CN109336955B

CN109336955B - Preparation method and application of goat pox virus recombinant protein antigens

Info

Publication number: CN109336955B
Application number: CN201811241377.0A
Authority: CN
Inventors: 景志忠; 陈国华; 贾怀杰; 何小兵; 房永祥; 牛贤云
Original assignee: Lanzhou Veterinary Research Institute of CAAS
Current assignee: Lanzhou Veterinary Research Institute of CAAS
Priority date: 2018-10-24
Filing date: 2018-10-24
Publication date: 2021-11-02
Anticipated expiration: 2038-10-24
Also published as: CN109336955A

Abstract

The invention discloses a preparation method and application of a group of goat pox subunit vaccine recombinant protein antigens. The invention provides a product with 1) or 2) functions, 1) preventing, diagnosing and/or treating goat pox of animals; 2) inhibiting or neutralizing goat pox virus; the product is any one of the following: protein ORF056, protein ORF112, and protein ORF 117; or a composition comprising protein ORF056, protein ORF112 and protein ORF 117; or a fusion protein comprising protein ORF056, protein ORF112 and protein ORF 117; and the like. Experiments prove that the subunit vaccine prepared by utilizing the IMV ORF056, ORF112 encoded protein and EEVORF117 encoded protein is higher in neutralizing antibody than a vaccine prepared by adopting single protein of ORF056, ORF112 or ORF117 for immunizing a mouse, and the subunit vaccine is an ideal vaccine with the potential of preventing capripox.

Description

Preparation method and application of goat pox virus recombinant protein antigens

Technical Field

The invention belongs to the fields of molecular biotechnology and immunity technology, and particularly relates to a preparation method and application of a group of goatpox subunit vaccine recombinant protein antigens.

Background

Goat pox (Goat pox) is a thermal, contact skin, mucosal disease caused by Goat pox virus of the genus Goat pox virus of the subfamily Chordopoxvirinae; the animal epidemic disease is widely popular in Africa, middle east, middle Asia, India, northwest and northeast of China, and the like, is listed as one of animal epidemic diseases which must be reported by the world animal health organization, and is listed as a legal animal epidemic disease in China. The sheep pox is endemic in China and regions such as Zhongya, India, Zhongfei, and Beifei, and is also endemic in provinces such as Heilongjiang, Jilin, Liaoning, Hebei, Gansu, Qinghai and inner Mongolia in China, which causes great economic loss to sheep raising industry.

At present, no specific treatment method for the disease exists, only clinical trauma treatment and supportive therapy can be adopted, and the method mainly adopts vaccination of goat pox attenuated live vaccine for prevention and control at home and abroad, but the vaccine can not completely eliminate and control the disease, and has certain defects in the aspects of immunity and safety, so that the creation of a safe and efficient novel vaccine is imperative. The subunit vaccine immune animal can avoid the risk of virulent return caused by gene rearrangement of attenuated vaccine strain, avoid the possibility of incomplete inactivated vaccine inactivation and virus dispersion, and induce comprehensive and lasting immune response.

The genus capripoxvirus includes, in addition to the capripoxvirus (GTPV), three viruses of Sheeppox virus (SPPV) and bovine nodular herpes disease virus (LSDV). The two major virion forms of GTPV virions are Intracellular Mature Virions (IMV) and Extracellular Enveloped Viruses (EEV), encoding about 140 proteins. Therefore, screening out antigens with good immunogenicity is very critical to the preparation of subunit vaccines.

Disclosure of Invention

It is an object of the present invention to provide a product.

The product provided by the invention is a substance shown in any one of the following A-K:

A. protein ORF056, protein ORF112, and protein ORF 117;

B. a composition comprising protein ORF056, protein ORF112, and protein ORF 117;

C. a fusion protein comprising protein ORF056, protein ORF112 and protein ORF 117;

D. compositions containing the fusion proteins;

E. a nucleic acid encoding protein ORF056, a nucleic acid encoding protein ORF112 and a nucleic acid encoding protein ORF 117;

F. a nucleic acid encoding the fusion protein;

G. expressing the biological material encoding the protein ORF056 nucleic acid, expressing the protein ORF112 nucleic acid and expressing the protein ORF117 nucleic acid;

K. biological material expressing the fusion protein;

the biological material is an expression cassette, a recombinant vector, a recombinant bacterium or a recombinant virus or a transgenic cell;

the protein ORF056 is a protein shown in the following 1) or 2):

1) protein consisting of amino acids shown in a sequence 2 in a sequence table;

2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein shown in 1) and has the same function;

the protein ORF112 is a protein shown in the following 3) or 4):

3) protein consisting of amino acids shown in a sequence 4 in a sequence table;

4) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein shown in the step 3) and has the same function;

the protein ORF117 is a protein shown in the following 5) or 6):

5) protein consisting of amino acids shown in a sequence 6 in a sequence table;

6) and (b) the protein with the same function is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence of the protein shown in the step 5).

The protein ORF056 can be a protein expressed by the full length of the gene of the encoding protein ORF056 and can also be a protein expressed by the extracellular region of the ORF056 gene;

the protein ORF117 may be a protein expressed in the entire length of the gene encoding the protein ORF117 or in the extracellular region of the ORF117 gene.

The product has the following functions of 1) or 2):

1) preventing, diagnosing and/or treating goat pox in an animal;

2) inhibit or neutralize a virus of the capripoxvirus genus.

In the above product, the composition comprising protein ORF056, protein ORF112 and protein ORF117 is a composition consisting of an antigen and an adjuvant, wherein the antigen consists of the protein ORF056, the protein ORF112 and the protein ORF 117;

the composition containing the fusion protein is a composition consisting of the fusion protein and an adjuvant;

or, the composition comprising protein ORF056, protein ORF112 and protein ORF117 is a composition consisting of the antigen and other proteins,

the composition containing the fusion protein is a composition consisting of the fusion protein and other proteins.

In the product, the other protein is a modified protein, a tag protein or a functional protein;

or, the adjuvant is Freund's adjuvant.

In the product, the mass ratio of the protein ORF056, the protein ORF112 and the protein ORF117 in the antigen is 1:1: 1.

In the above product, the virus of the genus capripoxvirus is capripoxvirus GTPV and/or capripoxvirus SPPV.

The nucleic acid of the coding protein ORF056 can be the whole length of the gene of the coding protein ORF056 and can also be the extracellular region of the ORF056 gene;

the nucleic acid encoding protein ORF117 may be the whole gene encoding protein ORF056 or the extracellular region of ORF117 gene.

In the above product, the nucleic acid encoding protein ORF056 is as follows:

a) the coding sequence comprises a sequence 1 in a sequence table;

b) DNA molecules which hybridize under stringent conditions with the DNA molecules defined under a) and which code for the same proteins;

c) DNA molecule with 80% or more or 90% or more homology with the DNA molecule defined in a) and encoding the same protein;

the nucleic acid encoding protein ORF112 is as follows:

d) the coding sequence comprises a sequence 3 in a sequence table;

e) DNA molecules which hybridize under stringent conditions with the DNA molecules defined under a) and which code for the same proteins;

f) DNA molecule with 80% or more or 90% or more homology with the DNA molecule defined in e) and encoding the same protein;

the nucleic acid encoding protein ORF117 is as follows:

g) the coding sequence comprises a sequence 5 in a sequence table;

h) DNA molecules which hybridize under stringent conditions with the DNA molecules defined under a) and which code for the same proteins;

i) DNA molecule which has more than 80% or more than 90% homology with the DNA molecule defined by g) and encodes the same protein.

The product is vaccine or medicine or kit.

The application of any one of the substances A-K in preparing products with the following functions 1) and/or 2):

1) preventing, diagnosing and/or treating goat pox in an animal;

2) inhibit or neutralize a virus of the capripoxvirus genus.

In the above application, the product is a vaccine or a medicament or a kit;

or, the virus of the capripoxvirus genus is the capripoxvirus GTPV and/or the capripoxvirus SPPV.

Experiments prove that the subunit vaccine prepared by utilizing the IMV ORF056, ORF112 encoded protein and EEVORF117 encoded protein is higher in neutralizing antibody than a vaccine prepared by adopting single protein of ORF056, ORF112 or ORF117 for immunizing a mouse, and the subunit vaccine is an ideal vaccine with the potential of preventing capripox.

The invention has the following advantages: (1) the expressed antigen protein can react with GTPV serum, which indicates that the antigen has good antigen reactivity; (2) the group of antigens has good immunogenicity and no toxic or side effect, and a single or three of the three protein antigens are mixed with an adjuvant to immunize a mouse, so that a high-titer specific antibody can be generated; the polyclonal antibody generated after the three are mixed has higher capability of neutralizing SPPV and GTPV viruses; the vaccine has better protective effect because the three components are mixed and matched with an adjuvant to generate a high-titer antibody which can neutralize GTPV and SPPV; this provides a firm foundation for eradication and prevention of capripox.

Drawings

FIG. 1 shows the result of Western blotting detection of recombinant protein expression.

FIG. 2 shows the result of detecting the reactogenicity of the recombinant protein by Western blotting.

FIG. 3 is a graph showing the results of three protein purifications.

FIG. 4 is a graph showing the results of measurement of antibodies specific to different proteins in the serum of immunized mice.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The following examples are presented to facilitate a better understanding of the present invention and are not intended to limit the invention thereto.

EXAMPLE 1 preparation of a goat pox subunit vaccine

Preparation of recombinant proteins of ORF056-C, ORF112 and ORF117-C

1. Cloning of ORF056, ORF117 extracellular region and ORF112 gene and construction of expression vector

Extracting genomic DNA of a goat pox virus GTPV/HBSZ strain (described in the following documents: Zhou T, Jia H, Chen G, et al, Phytoenic analysis of Chinese sheepppox and Goatpox virus isolates. Virol J, 20129: 25), PCR-amplifying the extracellular region of ORF056 gene, the extracellular region fragment of ORF117 gene and the full length of ORF112 gene, digesting the target fragment and prokaryotic expression vector by restriction enzyme, connecting the target gene and vector by ligase, transforming BL21 competent cells, inducing expression of the target protein by inducer, and purifying the expressed protein by nickel chromatography column. The method comprises the following specific steps:

1) primer design and PCR amplification

According to published ORF056, ORF112 and ORF117 gene sequences, the extracellular region sequences and ORF112 sequence expression primers of ORF056 and ORF117 genes are designed by using the Primer5.0 software, and the enzyme cutting sites of a prokaryotic expression vector pET-30a (+) and a target gene are analyzed by using the seman software:

ORF056 upstream primer: 5' -GCGAATTCATG GGA GCA GCC GCA AGT ATA C-3', the underlined part is EcoRI restriction enzyme cutting site; a downstream primer: 5' -CGCCTCGAGTTA TCC CGA ACT TTGA CTA GGG-3', the underlined part is the XhoI restriction site.

ORF112 upstream primer: 5' -CGGAATTCATG GAC AGA GCG TTA TCC A-3, wherein the underlined part is an EcoRI restriction enzyme cutting site; a downstream primer: 5' -CCAAGCTTTCAT AGT GTT GTA CTT CTT CCT GT-3', the underlined part is the HindIII restriction site.

ORF117 upstream primer: 5' -CCGCGGTACCTTA GCA TTT TTT AAT AATAAT ACA TGT G-3', the underlined part is a KpnI restriction enzyme site; a downstream primer: 5'-CCGCGCTCGAGTTA AAA AAA AGA TCT TACACA G-3', the underlined part is the XhoI restriction site.

And (3) taking the genome DNA of the GTPV/HBSZ strain as a template, and respectively carrying out PCR amplification by using an ORF056 upstream primer and a downstream primer, an ORF112 upstream primer and a downstream primer, and an ORF117 upstream primer and a downstream primer to obtain an extracellular region of the 546bp ORF056 gene, the total length of the 447bp ORF112 gene and an extracellular region of the 396bp ORF117 gene.

The total reaction volume of the ORF056PCR described above was 50. mu.L: sterilized deionized water 30.5. mu.L, 4 XPCR buffer 10. mu.L, 2.5mM dNTP mix 4. mu.L, upstream and downstream primers (50. mu. mol/L) each 1. mu.L; 3.5 μ L of DNA template; the PCR amplification conditions of the expression fragment ORF056 are as follows: 4min at 95 ℃; 15s at 94 ℃, 15s at 55 ℃, 1min at 68 ℃ and 35 cycles; 5min at 72 ℃.

The total reaction volume of the ORF112PCR described above was 50. mu.L: sterilized deionized water 30.5. mu.L, 4 XPCR buffer 10. mu.L, 2.5mM dNTP mix 4. mu.L, upstream and downstream primers (50. mu. mol/L) each 1. mu.L; 3.5 μ L of DNA template; the PCR amplification conditions of the expression fragment ORF056 are as follows: 4min at 95 ℃; 15s at 94 ℃, 15s at 53 ℃, 1min at 68 ℃ and 35 cycles; 5min at 72 ℃.

The total reaction volume of the ORF117PCR described above was 50. mu.L: sterilized deionized water 30.5. mu.L, 4 XPCR buffer 10. mu.L, 2.5mM dNTP mix 4. mu.L, upstream and downstream primers (50. mu. mol/L) each 1. mu.L; 3.5 μ L of DNA template; the PCR amplification conditions of the expression fragment ORF056 are as follows: 4min at 95 ℃; 15s at 94 ℃, 15s at 55 ℃, 1min at 68 ℃ and 35 cycles; 5min at 72 ℃.

The amplification products were sent for sequencing with the following results:

the nucleotide sequence of the extracellular region of the ORF056 gene is sequence 1, the coded protein is named as ORF056-C, and the amino acid sequence of the protein is sequence 2;

the nucleotide sequence of ORF112 gene is sequence 3, the coded protein is named ORF112, and the amino acid sequence of the protein is sequence 4;

the nucleotide sequence of the extracellular region of ORF117 gene is sequence 5, the encoded protein is named ORF117-C, and the amino acid sequence of the protein is sequence 6.

2) Construction of expression vector

The amplified fragment of the extracellular region (546bp) of ORF056 and pET-30a vector are cut by EcoRI and XhoI, the amplified fragment of ORF112(447bp) and pET-30a vector are cut by EcoRI and Hind III, the amplified fragment of the extracellular region (396bp) of ORF117 and pET-30a vector are cut by KpnI and XhoI, the fragments are recovered after cutting, the fragments are connected for 12-16 h at 16 ℃, the connection product is transformed into BL21(DE3) competence, single clone is picked for PCR, cutting and sequencing to identify the recombinant vector with correct sequencing as pET-30-ORF056, pET-30-ORF112 and pET-30-ORF117, and the recombinant bacteria containing the recombinant vectors are named as BL21(DE3)/pET-30-ORF056, BL21(DE3)/pET-30-ORF112 and 21(DE3)/pET-30-ORF 117.

The recombinant vector pET-30-ORF056 is a vector obtained by replacing DNA molecules between EcoRI and XhoI enzyme cutting sites of a pET-30a vector with an extracellular region of ORF056 gene shown in a sequence 1, the vector expresses recombinant protein ORF056, and the recombinant protein ORF056 is a protein obtained by fusing a his tag on a C-terminal fusion vector of ORF056-C shown in a sequence 2;

the recombinant vector pET-30-ORF112 is a vector obtained by replacing the ORF112 gene shown in the sequence 3 with a DNA molecule between EcoRI and Hind III enzyme cutting sites of a pET-30a vector, the vector expresses a recombinant protein ORF112, and the recombinant protein ORF112 is a protein obtained by fusing a his tag on the C-terminal of the ORF112 shown in the sequence 4;

the recombinant vector pET-30-ORF117 is a vector obtained by replacing DNA molecules between KpnI and XhoI enzyme cutting sites of pET-30a vector with an ORF117 gene extracellular region shown in a sequence 5, the vector expresses recombinant protein ORF117, and the recombinant protein ORF117 is a protein obtained by fusing a his tag on a C-terminal vector of ORF117-C shown in a sequence 6.

2. Expression and purification of recombinant protein ORF056, recombinant protein ORF112 and recombinant protein ORF117

1) Western blotting detection of reactogenicity of expressed protein

The recombinant bacteria BL21(DE3)/pET-30-ORF056, BL21(DE3)/pET-30-ORF112 and BL21(DE3)/pET-30-ORF117 are inoculated into LB culture medium containing 50ug/mL Kan according to the volume ratio of 1%, shaking for 2-3 h at the temperature of 37 ℃ by shaking in a shaking table at 200rpm/min, adding IPTG inducer with the final concentration of 1mM when the OD value reaches about 0.6, inducing for 6h, and collecting thalli by centrifugation. SDS-PAGE electrophoresis of the bacteria is carried out, then the bacteria are transferred to a Nitrocellulose (NC) membrane, goat pox positive serum (goat pox virus (Goatpox virus) CVCC AV40 introduced by Chinese veterinary medicine supervision is utilized to artificially infect a goat of 3 months old with a negative antibody detected, blood is collected from a vein after 35d infection, serum is separated) to serve as a primary antibody, rabbit anti-goat IgG (sigma, A5420) marked by HRP is utilized as a secondary antibody, and Western blotting is utilized to detect the expression and the reactogenicity of the recombinant protein.

The expression result of the recombinant protein detected by Western blotting is shown in figure 1,

FIG. 1A is a diagram showing the result of recombinant bacterium BL21(DE3)/pET-30-ORF056 expression of ORF056-C, wherein M is protein Marker, 1 is uninduced BL21(DE3)/pET-30-ORF056, 2 is empty vector BL21(DE3)/pET-30 (empty vector pET-30 is transferred into Escherichia coli), and 3 is BL21(DE3)/pET-30-ORF056, and it can be seen that BL21(DE3)/pET-30-ORF056 expression yields recombinant protein ORF056 with a size of about 26 kDa;

FIG. 1B is a diagram showing the result of recombinant strain BL21(DE3)/pET-30-ORF117 expressing ORF117-C, wherein M is protein Marker, 1 is uninduced BL21(DE3)/pET-30-ORF117, 2 is empty vector BL21(DE3)/pET-30 (empty vector pET-30 transferred into E.coli), and 3 is BL21(DE3)/pET-30-ORF117, and it can be seen that BL21(DE3)/pET-30-ORF117 is expressed to obtain recombinant protein ORF117 with a size of about 19 kDa;

FIG. 1C is a diagram showing the results of recombinant bacteria BL21(DE3)/pET-30-ORF112 expressing ORF112-C, wherein M is protein Marker, 1 is uninduced BL21(DE3)/pET-30-ORF112, 2 is empty vector BL21(DE3)/pET-30 (empty vector pET-30 is transferred into E.coli), and 3 is BL21(DE3)/pET-30-ORF 112. it can be seen that BL21(DE3)/pET-30-ORF112 is expressed to give recombinant protein ORF112 having a size of about 34kDa (actually, the predicted molecular weight is 23kDa, and the electrophoretogram shows that the actually expressed molecular weight is about 34Kd, but the protein sequence is correct by mass spectrometry, which may be caused by the dimer expressed by the protein).

The result of detecting the reactogenicity of the recombinant protein by Western blotting is shown in figure 2, M: protein molecular mass standard; 1. the recombinant protein ORF56 reacts with positive serum of goat pox virus, and 2, the negative serum reacts; 3. the reaction condition of the recombinant protein ORF117 and goat pox virus positive serum is shown in the specification, and 4, the reaction condition of the recombinant protein ORF117 and goat pox virus negative serum is shown in the specification: 5. the recombinant protein ORF112 reacts with negative serum, and 6, the recombinant protein ORF112 reacts with positive serum of goat pox virus, and it can be seen that the recombinant proteins ORF56, ORF112 and ORF117 all react with positive serum of goat pox virus to form corresponding bands, and do not react with negative serum or form bands.

2) Purification of protein antigens

(1) Carrying out amplification culture on recombinant bacteria BL21(DE3)/pET-30-ORF056, BL21(DE3)/pET-30-ORF112 and BL21(DE3)/pET-30-ORF117 according to the induction culture conditions in 1), and centrifuging to collect bacteria;

(2) the thalli is washed twice in PBS phosphate buffer solution, suspended by 20mL of buffer solution, repeatedly frozen and thawed twice in a refrigerator at the temperature of minus 70 ℃, and centrifuged at 12000rpm for 10min by using an ultrasonic disruptor for ultrasonic treatment (the power is 3800w, ultrasonic treatment is 5s, stopping for 5s and ultrasonic treatment is 1 h);

(3) washing the precipitate with 2M and 8M urea respectively for 2 times, dissolving the precipitate with 8M urea, standing at room temperature for 1h, centrifuging at 12000rpm for 30min, and collecting the supernatant;

(4) combining the supernatant with a purification column filled with nickel filler for 1h, washing for 5 times by using a phosphate buffer solution, controlling the flow rate (about 1mL/min), and washing off the foreign proteins;

(5) eluting the target protein with phosphate buffer solution containing 20mM, 50mM, 100mM, 200mM and 400mM imidazole respectively, and collecting the protein;

(6) then, proteins were dialyzed against phosphate buffers containing urea concentrations (6M, 4M, and 2M) which gradually decreased to remove urea at a large concentration, and the protein concentration was measured by BCA method to adjust the protein concentration to 1mg/mL, thereby obtaining the purification as shown in FIG. 3.

FIG. 3 is a graph showing the results of three protein purifications, 1 is a purified recombinant protein ORF 56; 2 is purified recombinant protein ORF 112; 3 is purified recombinant protein ORF 117.

Preparation of immune vaccine

ORF056+ ORF112+ ORF117 recombinant protein vaccine: mixing the purified recombinant protein ORF56 with the concentration of 1mg/mL, the purified recombinant protein ORF112 with the concentration of 1mg/mL and the purified recombinant protein ORF117 with the concentration of 1mg/mL according to the mass ratio of 1:1:1 to obtain an antigen; then the antigen is compatible with Freund's adjuvant (Sigma company), and the vaccine is obtained after stirring and mixing at low speed, and the volume ratio of the added adjuvant to the antigen is 1: 1.

ORF056 recombinant protein vaccine: the purified recombinant protein ORF056 with the concentration of 1mg/mL prepared in the previous step is used as an antigen to be compatible with Freund's adjuvant, the mixture is stirred and mixed at a low speed to obtain the vaccine, and the volume ratio of the added adjuvant to the antigen is 1: 1.

ORF112 recombinant protein vaccine: the purified recombinant protein ORF112 with the concentration of 1mg/mL prepared in the previous step is used as an antigen and is matched with Freund's adjuvant, the mixture is stirred at a low speed and mixed to obtain the vaccine, and the volume ratio of the added adjuvant to the antigen is 1: 1.

ORF117 recombinant protein vaccine: the purified recombinant protein ORF117 with the concentration of 1mg/mL prepared in the previous step is used as an antigen and is matched with Freund's adjuvant, the mixture is stirred at a low speed, and then the vaccine is obtained, wherein the volume ratio of the adjuvant to the antigen is 1: 1.

Example 2 recombinant protein antigen-specific antibody and neutralizing antibody assay

1. Test animals and immunizations

72 female BALB/C mice (18g) 6-8 weeks old were purchased from the university of Lanzhou laboratory animal center. The total number was 6 groups:

ORF056 recombinant protein vaccine group (GTPV-ORF 056): the ORF056 recombinant protein vaccine of example 1 was immunized by intramuscular injection into the legs of mice at 0d, 14d and 28d at a dose of 200. mu.L (50. mu.g protein/mouse).

ORF112 recombinant protein vaccine group (GTPV-ORF 112): the ORF112 recombinant protein vaccine prepared in example 1 was immunized by intramuscular injection into the legs of mice at 0d, 14d and 28d at a dose of 200. mu.L (50. mu.g protein/mouse).

ORF117 recombinant protein vaccine group (GTPV-ORF 117): the ORF117 recombinant protein vaccine prepared in example 1 was immunized by intramuscular injection into the legs of mice at 0d, 14d and 28d at a dose of 200. mu.L (50. mu.g protein/mouse).

ORF056+ ORF112+ ORF117 recombinant protein vaccine group (GTPV-ORF056+ ORF112+ ORF 117): the ORF056+ ORF112+ ORF117 recombinant protein vaccine prepared in example 1 was immunized by intramuscular injection into the legs of mice at 0d, 14d and 28d at a dose of 200. mu.L (total protein amount of 50. mu.g protein/mouse).

Freund's adjuvant control group: mice were immunized intramuscularly in the legs at 0d, 14d and 28d with Freund's adjuvant (Sigma) in an amount of 200. mu.L.

PBS control group: the mice were immunized intramuscularly in the legs at 0d, 14d and 28d with PBS at a concentration of 0.01 mmole pH 7.4 in an amount of 200. mu.L.

2. Indirect ELISA method for detecting level of specific antibody

After the immunization of the 6 groups of 1, tail vein blood was collected weekly, and serum was isolated to detect the level of specific antibody by indirect ELISA.

Detecting the specific antibody level and the typing antibody level of ORF056, ORF112 and ORF117 by an indirect ELISA method, comprising the following steps: coating 96-well ELISA plates with 5. mu.g/mL ORF056, ORF112 and ORF117 recombinant proteins, 100. mu.L each, and placing the plates in a wet box at 4 ℃ overnight; plates coated with different antigens were washed 5 times with PBST and the collected sera from each mouse were expressed as 1: 51200 diluting, adding 100 μ L of the diluted solution into each well, incubating in 37 deg.C incubator for 1h, and washing with PBST for 5 times; and adding 1: 20000 diluted anti-mouse IgG HRP secondary antibody, incubation in 37 deg.C incubator for 1h, PBST washing for 5 times, adding 100 μ L TMB, and measuring OD with microplate reader₄₉₀The value is obtained.

The results are shown in fig. 4, compared with the freund's adjuvant control group and the PBS control group, the specific antibodies in the four vaccine groups initially rise to the highest level in the 4 th week or the 5 th week after the initial immunization, which indicates that the antigens can generate specific antibodies, and the vaccine prepared by mixing the three protein antigens (GTPVORF056+ ORF112+ ORF117) has higher effect than the antibody level generated by the single antigen.

3. Determination of virus neutralizing antibodies

Inactivating each group of the serum collected at 5 weeks after 6 groups of the above 1 groups of the immunized serum in a constant temperature water bath at 56 ℃ for 30min, taking the inactivated serum, and diluting the inactivated serum on a 96-well micro-cell culture plate by using a diluent in a series of multiple ratios: 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1: 256.

The viruses GTPV and SPPV (described in Zhou T, Jia H, Chen G, et al, Phylogenetic analysis of Chinese sheepppox and Goatpox viruses, virol J, 20129: 25) stored at-80 ℃ in a refrigerator were diluted to 200TCID in accordance with the measured toxicity value₅₀(ii) a Equal volume of 200TCID₅₀Adding the diluted virus solution into the diluted serum solution, and inducing in a cell culture box at 37 ℃ and 5% CO2 for 1 h; adding 100 mu L of serum virus mixed solution into Vero cells cultured in a 96-well micro-cell culture plate with the cell density reaching about 80%, culturing in a cell culture box with the temperature of 37 ℃ and the concentration of 5% CO2 after culturing for 48h, and recording until the number of holes with CPE is not increased any more, wherein a virus control group, a serum toxicity control group and a normal cell control group are required to be arranged at the same time in order to ensure the accuracy test of the test result; the serum neutralizing antibody titer of the mice of the corresponding immunization group was calculated according to the Reed-Muench method.

As shown in tables 1 and 2, it can be seen that both the vaccine prepared from the single protein antigen and the vaccine prepared from the composite antigen can produce antibodies with the ability to neutralize GTPV and SPPV, and the vaccine prepared from the recombinant protein ORF056 antigen can produce antibodies with the ability to neutralize GTPV and SPPV at 1:32, the titer of the neutralizing GTPV and SPPV generated by the recombinant protein ORF112 antigen preparation vaccine is 1:19.03, and the titer of the neutralizing GTPV and SPPV generated by the ORF117 antigen preparation vaccine is 1:16, the vaccine group prepared by the recombinant protein ORF056+ ORF112+ ORF117 composite protein antigen and the adjuvant can effectively neutralize GTPV and SPPV, and the titer of the neutralizing GTPV antibody is 1: 44.67 (see Table 1), the potency of neutralizing SPPV was 1: 39.81, the titer of the neutralizing antibody is obviously higher than that of the vaccine group prepared by ORF112 single antigen and ORF117 protein antigen, and the sera of the PBS control group and the Freund's adjuvant control group can not neutralize GTPV and SPPV (see Table 2).

TABLE 1 serum GTPV neutralizing antibodies in mice of each immunization group

In the table, "+" shows cytopathy, and "-" shows no cytopathy.

TABLE 2 serum SPPV neutralizing antibodies in mice of each immunization group

In the table, "+" shows cytopathy, and "-" shows no cytopathy.

Sequence listing

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Met Gly Ala Ala Ala Ser Ile Gln Thr Thr Val Asn Thr Leu Asn Glu

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Lys Cys Asp Ile Glu Ile Gly Ser Ile Val Phe Arg Gln Asn Lys Gly

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Pro Asp Gln Lys Ala Tyr Val Pro Gly Leu Met Thr Ala Ala Leu Asn

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

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Lys Gln Lys Cys Thr Ser Lys Ser Val Ile Asp Asn Lys Leu Lys Ile

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His Asn Ile Phe Ile Asp Glu Cys Ala Ala Pro Thr Gly Thr Thr Thr

130 135 140

Asn Phe Glu Phe Ile Asn Ser Gly Thr Ser Gln Gly Ile Cys Ala Ile

145 150 155 160

Lys Thr Leu Met Asp Val Thr Thr Lys Ala Ser Thr Lys Ile Ser Pro

165 170 175

Ser Gln Ser Ser Gly

180

<210> 3

<211> 447

<212> DNA

<213> Artificial sequence

<400> 3

atggacagag cgttatccat ctttccaggc gacgatgatg aaaccaatga aagaaatata 60

aatcacatag agaaaactag tggtgatcac gggcattatg aagataaact tttggactta 120

agcgatgaag aacccaatat gataaaaata aaaaatgata ttaagaaaat aattaatgaa 180

agatatagca gttatatttc tatcaacgac gatgaaattt ctaacatttt aaaagattcg 240

ttcattagta acgaagaaat gcaaataaaa gattttgttt taagactgtt agtattagaa 300

aaactatttc aaacgtcagt aaaagaatgc aattcactaa aaaatattat taaaagatta 360

gaaaatcata tagaaactat tagaaaaaat atgattgttt taacaaaaaa ggtagatttt 420

caaacaggac gaagtacaac actatga 447

<210> 4

<211> 148

<212> PRT

<213> Artificial sequence

<400> 4

Met Asp Arg Ala Leu Ser Ile Phe Pro Gly Asp Asp Asp Glu Thr Asn

1 5 10 15

Glu Arg Asn Ile Asn His Ile Glu Lys Thr Ser Gly Asp His Gly His

20 25 30

Tyr Glu Asp Lys Leu Leu Asp Leu Ser Asp Glu Glu Pro Asn Met Ile

35 40 45

Lys Ile Lys Asn Asp Ile Lys Lys Ile Ile Asn Glu Arg Tyr Ser Ser

50 55 60

Tyr Ile Ser Ile Asn Asp Asp Glu Ile Ser Asn Ile Leu Lys Asp Ser Phe

65 70 75 80

Ile Ser Asn Glu Glu Met Gln Ile Lys Asp Phe Val Leu Arg Leu Leu

85 90 95

Val Leu Glu Lys Leu Phe Gln Thr Ser Val Lys Glu Cys Asn Ser Leu

100 105 110

Lys Asn Ile Ile Lys Arg Leu Glu Asn His Ile Glu Thr Ile Arg Lys

115 120 125

Asn Met Ile Val Leu Thr Lys Lys Val Asp Phe Gln Thr Gly Arg Ser

130 135 140 145

Thr Thr Leu

<210> 5

<211> 396

<212> DNA

<213> Artificial sequence

<400> 5

atgttagcat tttttaataa taatacatgt gaattaaatc aatttaagga acacaaaccg 60

tactttttaa aaaatccaaa tcctactaca tatagtgacg acgatactga atctgagtta 120

aatgtttata gatcatgtaa aggtattgtt tatagcggat actgttacac ttttaactta 180

gaacctaaaa gttttaatga tgcatacgat gattgtgaaa aaaaaaatag cgaattacca 240

tcaaataatt taatgaatga ttggataagt gactacttag atgggacgtg gggagaagac 300

ggtaacgtac tttttaaaga aaaaaatcaa aaacttgaag ctatagatat aagcgatgag 360

atgagaagct attactgtgt aagatctttt ttttaa 396

<210> 6

<211> 131

<212> PRT

<213> Artificial sequence

<400> 6

Met Leu Ala Phe Phe Asn Asn Asn Thr Cys Glu Leu Asn Gln Phe Lys Glu

1 5 10 15

His Lys Pro Tyr Phe Leu Lys Asn Pro Asn Pro Thr Thr Tyr Ser Asp

20 25 30

Asp Asp Thr Glu Ser Glu Leu Asn Val Tyr Arg Ser Cys Lys Gly Ile

35 40 45

Val Tyr Ser Gly Tyr Cys Tyr Thr Phe Asn Leu Glu Pro Lys Ser Phe

50 55 60 65

Asn Asp Ala Tyr Asp Asp Cys Glu Lys Lys Asn Ser Glu Leu Pro Ser

70 75 80

Asn Asn Leu Met Asn Asp Trp Ile Ser Asp Tyr Leu Asp Gly Thr Trp

85 90 95

Gly Glu Asp Gly Asn Val Leu Phe Lys Glu Lys Asn Gln Lys Leu Glu

100 105 110

Ala Ile Asp Ile Ser Asp Glu Met Arg Ser Tyr Tyr Cys Val Arg Ser

115 120 125

Phe Phe

130

Claims

1. A product which is a composition comprising protein ORF056, protein ORF112 and protein ORF 117;

the amino acid sequence of the protein ORF056 is SEQ ID NO. 2;

the amino acid sequence of the protein ORF112 is SEQ ID NO. 4;

the amino acid sequence of the protein ORF117 is SEQ ID NO 6.

2. The product of claim 1, wherein:

the composition containing the protein ORF056, the protein ORF112 and the protein ORF117 is a composition consisting of an antigen and an adjuvant, wherein the antigen consists of the protein ORF056, the protein ORF112 and the protein ORF 117.

3. The product of claim 2, wherein: the adjuvant is Freund's adjuvant.

4. The product of claim 2, wherein: in the antigen, the mass ratio of the protein ORF056, the protein ORF112 and the protein ORF117 is 1:1: 1.

5. The product according to claim 1 or 2, characterized in that: the coding sequence of the nucleic acid of the coding protein ORF056 is SEQ ID NO 1;

the coding sequence of the nucleic acid for coding the protein ORF112 is SEQ ID NO. 3;

the coding sequence of the nucleic acid for coding the protein ORF117 is SEQ ID NO: 5.

6. The product according to claim 1 or 2, wherein the product is a vaccine or a medicament or a kit.

7. Use of a substance according to any one of claims 1 to 6 in the manufacture of a product having the ability to neutralise a virus of the capripoxvirus genus.

8. Use according to claim 7, characterized in that: the product is a vaccine or a medicament or a kit.