CN115286699A - Goat pox virus monoclonal antibody and application thereof - Google Patents

Abstract

The invention relates to the technical field of medicines, and particularly relates to a goat pox virus monoclonal antibody and application thereof. A protein rGP32 delta with immunogenicity, wherein the coding sequence of the protein rGP32 delta is shown in the 4 th to 717 th positions of SEQ ID No. 1; the amino acid sequence of the protein rGP32 delta is shown as the 2 nd to the 239 th sites of SEQ ID No. 2. The invention uses the inactivated goat pox virus as immunogen to immunize a mouse, obtains the truncated protein rLP32 delta of P32 protein with strong LSDV immunogenicity through a prokaryotic system, and uses the rLP32 delta to screen LSDV monoclonal antibody hybridoma cell strains and establish a subsequent ELISA detection method, thereby greatly avoiding unnecessary biological potential safety hazards while maintaining the natural conformation of the virus antigen to the maximum extent.

Description

Goat pox virus monoclonal antibody and application thereof

Technical Field

The invention relates to the technical field of medicines, and in particular relates to a goat pox virus monoclonal antibody and application thereof.

Background

Goat pox virus (GTPV) belongs to Poxviridae (poxviridae) and Capripox (Capripoxvirus) and can infect sheep Yang and goats to cause malignant pox diseases, including kenyasheep Yang pox, goat pox, indian sheep dermatitis and the like. The fine wool sheep and the lamb are most susceptible to the virus, the diseased sheep generate fever, and the clinical manifestations of papules and herpes, lymph node enlargement, skin edema, emaciation of the diseased sheep, great reduction of milk production and the like occur at the skin mucous membrane of the hairless or hairless part, so that the production performance of the goat and the quality of wool and lamb skin products are seriously reduced, the mortality rate of adult diseased sheep can reach 42.06 percent, the mortality rate of diseased lambs reaches 100 percent, and once the diseased lambs are suffered from the disease, huge economic loss can be caused.

The disease has already occurred in many areas of the world, particularly, the epidemic is severe in parts of northern Africa, the middle east and Asia, and the epidemic is also existed in the peripheral countries of China, such as Nepal, russia, india and the like. The disease also occurs in Jiangsu, shaanxi, fujian, sichuan, yunnan, guangxi and other places in China, and the propagation and the prevalence of goat pox are mainly caused by the introduction and the flow of varieties in the development process of sheep raising industry.

The disease is a type A disease specified by OIE, and is classified as a national type I animal epidemic disease in China. In addition, goat pox virus can infect human, one goat pox virus occurring in Penghui county in Chongqing city has specific etiology diagnosis according to the goat pox infection case of human, and several reports of goat pox infection of human occur in other areas, all cases have contact history with diseased sheep and are contact infection, so that the research on the goat pox virus detection method has important public health significance.

Although China has commercial goat pox vaccines, the lack of effective GTPV antibody detection kits leads to the difficulty in screening goat pox antibody-negative animals in the process of developing related vaccines, and the evaluation of the immune effect of target animals can only adopt Virus Neutralization Test (VNT) related to live GTPV, so that great biological potential safety hazards exist. For this reason, there is an urgent need to establish a rapid, sensitive, specific GTPV antibody ELISA detection method.

The P32 protein of the goat pox virus and the antigenicity thereof have been disclosed in the prior art, but the obtained P32 protein has stronger hydrophobicity, which influences the biological application thereof. The P32 protein obtained by the prior art mostly exists in the form of inclusion bodies, the amount of soluble protein is less, the treatment process of an expression product is complex, and the expression amount is low.

Disclosure of Invention

The invention aims to provide a recombinant GTPV P32 protein truncation protein rGP32 delta, a hybridoma cell strain secreting anti-GTPV, a monoclonal antibody prepared by the hybridoma cell strain and a GTPV blocking ELISA detection method further established, so that technical support is provided for the research and development of a GTPV vaccine and the research and development of an efficacy test substitution method.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows.

In a first aspect, the invention provides a protein rGP32 delta with immunogenicity, wherein the coding sequence of the protein rGP32 delta is shown in the 4 th to the 717 th positions of SEQ ID No. 1;

in a specific embodiment, the coding sequence is the amino acid coding sequence of 1-238 th of the P32 protein of GTPV optimized according to the preferred codons of Escherichia coli, and a 6 XHis tag is introduced at the C-terminal end to synthesize the gene segment GGP32 delta.

Furthermore, the amino acid sequence of the protein rGP32 delta is shown as 2 nd to 239 th sites of SEQ ID No. 2.

In a specific example, the rGP32 delta protein is prepared from BL21 (DE 3) cell strain of E.coli, a host cell that recombinantly expresses rGP32 delta, as a production strain, which is the Escherichia coli BL322 strain.

Further, the protein rGP32 delta is a soluble protein;

in a specific embodiment, the soluble protein rGP32 delta is obtained by carrying out fermentation culture, induction expression, thallus disruption and soluble antigen protein separation and purification on a production strain Escherichia coli BL/GP32, wherein the preservation number of the Escherichia coli BL322 is CGMCC No. 245729.

In a second aspect, the invention provides a hybridoma cell strain secreting a monoclonal antibody against GTPV.

A hybridoma cell strain secreting monoclonal antibody against GTPV, wherein the hybridoma cell strain is GTPV hybridoma cell p32-11;

the preservation number is CGMCC No.45132.

In a specific embodiment, the hybridoma cell strain is prepared by the following method:

s101, taking the inactivated goat pox virus as an antigen to immunize an animal, and fusing splenocytes of the immunized animal with SP2/0 cells;

s102 fusion cell utilizes recombinant protein rGP32 delta to carry out screening of positive clone and subclone, and a hybridoma cell strain capable of stably secreting anti-GTPV monoclonal antibody is GTPV hybridoma cell p32-11.

In a third aspect, the invention provides a monoclonal antibody against GTPV, said monoclonal antibody being secreted by GTPV hybridoma cell p32-11;

the monoclonal antibody is a monoclonal antibody p32-11.

In a specific embodiment, the monoclonal antibody p32-11 is obtained from the culture of GTPV hybridoma p32-11.

In another specific embodiment, the monoclonal antibody p32-11 is obtained by inoculating hybridoma p32-11 strain into abdominal cavity of mouse to produce ascites.

In a fourth aspect, the invention provides a method for producing an anti-GTPV polyclonal antibody.

A preparation method of an anti-GTPV polyclonal antibody comprises the following steps: and immunizing the sheep with the GTPV inactivated vaccine, and separating serum of the diseased sheep as an anti-GTPV polyclonal antibody 14 days after challenge.

In a fifth aspect, the invention provides a product for detecting capripoxvirus and application thereof.

A product for detecting capripoxvirus, said product comprising a monoclonal antibody to GTPV.

Use of an anti-GTPV monoclonal antibody for the preparation of a medicament, a reagent, a test plate or a kit;

the reagent, the detection plate or the kit is used for detecting GTPV in a sample;

the agents are useful for screening of antibody negative animals for GTPV and/or vaccine efficacy testing.

The preparation of monoclonal antibody is the basis for establishing excellent ELISA detection method, and the immunogen is the key for preparing monoclonal antibody. Therefore, the invention uses inactivated GTPV as immunogen to immunize mice, obtains the truncated protein (rGP 32 delta) of P32 protein with stronger GTPV immunogenicity through a prokaryotic system, and uses the rGP32 delta to screen GTPV monoclonal antibody hybridoma cell strains and establish a subsequent ELISA detection method, thereby greatly avoiding unnecessary biological potential safety hazard while keeping the natural conformation of virus antigen to the maximum extent.

Meanwhile, the blocking ELISA detection method of the GTPV antibody is established by using the monoclonal antibody obtained by screening, so that technical support is provided for the research of GTPV related vaccines and the development of alternative methods for efficacy test.

The invention obtains GTPV truncated recombinant P32 protein rGP32 delta expressed in a soluble form through an escherichia coli expression system for the first time, and utilizes the protein as a coating antigen and inactivated goat pox virus as an immune antigen for screening anti-monoclonal antibody hybridoma cell strains and establishing GTPV antibody blocking ELISA for the first time. The invention has the following advantages:

(1) The soluble expression of the recombinant protein rGP32 delta of GTPV is realized through an escherichia coli expression system by codon optimization and artificial synthesis technology for the first time, so that the complexity of the purification of the inclusion body protein is avoided.

(2) The recombinant protein of GTPV and the inactivated goat pox virus are obtained by using an exogenous expression system for the first time to prepare the monoclonal antibody, so that the biological potential safety hazard of GTPV can be reduced.

(3) The rGP32 delta with the purity of more than 90 percent is used as a coating antigen for the first time to establish the GTPV antibody blocking ELISA, so that the purity of the coating antigen is ensured, and the potential safety hazard of GTPV is solved.

The invention establishes a GTPV antibody blocking ELISA detection method by using the anti-GTPV monoclonal antibody and the rGP32 delta for the first time, and the method has the characteristics of simple and convenient sample operation, low cost, quick reaction, strong specificity and the like, thereby providing a foundation for screening GTPV antibody negative animals and researching related vaccine efficacy test substitution methods.

Drawings

Fig. 1 shows the soluble expression identification of rGP32 Δ.

FIG. 2 shows the SDS-PAGE identification of rGP 32. Delta. Purification.

FIG. 3 shows the result of the identification of the purification effect of monoclonal antibody p32-11.

In FIG. 1, M is protein marker; PC1 is BSA (1. Mu.g); PC2 is BSA (2. Mu.g); 1 is uninduced cell lysate; 2 is cell lysate induced at 15 ℃ for 16 h; 3 is cell lysate induced at 37 ℃ for 4 h; 4 is uninduced cell lysis supernatant; 5, cell lysis supernatant induced at 15 ℃ for 16 h; 6 is cell lysis supernatant induced at 37 ℃ for 4 h; 7 is uninduced cell lysis precipitate; 8, cell lysis precipitation induced at 15 ℃ for 16 h; 9 is cell lysis sediment induced at 37 ℃ for 4h.

In FIG. 2, M is Protein marker; BSA was 1. Mu.g BSA; p32 is purified rGP32 Δ.

In FIG. 3, M is Protein marker;1 is purified p32-11.

Detailed Description

In order to make the technical solution of the present invention better understood by those skilled in the art, the following examples further describe the present invention in detail, and the following examples are only used for illustrating the present invention, but not to limit the scope of the present invention.

Example 1

Preparation of rGP 32. DELTA.

Construction of an expression vector for rGP32. DELTA.

(1) Gene synthesis

Amino acid coding sequence from 1 st to 238 th of GTPV (GenBank: MG 458384.1) P32 protein is optimized according to preferred codons of escherichia coli, a 6 XHis tag is introduced into the C terminal, and a gene segment GGP32 delta is artificially synthesized by a chemical synthesis method and contains 744 nucleotides in total. The specific nucleic acid sequence is shown as SEQ ID No. 1; the specific amino acid sequence is shown in SEQ ID No. 2.

(2) Construction of fusion expression vectors

Artificially synthesized GGP32 delta gene is used as a template, and a primer pair 1F/1R is adopted for PCR amplification.

Wherein the sequence of the upstream primer 1F is as follows: 5' cggccatatggcagatattcc-; the 5' end of the DNA is introduced with a restriction enzyme Nde I site and a protective base.

The sequence of the downstream primer 1R is as follows: 5 'ccgcaagctttatgtggtgatg-3'; the 5' end of the DNA is introduced with a restriction enzyme Hind III site and protective bases.

The PCR system is as follows:

Buffer(Mg ²⁺ plus) 10. Mu.L, dNTPs 4. Mu.L, upstream and downstream primers 1. Mu.L each,

HS polymerase 1. Mu.L, DNA template 2. Mu.L, supplemented with ddH ₂ O to 50. Mu.L system.

The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 1min; denaturation at 98 ℃ for 10s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1min for 33 cycles; finally, ring extension at 72 ℃ for 10min.

And recovering the target DNA band obtained by amplification, carrying out double digestion by Nde I/Hind III, connecting the target DNA band with a pET30a vector digested by the same digestion to obtain a positive clone pET30a-GGP32 delta inserted into a GGP32 delta gene, converting the obtained prokaryotic expression plasmid pET30a-GGP32 delta into a DH5 alpha competent cell, picking a single clone into an LB liquid culture medium containing kanamycin, carrying out shaking culture at 37 ℃ overnight, and extracting the plasmid for later use.

Expression and purification of rGP32. DELTA.

(1) Construction of genetic engineering strain of recombinant GGP32 delta gene

The extracted plasmid is transformed into Escherichia coli BL21 (DE 3) competent cells, a single clone is selected to be put into an LB liquid culture medium containing kanamycin, the culture is carried out overnight under shaking at 37 ℃, after the identification of the DNA fragment containing the target DNA through PCR, the plasmid is named as Escherichia coli (Escherichia coli) BL322 strain, and the equal volume of 50 percent of glycerol LB is added for freezing and storing at-70 ℃.

(2) Expression and purification of rGP 32. DELTA

Recombinant Escherichia coli (E.coli) BL322 strain was inoculated into 1L LB liquid medium containing kanamycin, fermented and cultured, and shake-cultured at 37 ℃ to OD ₆₀₀ When the concentration is 0.6-0.8, IPTG solution with the final concentration of 0.5mM is added for induced culture for 4 hours.

Centrifugally collecting thalli after bacterial liquid culture, adding 10mL of lysate [0.02mol/L Tris buffer solution (pH value 7.2) and 0.3mol/L NaCl ] into each gram of thalli, re-suspending the thalli according to the proportion, and ultrasonically crushing the thalli for 30min in an ice water bath, wherein the crushing conditions are as follows: the working time is 9s, the pause time is 9s, the ultrasonic power is 400W, and the soluble expression identification result is shown in figure 1.

The crushed bacterial liquid is centrifuged at 12000r/min for 10min at 4 ℃, and the supernatant is collected. According to the instruction of Ni-IDA affinity chromatography medium kit (Nanjing Kingsrey company), the target protein which is expressed in a soluble way in the thalli lysis supernatant is purified and filtered by a filter membrane with the aperture of 0.22 mu m, thus obtaining the primarily purified target protein. The SDS-PAGE identification of rGP 32. Delta. Purification is shown in FIG. 2.

Example 2

Screening of P32 protein monoclonal antibody hybridoma cell strain secreting GTPV and preparation of monoclonal antibody ascites

BALB/c mice immunization and antibody titer determination

The inactivated goat pox virus is used as an immunizing antigen to immunize 3 BALB/c mice of 6-8 weeks for 4 times, and the interval of each time is 2 weeks.

Adding equal volume of Freund complete adjuvant into protein for the first immunization, emulsifying, and immunizing mice by multi-point subcutaneous injection at neck and back, wherein each mouse is 50 mu g; then, the adjuvant is replaced by Freund incomplete adjuvant after immunization, and the rest is the same as the initial immunization; 7-10 days after the three-immunization, a small amount of blood is collected at the tail part, the serum titer of the purified rGP32 delta is measured by an indirect ELISA method by using a 2 mu g/mL coated ELISA plate, a mouse with the titer higher than 1. Taking the splenocytes of the mice with the highest titer 3-5 days after the last immunization for cell fusion.

2. Cell fusion and screening of hybridoma cell lines

Taking myeloma cells SP2/0 with good growth state and splenocytes of mice with highest ELISA detection titer to perform chemical fusion by PEG1450, screening positive hybridoma cells by using an indirect ELISA method taking rGP32 delta as a coating antigen, performing subcloning for 3-5 times by using a limiting dilution method to obtain 1 hybridoma cell strain P32-11 secreting anti-GTPV P32 protein, and identifying according to the specification of an IsoQuickTMStreps and Kits for Mouse Monoclonal Isotyping kit, wherein the subtype of a Monoclonal antibody secreted by the hybridoma cell strain is G2a.

3. Preparation of anti-GTPV P32 protein monoclonal antibody ascites

Healthy multiparous BALB/c mice of 6-8 months of age are selected, intraperitoneal injection of Freund's incomplete adjuvant is carried out for sensitization, 0.5mL of mice is used, and hybridoma cells are injected after 7 days. Collecting the three hybridoma cells in the logarithmic growth phase and SP2/0 cells, centrifuging at 800r/min for 10min, washing the cells once by using a DMEM culture solution, resuspending, adjusting the cell density to 106/mL, and injecting 0.5mL into the abdominal cavity of each mouse. Gently kneading the abdomen of the mouse with alcohol cotton to uniformly disperse cells in the abdominal cavity; after 7 to 14 days, when the abdomen of the mouse is obviously swollen, aseptically puncturing the lower abdominal circumference swelling part of the mouse by using a needle head of a 20ml syringe, and gently kneading and pressing to ensure that ascites flows out or drips out; ascites is collected in a 15ml centrifuge tube, centrifuged at 3000r/min for 10min, marked as p32-11 and ascites contrast (S) of myeloma cell SP2/0, subpackaged and stored at-20 ℃ for later use.

Example 3

Purification and detection of monoclonal ascites

The Protein A (GE Healthcare 17-5079-01) affinity column purification method is adopted for purification, and the purification steps are as follows:

sample pretreatment: ascites fluid was diluted with a coupling buffer (20 mM sodium phosphate buffer, pH 7.0) at 1.

Balancing: the column was equilibrated with 5-10 column volumes of coupling buffer, maintaining a flow rate of 2 s/drop.

Loading: the sample 4 was injected into the upper port of the column using a syringe and the effluent collected in a 50ml centrifuge tube, maintaining a flow rate of 4 s/drop.

Impurity washing: the column was run with 5 column volumes of coupling buffer, maintaining a flow rate of 2 s/drop.

And (3) elution: the antibody was eluted with 5 column volumes of elution buffer (0.1M sodium citrate buffer, pH 9.0) and collected in the above-mentioned EP tube at a flow rate of 4 s/drop.

And (3) detection of the sample: performing SDS-PAGE identification on the purified monoclonal antibody, and obtaining the monoclonal antibody with high purity by a Protein A affinity column purification method; the BCA method is adopted to measure the concentration of the monoclonal antibody, and the concentration can reach 2.62mg/mL.

The result of the identification of the purification effect of the monoclonal antibody p32-11 is shown in FIG. 3.

Example 4

Establishment of blocking ELISA detection method of goat pox virus (GTPV) antibody based on monoclonal antibody p32-11

1. Preparation of anti-GTPV polyclonal antibody:

selecting sheep as an immune animal, immunizing for 3 times by using 2 parts of GTPV inactivated vaccine, spacing for 14 days before and after immunization, and collecting sheep serum for later use after 14 days after the third immunization.

Determination of blocking ELISA detection method conditions for GTPV antibody

(1) Optimal working concentration of goat anti-GTPV polyclonal and monoclonal antibodies

Diluting purified rGP32 delta with PBS to 2 mug/ml, 100 mug/well, blocking overnight at 4 ℃, washing three times with PBST, adding PBST solution containing 5% skimmed milk powder as blocking solution, 200 mug/well, blocking overnight at 4 ℃;

washing with PBST solution for 3 times, patting the 96-well enzyme-labeled plate dry, and storing at-20 ℃ for later use.

And (2) taking out the coated 96-well elisa plate from a refrigerator at the temperature of-20 ℃, placing the plate at room temperature for 30min, adding the obtained goat anti-GTPV polyclonal antibody, selecting PBS as a diluent, and adding negative serum with corresponding dilution in the following dilution levels of 1.

Washing with PBST solution for 3 times, adding p32-11, diluting with PBS at concentration of 2000 times to 1.6 ten thousand times, 100 μ L/well, and incubating at 37 deg.C for 30min.

Washing with PBST solution for 3 times, adding soluble TMB substrate developing solution 50 μ L/well, reacting at room temperature in dark place for 15min, and reacting with 2M H ₂ SO ₄ The reaction was stopped and absorbance was read at 450 nm.

And selecting the OD value of the negative control hole to be close to 1.0, and setting the corresponding dilution as the optimal working concentration when the positive serum blocking rate is more than or equal to 50 percent.

Finally, the working dilution of the goat anti-GTPV polyclonal antibody is determined to be 1.

(2) Determination of criteria for decision of results

Detecting 63 sheep serum samples by using the established antibody blocking ELISA, setting positive and negative controls at the same time,

ˉ

determination of OD ₄₅₀ The average (x) =5.619% and the standard deviation (S) =6.108% were calculated. When the sample blocks the rate

ˉˉ

If PI is more than or equal to x +3s (23.9%), the serum is judged to be positive; when PI is less than or equal to x +2s (17.8 percent), the PI is negative, and if the PI is still suspicious, the PI is determined to be positive.

(3) Specific detection of GTPV antibody blocking ELISA method

Diluting the goat colibacillosis antibody positive serum, the goat aphtha virus positive serum, the goat clostridium putrefying toxin, the A and D type clostridium perfringens toxin antibody positive serum by 1. The result shows that the above serums are all negative, which indicates that no cross reaction exists, and indicates that the method has good specificity.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the details of the above embodiments, and various modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the scope of the present invention.

It should be noted that, in the foregoing embodiments, various specific technical features and steps described in the above embodiments can be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations of the features and steps are not described separately.

In addition, any combination of the various embodiments of the present invention can be made, and the same should be considered as the disclosure of the present invention as long as the idea of the present invention is not violated.

Sequence listing

<110> China institute for veterinary drug inspection

<120> goat pox virus monoclonal antibody and application thereof

<141> 2022-04-22

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 744

<212> DNA

<213> Artificial Sequence

<400> 1

catatggctg acatcccgct gtatgtgatc ccgattgttg gtcgtgaaat ctctgacgtg 60

gtcccggaac tgaaatctgg taatgatatt ttttataaaa aagtcgatac cgtgaaagac 120

ttcaaaaact ctgatgtcaa atttttcctg aaagataaaa aagacatcag cctgtcttac 180

aaattcctga tctgggaaaa ggtagaaaaa tcaggaggtg ttgaaaattt tacagaatat 240

ttttctggat tatgtaatgc tctttgtaca aaagaggtaa aaagttctat tgcaaaacac 300

tttagtttat ggaaatcgta tgccgatgcg gatataaaaa attctgagaa taagtttatt 360

gttgttatag aagatgataa cacattaaaa gatttaataa caatatataa cattataatt 420

gaaatgcaag aaaaaaatat agacattttc caattacgtg aaacttttca taatagtaat 480

tctagaatat tgttcaatca agaaaataat aattttatgt attcgtacac agggggatat 540

gattttacct tatctgcata cgtaattaga ttatcgtctg ccataaaaat aataaacgaa 600

attataaaaa ataaaggtat ttctaccagt ttgagttttg aaatgtataa gttggaaaaa 660

gaattaaaac tcaatagaca agttttaaat gactcatcta agtatatact tcacaatcat 720

catcaccatc accactaaaa gctt 744

<210> 2

<211> 245

<212> PRT

<213> recombiant p32 of lumpy skin disease virus

<400> 2

His Met Ala Asp Ile Pro Leu Tyr Val Ile Pro Ile Val Gly Arg Glu

1 5 10 15

Ile Ser Asp Val Val Pro Glu Leu Lys Ser Gly Asn Asp Ile Phe Tyr

20 25 30

Lys Lys Val Asp Thr Val Lys Asp Phe Lys Asn Ser Asp Val Lys Phe

35 40 45

Phe Leu Lys Asp Lys Lys Asp Ile Ser Leu Ser Tyr Lys Phe Leu Ile

50 55 60

Trp Glu Lys Val Glu Lys Ser Gly Gly Val Glu Asn Phe Thr Glu Tyr

65 70 75 80

Phe Ser Gly Leu Cys Asn Ala Leu Cys Thr Lys Glu Val Lys Ser Ser

85 90 95

Ile Ala Lys His Phe Ser Leu Trp Lys Ser Tyr Ala Asp Ala Asp Ile

100 105 110

Lys Asn Ser Glu Asn Lys Phe Ile Val Val Ile Glu Asp Asp Asn Thr

115 120 125

Leu Lys Asp Leu Ile Thr Ile Tyr Asn Ile Ile Ile Glu Met Gln Glu

130 135 140

Lys Asn Ile Asp Ile Phe Gln Leu Arg Glu Thr Phe His Asn Ser Asn

145 150 155 160

Ser Arg Ile Leu Phe Asn Gln Glu Asn Asn Asn Phe Met Tyr Ser Tyr

165 170 175

Thr Gly Gly Tyr Asp Phe Thr Leu Ser Ala Tyr Val Ile Arg Leu Ser

180 185 190

Ser Ala Ile Lys Ile Ile Asn Glu Ile Ile Lys Asn Lys Gly Ile Ser

195 200 205

Thr Ser Leu Ser Phe Glu Met Tyr Lys Leu Glu Lys Glu Leu Lys Leu

210 215 220

Asn Arg Gln Val Leu Asn Asp Ser Ser Lys Tyr Ile Leu His Asn His

225 230 235 240

His His His His His

245

Claims (10)

1. An immunogenic protein, rGP 32. DELTA., characterized by: the coding sequence of the protein rGP32 delta is shown in the 4 th to 717 th positions of SEQ ID No. 1;

the amino acid coding sequence of 1-238 sites of the P32 protein of GTPV is optimized according to the preferred codon of Escherichia coli, and a 6 XHis tag is introduced into the C terminal to synthesize a gene segment GGP32 delta.

2. An immunogenic protein rGP32 Δ according to claim 1, wherein: the amino acid sequence of the protein rGP32 delta is shown as the 2 nd to the 239 th sites of SEQ ID No. 2.

3. The immunogenic protein rGP32 Delta according to claim 1, wherein said protein rGP32 Delta is a soluble protein;

the strain is obtained by carrying out fermentation culture, induction expression, thallus breakage and soluble antigen protein separation and purification on a production strain Escherichia coli BL322, wherein the preservation number of the Escherichia coli BL322 is CGMCC No. 245929.

4. A hybridoma cell strain secreting monoclonal antibodies against GTPV is characterized in that the hybridoma cell strain is GTPV hybridoma cell p32-11;

the preservation number is CGMCC No.45132.

5. The hybridoma cell strain secreting anti-GTPV monoclonal antibody of claim 4, wherein: the hybridoma cell strain is prepared by the following method:

s101, taking the inactivated goat pox virus as an antigen to immunize an animal, and fusing splenocytes of the immunized animal with SP2/0 cells;

s102 fusion cell utilizes recombinant protein rGP32 delta to carry out screening of positive clone and subclone, and a hybridoma cell strain capable of stably secreting anti-GTPV monoclonal antibody is GTPV hybridoma cell p32-11.

6. An anti-GTPV monoclonal antibody characterized by: the monoclonal antibody is secreted by a GTPV hybridoma cell p32-11;

the monoclonal antibody is a monoclonal antibody p32-11.

7. The anti-GTPV monoclonal antibody of claim 11, wherein the monoclonal antibody p32-11 is obtained from the culture of GTPV hybridoma p32-11 or is obtained by inoculating a hybridoma cell line into the abdominal cavity of a mouse to produce ascites.

8. A preparation method of an anti-GTPV polyclonal antibody is characterized by comprising the following steps: and (3) immunizing the sheep with the GTPV inactivated vaccine, and separating serum of the diseased sheep 14 days after the virus challenge to serve as an anti-GTPV polyclonal antibody.

9. A product for detecting capripoxvirus, which is characterized in that: the product comprises a monoclonal antibody to GTPV.

10. Use of an anti-GTPV monoclonal antibody for the preparation of a medicament, reagent, test panel or kit;

the reagent, the detection plate or the kit is used for detecting GTPV in a sample;

the agents are useful for screening of antibody negative animals for GTPV and/or vaccine efficacy testing.

Images