCR amplification system which is subjected to amplification culture, carrying out oscillation and identification on a pUC 2.5, and carrying out DNA ligase analysis, wherein the PCR amplification reaction results are shown by adding a DNA ligase DNA fragment with correct DNA insert DNA, and carrying out electrophoresis analysis, and carrying out electrophoresis on a DNA electrophoresis result, and carrying out an electrophoresis on a DNA recombination on a DNA fragment with a DNA insert enzyme digestion result which is shown in a DNA fragment with 10 mH 2. mu. 5, and a PCR analysis, and is carried out an electrophoresis, wherein the PCR amplification reaction system which is carried out an electrophoresis, and is carried out on a PCR amplification reaction system which is carried out on a PCR amplification.

Induced expression of gene III, K3L

Taking 50 mu L positive bacteria liquid overnight culture to 5m L L B culture medium, carrying out shake culture at 37 ℃ until OD600=0.6, adding IPTG (isopropyl-beta-thiogalactoside) to the final concentration of 1 mM for induced expression for 4h, sucking 1m L induced bacteria liquid, centrifuging at 5500 r/min for 10min to collect thalli, adding 80 mu L PBS for resuspending the thalli, adding 20 mu L5 × SDS-PAGE loading buffer, mixing uniformly, placing in boiling water for treatment for 5 min, and detecting the target protein expression condition by SDS-PAGE, wherein the size of the recombinant protein is about 10 ku and is consistent with the expected size as shown in figure 10.

Four, antigenicity analysis of SPV K3L recombinant protein

The protein sample is subjected to SDS-PAGE electrophoresis, and then transferred to an NC membrane by a semidry method, the membrane is soaked in a confining liquid for overnight sealing at 4 ℃, SPV standard positive serum is used as a primary antibody, HRP-labeled rabbit anti-goat IgG is used as a secondary antibody for Western-blot analysis, and finally, a target strip is developed and the anti-antigenicity of the recombinant protein is analyzed by referring to the operating instruction of SuperSignal West Femto chemiluminescence substrate of Thermo Fisher company, the result is shown in figure 12, the SPV standard positive serum can identify the SPV K3L recombinant protein, and the SPV K3L protein can stimulate an animal body to generate a specific antibody, namely SPV K3L has antigenicity and can be used as a candidate protein for detecting SPV infection.

Fifthly, purification of SPV K3L recombinant protein

Inoculating 1m L recombinant strain into 100 ml L B medium containing AMP resistance, and shake-culturing at 37 deg.C to OD₆₀₀Carrying out induction expression according to optimized expression conditions, centrifuging at 5500 r/min for 10min to collect thalli, abandoning supernatant, carrying out ultrasonic disruption after the thalli is resuspended by 10m L PBS, stopping the ultrasonic treatment for 8 s at 5 s, centrifuging at 12000 r/min for 10min by 1m L thalli lysate after the ultrasonic treatment is finished, respectively collecting supernatant and precipitate, resuspending the precipitate by PBS with the same volume as that of the supernatant, and analyzing the solubility of the SPV K3L recombinant protein by SDS-PAGE, respectively taking 10 mu L supernatant and precipitate to carry out SDS-PAGE gel electrophoresis analysis, wherein the result is shown in figure 10, and most of the recombinant protein exists in the supernatant in a soluble formThe recombinant protein was purified using His-binding resin instructions from Novagen, and 10 μ L samples were each analyzed by SDS-PAGE gel electrophoresis, the results of which are shown in fig. 11, the purified recombinant protein band was relatively single and free of contamination by other foreign proteins.

Sixthly, immunizing BA L B/c mice by using recombinant protein SPV K3L

Selecting a BA L B/c mouse with the age of 6-8 weeks, and carrying out the following immunization program, wherein during primary immunization, 100 mu g of purified recombinant protein SPV K3L is mixed with an equivalent amount of Freund's complete adjuvant, and subcutaneous multi-point injection is carried out after emulsification, 14 days after primary immunization, the same amount of recombinant protein is mixed with Freund's incomplete adjuvant, the immunization is strengthened after emulsification, blood is collected and serum is separated 14 days after secondary immunization, 0.2 mu g of purified recombinant protein SPV K3L is used for coating an E L ISA plate, an indirect E L ISA test is carried out, the serum titer is measured, and the result shows that the titer of the prepared mouse antiserum is 1: 100000.

The method comprises the steps of carrying out SDS-PAGE electrophoresis on purified recombinant protein SPV K3L, transferring the recombinant protein SPV K3L to an NC membrane by a semidry method, immersing the membrane in a confining liquid, sealing the membrane at 4 ℃ overnight, respectively using prepared mouse antiserum as a primary antibody and goat anti-mouse IgG marked by HRP as a secondary antibody to carry out Western-blot analysis, finally developing a target band by referring to a SuperSignal West Femto chemiluminescent substrate operation instruction of Thermo Fisher company, and identifying the prepared SPV K3L protein mouse antiserum, wherein the result is shown in figure 12.

Seventhly, cell fusion

Cell fusion was performed 3d after boosting mice. Taking out eyeballs of the mice, taking blood, dislocating and killing the mice, placing the mice in a 70% alcohol bottle for 2 min, fixing the mice on a foam plate in an ultra-clean workbench, unfastening the abdominal skin to find spleen, taking the mice down with tweezers, placing the mice in a 200-mesh stainless steel filter membrane for slight grinding, slightly washing the cells with PBS, then placing the cells in a centrifuge at 37 ℃ for centrifugation at 3000 rpm for 10min, and discarding the supernatant for later use; when preparing feeder cells, mice were killed by dislocation, placed in a 70% alcohol bottle for 2 min, and then fixed to foam in a clean benchOn the plate, unfastening the abdominal skin, sucking PBS with a syringe, gently injecting the PBS under the abdominal membrane, washing out the liquid containing the feeder cells from the other side, then placing the liquid into a centrifuge at 37 ℃ for 10min at 3000 rpm, discarding the supernatant for later use, placing the liquid in a water bath beaker at 40 ℃, slowly dropping PEG2000 into a fusion centrifugal tube gently shaking clockwise for 1 min in order to reduce the toxicity of PEG2000, then adding 20% FBS 1640 cell culture medium, gently blowing up the cells, spreading the cells into 6 96 empty culture plates with each well being 100 mu L, marking the culture plates, then placing the plates into a culture plate at 37 ℃ containing 5% CO₂The cell culture box is used for culturing, a large number of tumor cells die within 1-2 days of HAT selective culture, the tumor cells disappear after 3-4 days, the hybrid cells form small colonies, the HAT selective culture solution is maintained for 7-10 days, then the HT culture solution is used, and the ordinary culture solution is used again after the HAT selective culture solution is maintained for 2 weeks. During the selective culture, when the hybridoma cells spread over the area of the well bottom 1/10, the detection of the specific antibody is started, and the desired hybridoma cell line is selected. During the selective culture period, half of the culture solution is generally changed every 2 to 3 days.

The obtained hybridoma cell line is delivered to China center for type culture collection to be preserved, and the preserved material is named as hybridoma cell line K3L 35 with the preservation number of CCTCC No. C2015223.

Eight, screening and subcloning culture of positive hybridoma cell strain

Firstly, K3L is determined as the best coating amount of antigen through a square experiment, 0.5, 1.0, 2.0 and 4.0 mu g of K3L purified protein is coated on a 96-well plate, each well with the concentration of 6 is respectively set to be 3 positive and 3 negative, square titration is carried out by using K3L purified protein immune mouse positive serum with different dilution times, and meanwhile, non-immune mouse negative serum is used as a negative control.

Coating a 96-well E L ISA plate with 0.5 mu g of purified K3L protein per well, standing overnight at 4 ℃, washing 3-4 times for 3-5 min each time, adding 200 mu L of PBST of 5% skimmed milk powder into each well, sealing for 2h at 37 ℃, washing 3-4 times for 3-5 min each time, patting on folded gauze, adding 0.1ml of a sample to be detected into each reaction well, incubating at 37 ℃ for 1-2 h, washing (simultaneously adding blank wells (without adding the sample), negative control wells (wild type) and positive control wells), adding 0.1ml of a newly diluted antibody into each reaction well, incubating at 37 ℃ for 1-2 h, washing, adding an enzyme-labeled secondary antibody, adding 0.1ml of a newly diluted enzyme-labeled antibody into each reaction well, incubating at 37 ℃ for 45-1 h, washing for 5 times, adding a substrate solution into each reaction well, adding 0.1ml of the prepared OPD solution into each reaction well, adding 0.1ml of the newly diluted enzyme-labeled antibody into each reaction well, incubating at 37 ℃ for 45 min, adding a corresponding substrate solution, adding a corresponding protein after the detection result of the blank wells, and detecting the positive control cells, wherein the OD value of the detected by adding sulfuric acid is more than 7 nm, and the OD is calculated by adding the OD of the detection of the antibody, and the antibody, wherein the antibody is more than 7 mm, the.

The positive hybridoma cells obtained by screening were subcloned by the method described above, and the original wells were diluted with HAT selection medium by the limiting dilution method and then replated into 96-well culture plates, and then the morphology and number of cells were observed. Adjusting the cell number to 3-10 cells/ml. The cell culture plate of the feeder cell layer prepared the first day was taken, and 100. mu.l of the diluted cells were added to each well. Incubate at 37 ℃ in a 5% CO2 incubator. Changing the liquid on the 7 th day, and changing the liquid 1 time every 2-3 days later. Cell clone formation can be seen in 8-9 days, and the activity of the antibody can be detected in time. Cells from positive wells were transferred to 24-well plates for expanded culture. Each clone should be frozen as soon as possible.

Preparation of monoclonal antibody in large scale and determination of antibody titer

Intraperitoneal injection of 0.5ml of pristane into BA L B/C mice with age of 8 weeks, and intraperitoneal injection of 1 × 10 after 2 weeks⁶And (3) inoculating the hybridoma cells to generate ascites after 7-10 days, closely observing the health condition and ascites symptoms of the animal, killing the mice when the ascites is as much as possible and before the mice die frequently, sucking the ascites into a test tube by using a dropper, and obtaining 5-10 ml of ascites from one mouse. Or extracting ascites with syringe, and collecting repeatedly. Centrifuging the obtained ascites at 1000g for 10min, removing upper layer oil and bottom precipitate, collecting supernatant, packaging at-20 deg.C, and measuring monoclonal antibody content in ascites to 8 mg/ml.

Ten, biological analysis of monoclonal antibodies

(1) Monoclonal antibody subtype identification

The immunoglobulin standard subclass identification kit of Sigma company is adopted for identification, and the operation is mainly carried out according to the kit instruction. The identification result is shown in FIG. 13, which indicates that the monoclonal antibody obtained by the present invention is IgG2 b.

(2) Immunological Activity assay (WB)

The C terminal of K3L (synthetic mass spectrogram shown in figure 14), N terminal synthetic peptide (synthetic mass spectrogram shown in figure 15), recombinant purified K3L protein, cell collection lysate of 12h and 24h infected by the sheep pox virus and uninfected cell protein are subjected to SDS-PAGE electrophoresis, then transferred to an NC membrane, the membrane is immersed in a sealing solution and sealed overnight at 4 ℃, the monoclonal antibody diluted 1:100 respectively induces the ascites of a mouse to be primary antibody, goat anti-mouse IgG marked by HRP is taken as a secondary antibody to be subjected to Western-blot analysis, and finally, the target band is developed according to the operating instruction of a SuperSignal West Femto chemiluminescence substrate of Thermo Fisher company, and the prepared SPV K3L protein monoclonal antibody is identified as shown in figure 16.

(3) Assays at various times after detection of virus-infected cells by K3L antibody

The method comprises the steps of inoculating a capripoxvirus to Vero cells, collecting cells and culture solution at 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 24 hours and 48 hours after infection respectively, then coating an E L ISA plate, using anti-capripoxvirus K3L protein hybridoma cell supernatant as an antibody, and detecting the expression quantity of K3L protein after SPV infection of the cells, wherein the result is shown in figure 17.

(4) Immunofluorescence analysis subcellular localization

The Vero cells of a control group and Vero cells infected by a goat pox virus GY strain are fixed with 4% paraformaldehyde for 20 min, dried at room temperature, washed 3 times with PBS containing 0.1% Tween-20 and pH7.4 for 10 min/time, incubated for 30 min with 5% BSA, added with goat pox virus K3L protein C-terminal monoclonal antibody diluted 100 times with PBS 1: 0.1% Tween-20 and pH7.4 to induce mouse ascites to be primary antibody, washed with the method after 1 h at 37 ℃, added with 1:100 times diluted rhodamine-labeled goat anti-mouse IgG antibody, washed with the same method after 30 min at 37 ℃, finally sealed with 90% glycerol, and observed by a fluorescence microscope, the results of indirect immunofluorescence detection are shown in figures 18 to 20, wherein the goat pox virus K3L protein detected in figure 18 expresses, PI 19 stains cell nuclei, the results of overlapping K3L after figures 20 and show that the fluorescence of the red cell nuclei appears obviously, and the fluorescence polymerization degree of the fluorescence of the cells is higher.

(5) Antibody specificity assay

E L ISA plates are respectively coated with four pathogens of A type foot-and-mouth disease virus (FMDV-A), orf virus (ORFV), peste des petits ruminants virus (PPRV) and SPV, and the detection is carried out by using anti-capripoxvirus K3L protein hybridoma cell supernatant as an antibody, the detection results are shown in Table 1.

Conclusion

The recombinant K3L protein is used for immunizing a BA L B/c mouse, the prepared monoclonal antibody can specifically recognize the SPV K3L protein, and the detection of K3L protein at different time points after the virus infects cells finds that the K3L protein can be detected at the earliest in 2 hours of cell inoculation, which indicates that the recombinant K3L protein can be used for detecting the SPV K3L protein in basic tests.

<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences

<120> monoclonal antibody against capripoxvirus K3L protein C terminal and application thereof

<160>3

<210>1

<211>267

<212>DNA

<213> SPV K3L Gene sequence before optimization

<400>

atgtcatcga atagcgattt ggcattttgt tacgttttac ctaacattaa tgaagtaaca 60

gatggtattg tgtgtataag agataacatt gtatatgtaa aactaattaa ctatggtttg 120

gaagcacttg taatagatta tgttaatata aacatggatc aaatgaataa tataaaaaaa 180

acattagtta ataaattaat taatgtgcaa attataagga tgaacaaaat aaaaggatat 240

attgatgtaa aaatttataa taacaac 267

<210>2

<211>267

<212>DNA

<213> optimized SPV K3L Gene sequence

<400>

atgtcaagta actccgacct ggcgttttgc tatgtgctgc cgaatatcaa cgaagtcacg 60

gatggtattg ttttcattaa agataacatt gtgtatgtta aactgatcaa ctacggcctg 120

gaagcgctgg tcattgatta tgtggacatc aacatggatc agatgaacaa cattaagaaa 180

accctggtta acaaactgat caatgtccaa atcgtgcgta tgaataaaat caaaggctac 240

atcgacgtga aagtccacaa taataat 267

<210>3

<211>89

<212>PRT

<213> sequence encoding SPV K3L protein

<400>

Met Ser Ser Asn Ser Asp Leu Ala Phe Cys Tyr Val Leu Pro Asn

1 5 10 15

Ile Asn Glu Val Thr Asp Gly Ile Val Cys Ile Arg Asp Asn Ile

16 20 25 30

Val Tyr Val Lys Leu Ile Asn Tyr Gly Leu Glu Ala Leu Val Ile

31 35 40 45

Asp Tyr Val Asn Ile Asn Met Asp Gln Met Asn Asn Ile Lys Lys

46 50 55 60

Thr Leu Val Asn Lys Leu Ile Asn Val Gln Ile Ile Arg Met Asn

61 65 70 75

Lys Ile Lys Gly Tyr Ile Asp Val Lys Ile Tyr Asn Asn Asn

76 80 85 89

Claims (6)

1. A hybridoma cell strain K3L 35 capable of secreting the C-terminal monoclonal antibody of the anti-capripoxvirus K3L protein has a preservation number of CCTCC NO: C2015223 in China center for type culture collection.

2. The monoclonal antibody secreted by the hybridoma cell line of claim 1.

3. The use of the hybridoma cell line of claim 1 in the preparation of an early diagnosis reagent or an early detection reagent for capripoxvirus infection.

4. The use of the monoclonal antibody of claim 2 for the preparation of a reagent for the early diagnosis or the early detection of capripoxvirus infection.

5. Use of the hybridoma cell line K3L 35 according to claim 1 for the preparation of a reagent for sheep pox virus testing.

6. Use of the monoclonal antibody secreted by hybridoma cell line K3L 35 according to claim 1 for the preparation of a reagent for the sheep pox virus assay.

Images