CN109705223B

CN109705223B - Recombinant subunit vaccine of orf virus and production method thereof

Info

Publication number: CN109705223B
Application number: CN201811628927.4A
Authority: CN
Inventors: 冯平; 陈瑞; 张磊
Original assignee: Yulin University
Current assignee: Yulin University
Priority date: 2018-12-29
Filing date: 2018-12-29
Publication date: 2023-05-12
Anticipated expiration: 2038-12-29
Also published as: CN109705223A

Abstract

The invention relates to an orf virus fusion protein rB2LcF L, wherein the amino acid sequence of the orf virus B2L and F1L fusion proteins is SEQ ID No.2, compared with the wild type orf virus F1L protein, the orf virus fusion protein rB2LcF L lacks the 1-70 site amino acid sequence, which is favorable for the expression of the fusion proteins, improves the expression quantity, and meanwhile, the C end of the fusion proteins is added with a 6 histidine tag, so that the fusion proteins are convenient for purification in subsequent production, and in the amino acid sequences, compared with the B2L amino acid sequence of the traditional isolate strains, the V/L mutation of the 9 th site amino acid is G, namely the IPVG or IPLG mutation is IPGG, and the 35 th site LDCF mutation is LYCF, thus the expression quantity and the immunogenicity of the fusion proteins are remarkably improved.

Description

Recombinant subunit vaccine of orf virus and production method thereof

Technical Field

The invention belongs to the field of biological products for animals, and particularly relates to an orf virus recombinant subunit vaccine and a production method thereof.

Background

The sheep aphtha is also called sheep infectious pustule, is a contact and epitheliophilic infectious disease which seriously threatens the sheep industry, and mainly endangers lambs and partial adult sheep of 3-6 months old. The clinical symptoms of the disease are red rash, pustule, ulcer and crusting of lips and mucous membrane parts, lambs are broken lips, eating is impossible, the morbidity is up to 100%, and the mortality rate of other pathogenic microorganisms is up to 15% if the secondary or mixed infection is carried out. The chilblain occurs worldwide and is widely prevalent. In recent years, along with the development of sheep raising industry and the change of raising modes in China, the occurrence risk of the disease is increased, the affected area and the serious degree of hazard are also higher and higher, and huge economic loss is brought to the sheep raising industry. Meanwhile, as a zoonosis, the virus also endangers human health. The disease is outbreak and epidemic in partial areas of China, wherein the occurrence and epidemic of the disease are reported in areas of Gansu, ningxia, sichuan, jiangsu, yunnan, qinghai, shaanxi, shandong, inner Mongolia, xinjiang, guizhou, heilongjiang and other provinces. The clinical symptoms of the chilblain are easy to diagnose, no targeted drug is used for treatment at present, antibiotics are generally used for preventing secondary or mixed infection treatment, no specific drug is used, and vaccination is considered to be a reliable and effective means for preventing and controlling the disease. Currently, weak-toxin vaccines are mainly used for preventing the aphtha abroad, and no weak-toxin vaccine for aphtha is currently supplied in the market of China for various reasons, and the weak-toxin vaccine may have the defects of strong virulence return, virus diffusion and the like. Therefore, development of the genetic engineering subunit vaccine for the aphtha, which is simple and convenient to use and has a definite immune effect, has become a necessary trend for preventing and controlling aphtha epidemic diseases.

The Orf virus (ORFV) belongs to the Parapoxvirus genus (Parapoxvirus) of the subfamily Poxviridae (chord) of the Poxviridae family (Poxviridae) and has a genome of about 135 genes capable of encoding a number of different proteins. Among them, 42 kU envelope protein encoded by Orf 011 (B2L) gene is an important immunogenic protein. In addition, the gene is widely used in laboratory detection of parapoxvirus viruses because it is capable of detecting the lowest copy number of the viral particles. After the stimulation of the orf virus, the organism can generate cellular immune response and humoral immune response, wherein the cellular immune response is mainly the cellular immune response, but antibodies generated by the humoral immunity play a certain role in resisting viruses. The research shows that the B2L protein is one of the components of the surface envelope of the virus, can stimulate the organism to generate strong humoral immune response, and can also cause proliferation and differentiation of lymphocytes. In view of this property of B2L proteins, expression has been obtained in different systems, such as prokaryotic systems, eukaryotic expression vectors pcdna3.1, pVAX1, etc.

The inventor also carries out related researches, namely, the isolation and identification of an ORFV-Yulin strain and the expression of a B2L gene thereof in an insect baculovirus system (university of North-west agriculture and forestry science and technology report (natural science edition), 2017 45 (9)) are used for successfully isolating 1 strain of the orf virus, the strain is named as an ORFV-Yulin strain, the length of the amplified B2L full-length gene is 1137 bp, the affinity relationship with Hub13 and GY-AHF10 strains is nearest, the similarity of a nucleotide sequence and an amino acid sequence is 99% and 99.2%, and the B2L gene is successfully expressed through an insect baculovirus expression system, so that recombinant proteins are obtained. However, serological experiments show that the protective power of the vaccine to immunized mice only reaches 60 percent, and the vaccine production requirement cannot be met. In addition, liu et al ("construction of eukaryotic expression plasmids for the genes F1L and B2L of the orf virus and their expression in MDBK cells", "Probezoonotic journal of Chinese, 201231 (12)) used pVAX1 as eukaryotic expression vector, constructed eukaryotic expression plasmids pVAX1-F1L and pVAX1-B2L, transfected into MDBK cells, RT-PCR and IFA simultaneously verified that the target gene could be transiently expressed in MDBK cell slurry, in which the expression vector was constructed separately, on the one hand, the operation was relatively complicated, on the other hand, only transient expression was achieved in eukaryotic cells, just as it was pointed out in the literature that" since pVAXL vector could only transiently express a cell line incapable of stable expression was selected on the basis of pCDNA3.1 vector ", it could not obtain a cell line incapable of stable expression, therefore, only was considered for use in a double gene nucleic acid vaccine, but could not be used in a subunit vaccine for industrial production, and its immunization effect was to be verified. The invention patent application CN201710138033.6 is an immunogenic composition composed of eukaryotic expression plasmids pVAX1-FIL, pVAX1-B2L and pVAX1-IL-2, wherein the orf vaccine contains eukaryotic expression plasmids capable of expressing two highly conserved orf virus genes and eukaryotic expression plasmids capable of expressing cytokine IL-2 genes as vaccine antigens, and the orf can be prevented by constructing eukaryotic expression plasmids as DNA vaccines, and the vaccine can effectively stimulate organisms to generate specific humoral immunity and cellular immune responses, so that the invention has important clinical significance for the research of novel orf vaccines. However, as reported in Liu Yuan et al, the method of the patent application of the invention only achieves transient expression in eukaryotic cells, does not allow for stable expression of subunit vaccines, and involves transformation of multiple plasmids, which are also susceptible to plasmid loss during passage, and does not allow for genetically stable expression cell lines.

Based on the technical research, the traditional orf virus is difficult to culture and proliferate on a conventional cell line, is not suitable for preparing an inactivated vaccine or a attenuated vaccine, and has certain defects in the research of the traditional subunit vaccine or the nucleic acid vaccine, such as poor genetic stability, inability to obtain a genetically stable expression cell line, poor immunogenicity or low protection rate. Therefore, development of an orf subunit vaccine capable of stable expression and production is needed.

Disclosure of Invention

In order to solve the technical problems, the invention aims to prepare the recombinant subunit vaccine of the orf virus, which is used for preventing diseases caused by the orf virus. The recombinant subunit vaccine of the orf virus prepared by the invention takes B2L and F1L of an orf virus ORFV-Yulin strain separated from the disease materials of Shanxi white down goats suffering from Shanxi elm as templates, and adopts a baculovirus expression system to carry out tandem expression on the B2L and the F1L through codon optimization. Meanwhile, the vaccine has the advantages of simple preparation process, low immune dose, good immune efficacy and the like, and is an ideal candidate vaccine of the traditional Chinese aphtha virus.

In order to solve the technical problems, the invention adopts the following technical means:

the fusion protein rB2LcF L of the orf virus is characterized in that the amino acid sequence of the fusion proteins of B2L and F1L of the orf virus is SEQ ID No. 2.

The nucleic acid sequence of the gene encoding the orf virus fusion protein rB2LcF1L is SEQ ID No. 1.

In the amino acid sequence, compared with the B2L amino acid sequence of the traditional isolated strain, the V/L of the 9 th amino acid is mutated into G, namely IPVG or IPLG is mutated into IPGG, and the 35 rd LDCF is mutated into LYCF, so that the expression quantity and immunogenicity of the fusion protein are obviously improved.

Compared with wild type orf virus F1L protein, the orf virus fusion protein rB2LcF L lacks the 1-70 amino acid sequence, which is beneficial to the expression of the fusion protein and improves the expression quantity. Meanwhile, a 6 histidine tag is added at the C end of the fusion protein, so that the purification in the subsequent production is facilitated.

A recombinant noctiluca californica nuclear polyhedrosis virus Bac-ORFV-01 strain which has been delivered 22 days in 2018 to the China general microbiological culture Collection center of the national institute of microbiology, national institute of sciences, national institute of microbiology, national academy of sciences, no. 3, north chen west way No.1, dynasty, and has a deposit number of: CGMCC No.15494; the strain can stably and efficiently express the fusion proteins of the B2L and the F1L of the orf virus.

The amino acid sequence of the fusion protein of the orf virus B2L and F1L is SEQ ID No. 2.

The preparation method of the recombinant noctuid california nuclear polyhedrosis virus Bac-ORFV-01 strain comprises the following steps:

step one, synthesizing a gene sequence with a sequence of SEQ ID No.1 by a chemical synthesis method, wherein the gene sequence contains 1908 nucleotides;

step two, constructing a fusion expression vector: PCR amplification is carried out by taking the artificially synthesized gene sequence of SEQ ID No.1 as a template and adopting a primer pair 1F/1R (sequence 3/sequence 4); wherein the upstream primer 1F sequence is: 5'-CGCGCTAGCA TGTGGCCGTT CTCCTCC-3' (SEQ ID No: 3) having a restriction enzyme introduced at its 5' endNheII site and protective base; the sequence of the downstream primer 1R is as follows: 5'-CCGAAGCTTTTAGTGGTGAT GGTG-3' (SEQ ID No: 4) having a restriction enzyme introduced at its 5' endHind IIIA site and a protective base; amplifying the obtained target DNA band, recovering the target DNA band, and adoptingNheⅠ/Hind IIIDouble digestion, and connection with insect baculovirus expression system plasmid pBac5 vector with the same digestion to obtain positive clone pBac5-GB2LcF L inserted with full-length gene of the aphtha virus; transforming the ligated plasmidSelecting a monoclonal DH5 alpha competent cell into an LB liquid medium containing kanamycin, carrying out shake culture at 37 ℃ overnight, and extracting plasmids for later use;

step three: construction of recombinant expression of the fusion protein rB2LcF L gene engineering strain of the orf virus: 6-well cell culture plates were taken at 1X 10 ⁶ ml ^-1 Inoculating sf9 cells with good growth state at the cell density, culturing at 27 ℃ for 2 h, transfecting when the fusion degree of the cells reaches about 80%, and uniformly mixing 3 mug pBac5-GB2LcF1L and 3 mug linearized Bacmid by using 100 mug serum-free culture medium to prepare solution I; diluting 5 mu L of cations (the tattoo transfection reagent) with 100 mu L of serum-free culture medium, slowly dripping the diluted cations into the solution I, uniformly mixing, and standing for 20min; adding the mixed solution into the cell plate of sf9, incubating at 27 ℃ for 5-7 d, observing the expression condition of a reporter gene cGFP under a fluorescence microscope, and harvesting cell supernatant in a 1 mL centrifuge tube after the diseased cells reach more than 90%, wherein the cell supernatant is marked as P1 generation recombinant baculovirus, and storing at-80 ℃ for a long time; the strain is delivered to China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, which is the national academy of sciences of China, including No. 3, of the national academy of sciences of China, including the Korean area of Beijing, at 22 days 2018, and has a collection number of: CGMCC No.15494.

The invention also claims the application of the recombinant alfalfa silver vein moth nuclear polyhedrosis virus Bac-ORFV-01 strain in preparing the recombinant subunit vaccine of the aphtha virus.

An orf virus recombinant subunit vaccine, characterized in that the vaccine comprises recombinant orf virus B2L and F1L fusion protein rB2LcF L expressed by insect baculovirus; the strain for preparing the seedlings is a baculovirus for recombinant expression of fusion proteins of the sheep sore viruses B2L and F1L, is named as a recombinant alfalfa silver vein noctuid nuclear polyhedrosis virus Bac-ORFV-01 strain, and is delivered to China general microbiological culture Collection center for culture Collection, china institute of microbiological culture Collection, national institute of China, and the national institute of sciences of China, no. 3, the university of microorganisms, and the accession number is: CGMCC No.15494.

The recombinant subunit vaccine of the orf virus is characterized in that the alfalfa silver vein noctuid nuclear polyhedrosis virus (Bac-ORFV-01) strain expressing the recombinant fusion protein of the orf virus is used as a vaccine production strain, and is prepared by culturing, expressing the target protein, separating and purifying, and adding a biphase oil adjuvant for mixing.

A method of preparing a recombinant subunit vaccine for an orf virus comprising the steps of:

(1) Strains: the strain for preparing the seedlings is a strain of the alfalfa silver vein moth nuclear polyhedrosis virus Bac-ORFV-01 which recombinant expresses fusion proteins of the sheep sore viruses B2L and F1L, and is delivered to China general microbiological culture Collection center (China Committee for culture Collection) of the microbiological culture Collection, china academy of China, national academy of sciences of China, including No. 3, of the Korean area, beijing, city, for 22 days, and has a preservation number of: CGMCC No.15494;

(2) First-stage seed propagation and identification: sf9 cells were cultured according to 2.5X10 ⁶ mL ^-1 Spreading the total number of cells in a T25 square bottle, after the cells are attached to 2 h, inoculating 100 mu L P generation 1 virus into the T25 square bottle by using a 1 mL injector, culturing for 5-7 d, and freezing at minus 80 ℃ after pure inspection to be qualified as primary seeds for preparing seedlings;

(3) And (3) secondary seed propagation and identification: inoculating the first-stage seeds into a primary fermentation tank, culturing for 3 d, and obtaining the second-stage seeds after pure inspection;

(4) Preparation of antigen for seedling preparation: taking qualified secondary seeds, and culturing the secondary seeds in a fermentation tank for production for 24-48 hours;

(5) Sample collection: centrifugally collecting and respectively collecting cells and supernatant;

(6) Purifying: carrying out affinity chromatography by using His antibody by adopting a nickel column method, detecting rB2LcF L finally obtained by gray scanning through SDS-PAGE and carrying out gray scanning on the band, wherein the protein purity is not lower than 75%;

(7) Protein content detection: detecting protein content by BCA method, wherein the protein content is not lower than 0.2mg/mL;

(8) And (3) sterile inspection: according to the appendix of the current "Chinese animal pharmacopoeia" (Chinese animal pharmacopoeia Committee, chinese people's republic of China, second good, first five years edition, third good, chinese agricultural press, 2011, hereinafter "Chinese animal pharmacopoeia");

(9) Preparing a vaccine: introducing a biphasic oil adjuvant (such as 201 adjuvant) into an oil phase tank, sterilizing at least 121 ℃ for 30 minutes under high pressure, cooling to room temperature for standby, properly diluting and uniformly mixing the qualified purified protein with PBS (pH 7.2.01 mol/L) according to the protein content measurement result, adding an aqueous phase into an emulsifying tank, stirring at 80-100 r/min, slowly adding the oil phase according to the ratio of 1:1 (V/V), and stirring for 20-30 min after the completion of the addition. Sampling after emulsification, inspecting, and sub-packaging after passing.

Based on the technical scheme, the invention has the following advantages and beneficial effects;

firstly, the fusion expression technology is adopted, so that the vaccine prepared by singly expressing B2L, F1L in the prior art is broken through, and the immunogenicity of the prepared protein is low and is insufficient to meet the titer requirement for preparing the vaccine; the defects that the cell/strain with genetic stability can not be obtained and the industrialized production can not be realized by adopting multi-vector expression to reach B2L, F L are overcome. The recombinant noctiluca californica nuclear polyhedrosis virus Bac-ORFV-01 strain is constructed by designing and synthesizing a gene for fusion expression of rB2LcF L, has good genetic stability, can be stably proliferated on sf9 cells in a large quantity, and produces fusion protein rB2LcF L; and serial fusion expression is adopted, so that the cost and the vaccine effect in the vaccine production process are greatly reduced.

Secondly, the invention is based on B2L and F1L of the domestic isolated virulent strain, namely the orf virus ORFV-Yulin strain, as templates, and is easier to realize high expression in the noctuid nuclear polyhedrosis virus of the alfalfa silver vein moth through codon optimization. The technology of point mutation is adopted to mutate part of amino acid sites of B2L, the mutation obviously improves the expression quantity and immunogenicity of fusion protein, part of amino acids of F1L are truncated and deleted, compared with wild type orf virus F1L protein, 1-70 amino acid sequences are deleted, and the deletion is favorable for the expression of fusion protein and improves the expression quantity. And 6 histidine tags are added at the C end of the fusion protein, so that the purification in the subsequent production is facilitated.

In the process of designing protein sequence, the invention adopts point mutation technology, makes targeted mutation on certain sites, mainly makes mutation on 9 th and 35 th amino acids, and compared with the traditional B2L amino acid sequence of the isolated strain, makes V/L mutation of 9 th amino acid G, namely 'IPVG or IPLG mutation to IPGG', and makes LDCF mutation of 35 th site LYCF, and the mutation obviously improves the expression quantity and immunogenicity of fusion protein. The technical effect achieved is not to be expected.

In summary, the recombinant baculovirus expression system is constructed by adopting the fusion expression technology through codon optimization and amino acid fragment optimization, so that the expression of high expression quantity in sf9 can be realized, the recombinant baculovirus expression system can be used for producing the recombinant subunit vaccine of the orf virus, and the produced subunit vaccine has good immunogenicity, can produce antibodies with high titer and has higher protection efficacy. The recombinant expressed protein rB2LcF L is completely nontoxic in mice, and has good immunogenicity and immunoprotection in a mouse model. The recombinant subunit vaccine for the orf virus provided by the invention greatly reduces the biosafety risk in the vaccine production process. In addition, by virtue of the advantage of high protein concentration of the semi-finished vaccine product, when the vaccine is prepared together with other antigens, the vaccine can be prepared without increasing the dosage of the vaccine, and the development of the vaccine is greatly facilitated.

Drawings

Fig. 1: expression of GFP in insect viruses expressing the rB2LcF1L fusion protein.

Fig. 2: identification of expression of rB2LcF L in sf9 cells in the map: m is Protein marker; lane 1, cell supernatant; lane 2 cell pellet

Fig. 3: in the western blot identification map of rB2LcF1L in sf9 cells: m is Protein marker; lane 1, cell supernatant; lane 2 cell pellet

Fig. 4: detection result of anti-ORFV antibody in mouse serum

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following examples are given to better illustrate the technical aspects of the present invention, but are not to be construed as limiting the technical aspects of the present invention.

EXAMPLE construction, expression and identification of the fusion protein vectors of the B2L and F1L Caprae Seu Ovis

1. Gene synthesis

According to the sequences of B2L and F1L natural protein genes of an ORFV-Yulin strain of the orf virus, after codon optimization, a tandem fusion gene expressing the B2L full-length gene and the F1L truncated gene (deleting 1-70 amino acids) is synthesized, so that the fusion protein for the orf virus B2L and F1L is obtained. Meanwhile, the C-terminal of the mutein is tagged with 6 histidine. The gene sequence was synthesized by chemical synthesis and contained 1908 nucleotides in total. Wherein, the 1 st to 1134 th are the full-length sequence of the orf virus B2L, and the 1147 th to 1908 th are F1L truncated genes (deleting 1-70 th amino acids). The specific nucleic acid sequence is shown as SEQ ID No.1, and the amino acid sequence is shown as SEQ ID No. 2.

2. Construction of fusion expression vectors

The tandem fusion gene of the artificially synthesized full-length gene B2L of the orf virus and the F1L truncated gene (deleting 1-70 amino acids) is used as a template, and a primer pair 1F/1R (sequence 3/sequence 4) is adopted for PCR amplification. Wherein the upstream primer 1F sequence is: 5'-CGCGCTAGCATGTGGCCGTT CTCCTCC-3' 27nt (SEQ ID NO: 3), 5' -end of which was introduced with restriction enzymeNheII site and protective base; the sequence of the downstream primer 1R is as follows: 5'-CCGAAGCTTT TAGTGGTGAT GGTG-3' 24nt (SEQ ID NO: 4), 5' -end of which was introduced with restriction enzymeHind IIIA site and a protective base.

The PCR system is as follows: 10X EX Taq Buffer 5 mu l, dNTPs 4 mu l, each 2 mu l of upstream and downstream primers, 0.5 mu l of EX Taq enzyme, 2 mu l of full-length gene DNA template and ddH supplementing ₂ O to 50 μl system. The PCR reaction conditions were: pre-denaturation at 94℃for 10min; denaturation at 94℃for 30s, annealing at 53℃for 30s, extension at 72℃for 2min10s for 33 cycles; finally, the ring is extended for 10min at 72 ℃.

Amplifying the obtained target DNA band, recovering the target DNA band, and adoptingNheⅠ/Hind IIIDouble digestion, and connection with insect baculovirus expression system plasmid pBac5 carrier through the same digestion to obtain positive clone pBac5-GB2 inserted with full-length gene of aphtha virusLcF1L. The DH5 alpha competent cells are transformed by the connected plasmids, the monoclonal is selected and put into LB liquid medium containing kanamycin, and the plasmids are extracted for standby after shaking culture at 37 ℃ for overnight.

3. Construction of recombinant expressed full-length gene B2L of orf virus and F1L truncated gene fusion gene engineering strain

6-well cell culture plates were taken at 1X 10 ⁶ ml ^-1 The cell density of (2) is used for inoculating sf9 cells with good growth state, and the cells are placed at 27 ℃ for 2 h, and when the fusion degree of the cells reaches about 80%, transfection is carried out. Uniformly mixing 3 mu g of pBac5-GB2LcF L and 3 mu g of linearized Bacmid by using 100 mu L of serum-free culture medium to prepare a solution I; after diluting 5 mu L of cations (the tattoo transfection reagent) with 100 mu L of serum-free culture medium, slowly dripping the diluted cations into the solution I, uniformly mixing, and standing for 20min. Adding the mixed solution into the cell plate of sf9, incubating at 27 ℃ for 5-7 d, observing the expression condition of a reporter gene cGFP under a fluorescence microscope, and harvesting cell supernatant in a 1 mL centrifuge tube after the diseased cells reach more than 90%, wherein the cell supernatant is marked as P1 generation recombinant baculovirus, and storing at-80 ℃ for a long time. The strain is delivered to China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, which is the national academy of sciences of China, including No. 3, of the national academy of sciences of China, including the Korean area of Beijing, at 22 days 2018, and has a collection number of: CGMCC No.15494.

4. Expression and identification of proteins of interest

Sf9 cells were cultured according to 2.5X10 ⁶ ml ^-1 After the cells are attached to the wall of 2 h, 100 mu L P generation 1 virus is blindly connected to the T25 square bottle by a 1 mL injector, 5-7 d is cultivated, fluorescence is observed, cell supernatant and cells are collected as subsequent test samples, and part of the cells are subjected to ultrasonic disruption (ultrasonic for 1s, interval for 5s, total ultrasonic for 1 min) and then the supernatant and the sediment are collected for later use. 12% SDS-PAGE was performed on the cell supernatant and the supernatant and pellet samples after sonication to examine the expression status and the solubility of the recombinant protein. And cutting a target strip on the gel protein, and delivering to China large gene company for mass spectrum identification. At the same time, the samples were transferred to nitrocellulose membranes (NC membranes) after SDS-PAGE, and His-tagged antibodies were used as the respective antibodiesIncubation is carried out by using the primary antibody and the goat anti-mouse IgG marked by HRP as secondary antibodies, color development is carried out by using ECL color development liquid, and finally exposure is carried out in a chemiluminescent instrument. And simultaneously taking loading Buffer, sf9 cell supernatant and baculovirus expressed 40 kU protein as blank, negative and positive controls.

5. Purification of the protein of interest

After filling the purification column with resin, the resin was equilibrated with 1X Extraction Buffer (pH 7.0). The supernatant was added to the equilibrated resin and bound on ice for 30-60min, and the equilibrated column was shaken at intervals to allow more complete binding of the resin to protein, washed with 10 column volumes of 1 XWash Buffer (10 mL), and then added to a volume of 1 XElutation Buffer (pH 7.0) and bound on ice for 30min.

And collecting effluent liquid to obtain purified rB2LcF L protein.

Example preparation of recombinant subunit vaccine against Di-oral sore Virus

(1) Strain culture: and (3) inoculating secondary seeds of the noctuid nuclear polyhedrosis virus (Bac-ORFV-01) strain into a suspension culture tank according to 1% of the total amount of the culture medium, and culturing for 48 hours.

(3) Purifying: by adopting a nickel column method, carrying out affinity chromatography by utilizing a His antibody, carrying out gray scale scanning, detecting the obtained rB2LcF L by SDS-PAGE and carrying out gray scale scanning on the band, wherein the protein purity is not lower than 75%.

(4) Protein content detection: protein content was measured by BCA method. As a result, the protein content was 0.65mg/mL. The protein purity was 85% as detected by SDS-PAGE and the bands were gray scanned.

(5) And (3) sterile inspection: according to the annex of the current Chinese animal pharmacopoeia. The results were all aseptically grown.

(6) Preparing a vaccine: a biphasic oil adjuvant (e.g. 201 adjuvant) is introduced into the oil phase tank, autoclaved at least 121 ℃ for 30 minutes and cooled to room temperature for use. According to the protein content measurement result, PBS (pH 7.2.01 mol/L) is used for diluting the purified protein which is qualified by inspection to a final concentration of 500 mu g/ml, and then the purified protein is uniformly mixed. Adding the water phase into an emulsifying tank, stirring at 80-100 r/min, slowly adding the oil phase according to the ratio of 1:1 (V/V), and stirring for 20-30 min after the addition is finished. Sampling after emulsification, inspecting, and sub-packaging after passing.

Inspection of recombinant subunit vaccine of orf virus

(1) Traits (3)

The appearance of the emulsion is milky white.

The dosage form is water-in-oil-in-water (W/O/W). A cleaning straw is taken, a small amount of vaccine is sucked and dripped on the surface of the cleaning cold water, and the vaccine is required to be dispersed in a cloud form.

10ml of the stable aspirated vaccine is added into a centrifuge tube, and centrifuged at 3000r/min for 15min, no demulsification should be performed, and no more than 0.5ml of water is precipitated at the bottom of the tube.

The viscosity is measured according to the annex of the Chinese animal pharmacopoeia, and the viscosity meets the regulations.

(2) The filling quantity inspection is carried out according to the annex of the Chinese animal pharmacopoeia, and the filling quantity inspection meets the regulations.

(3) The sterility test is carried out according to the annex of the Chinese animal pharmacopoeia, and the sterility test should be carried out.

(4) Safety test 10 Balb/c mice of 6-8 weeks of age, 1.0ml of vaccine was injected into each muscle or subcutaneous, and observed for 10 days. All healthy and alive.

(5) Efficacy test 20 Balb/c mice were randomly divided into 2 groups of 10. The rB2LcF L and PBS controls were immunized subcutaneously with 100 ug antigen/dose for 2 total immunizations at 2 weeks intervals. On day 7 after the 2 nd immunization, the cells were cultured with the orf virus (1 x 10 ⁷ TCID 50/mL), and is subjected to a toxicity attack protective test, and the toxicity attack amount is as follows: subcutaneous inoculation 600 μl/min. After the toxicity is removed, isolated feeding is carried out, main clinical indexes such as spirit, appetite and the like of animals after the toxicity is removed are observed and recorded daily, and the morbidity and mortality of mice in each immune group are recorded and counted. The death rate of the control mice is not lower than 50 percent, the protection rate of the immune group is not lower than 80 percent, and the mice are judged to be qualified.

Example test of virulence of the three Caprae virus B2L and F1L fusion proteins rB2LcF1L on mice

The actual attenuation effect of the mutant in vivo was verified by determining the virulence of the fusion proteins rB2L and F1L, rB2LcF L, of the orf virus prepared in example one, to mice. The fusion proteins rB2 and F1L of the orf virus B2L and F1L of rB2LcF L were subcutaneously injected into 6-8 week old Balb/c mice at a dose of 300 μg/mouse for a total of 10 mice. As a result, it was found that 100% of mice survived, and that no organ abnormality was observed by dissection after sacrifice by cervical dislocation. The results indicate that fusion protein rB2LcF1L is non-toxic in mice.

Example detection of OrfV-specific antibodies in serum after immunization of mice with the fusion proteins rB2L and F1L, rB2LcF L

To verify the immunogenicity of the fusion protein rB2LcF L of example one of the present invention, experiments were performed using mice. 100 mice of the Kunming series 6 weeks old are selected and randomly divided into 5 groups, 20 mice in each group are respectively a control group (PBS), and 201 adjuvant of PBS+1 head is injected in a subcutaneous injection mode; the B2L+F1L group adopts a subcutaneous injection mode, B2L recombinant protein and F1L recombinant protein are injected, the specific preparation method of the recombinant B2L protein is referred to in the separation and identification of ORFV-Yulin strain and the expression of B2L gene in insect baculovirus system, the preparation of the recombinant F1L protein is referred to in the same method in the above document, the adopted sequence is the F1L sequence (full length, not truncated) of Yulin strain, and the antigen inoculation dose is 60 mug/1 head part of 201 adjuvant of each B2L, F L protein; a control group 1, wherein the recombinant B2L+F1L fusion protein prepared in the following control group 1 is adopted, and the antigen inoculation amount is 100 mug/dose; the weak-toxicity commercial vaccine group adopts the sheep mouth sore weak-toxicity cell freeze-dried live vaccine produced by the livestock organisms of Tiankang in Xinjiang, and the inoculation amount is 1 part per animal; rB2LcF L group, 1 part of the recombinant subunit vaccine of the orf virus prepared in the second example is used, immunization is carried out by subcutaneous injection, and the antigen inoculation amount is 100 mug/dose. The mice were subjected to eyeball blood collection before immunization (day 0), 7 days after the first immunization, 14 days, 21 days, 28 days, 42 days, and 70 days, serum was collected, and stored at-20 ℃ for later use, and orfV-specific antibodies in the serum of the mice were measured using an orf virus antibody (OrfV-Ab) ELISA detection kit (commercially available). The microplate reader determines the OD450 value. See fig. 4 for specific results.

Based on the detection results of fig. 4, it can be known that the fusion protein rB2LcF1L according to the embodiment 1 of the invention generates specific antibodies in the mice after 7 days of the first immunization, and the specific antibodies in the mice reach the highest level after two weeks of the second immunization, namely, on day 28, and still maintain a higher antibody level on days 42 and 70; compared with the commercial attenuated vaccine group, the specific antibody level generated by the subunit vaccine of the second embodiment of the present invention is close to the specific antibody level, but the time for reaching the highest level of the specific antibody is earlier, the commercial attenuated vaccine group reaches the highest level about 42 days, and the vaccine of the present invention can reach the highest antibody level on the 28 th day, which has great significance for animal protection, especially for preventing and controlling the spread of the oral ulcer in the case of regional epidemic outbreak, the higher immune level can be quickly reached to more effectively control the spread of the epidemic situation, and compared with the commercial vaccine group, the antibody level of the present invention is maintained for a longer time, and as can be seen from fig. 4, the subunit vaccine of the present invention still maintains a higher antibody level on the 42 th day and the 70 th day after the first immunization, which indicates that the subunit vaccine of the present invention can maintain a longer immune protection period. Compared with the B2L+F1L group, the fusion protein rB LcF1L prepared by adopting a fusion expression mode can stimulate the organism to produce higher and more continuous specific antibodies compared with the combination of the independent recombinant B2L protein and the recombinant F1L protein; compared with the control group 1, the fusion protein rB2LcF1L provided by the invention has better immunogenicity.

The difference between the control group 1 and the first and second embodiments of the present invention is that the sequence adopted in the control group 1 is a conventional sequence, i.e., the amino acid at position 9 is V, which is not the mutation of the present invention to G, i.e., it is still "IPVG", but not IPGG, and the 35 rd is still LDCF, which is not the mutation of the present invention to LYCF (see SE1 ID No: 5). Methods for the expression and preparation of specific proteins and for the production of vaccines are described in examples 1 and 2.

Examples immunoprotection test of the fusion proteins rB2LcF1L of the five-port sore Virus B2L and F1L

60 Balb/c mice were randomly divided into 3 groups of 20 mice each.

Groups

1 and 2 were subcutaneously immunized with rB2LcF L (100. Mu.g/dose of antigen) respectively, PBS control, and group 3 was used with oral sores produced by Sankang livestock organisms in XinjiangThe attenuated cells are freeze-dried to obtain live vaccine, the inoculation amount is 1 head part/one, the total immunization is 2 times, and the immunization interval is 2 weeks. On day 7 after the 2 nd immunization, cultures of cells of the strains Yulin of the orf virus (1 x 10 ⁷ TCID 50/mL), and is subjected to a toxicity attack protective test, and the toxicity attack amount is as follows: subcutaneous inoculation 600 μl/min. After the toxicity is removed, isolated feeding is carried out, main clinical indexes such as spirit, appetite and the like of animals after the toxicity is removed are observed and recorded daily, and the morbidity and mortality of mice in each immune group are recorded and counted.

The tested secondary immunization is used for attacking the virus, the death rate of a control mouse is 75%, the protection rate of an rB2LcF L immune group is 90%, and the protection rate of a attenuated cell freeze-dried live vaccine group is 90%, so that the subunit vaccine has good immunogenicity and is close to the protection efficacy of commercial attenuated vaccine.

In view of the current provision of the thrush vaccine in the market of China, the vaccine produced by the method is an ideal candidate vaccine for the current thrush vaccine in China.

Sequence listing

<110> Ulmin college

<120> an orf virus recombinant subunit vaccine and method for producing the same

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1914

<212> DNA

<213> tandem genes of full-length gene of Caprae Seu Ovis B2L and truncated gene of F1L (Artificial synthesis)

<400> 1

atgtggccgt tctcctccat ccccgggggc gccgactgcc gcgtcgtgga gacgctgccc 60

gcagaggtgg cgtccctggc gcagggcaac atgagcaccc tctactgctt caccgccatc 120

gccgagtccg cgaagaagtt tttgtacatc tgcagcttct gctgcaacct gagctccacc 180

aaggagggcg tcgacgtcaa ggacaagctc tgcacgctcg ccaaggaggg cgtcgacgtc 240

acgctgctcg tggacgtgca gagcaaggac aaggacgcgg acgagctgcg cgaggcgggc 300

gtcaactact acaaagtcaa ggtgtccacg cgggaaggcg tcggcaacct tctcggcagc 360

ttctggctct cggacaccgg gcactggtac gtgggcagcg cctcgctcac gggcgggtcc 420

gtgtccacca tcaagaacct cgggctctac tccaccaaca agcacctggc ctgggacctc 480

atgaaccgct acaacacctt ctactccatg atagtggagc cgaaggtgcc gttcacgcgg 540

ctctgctgcg ccgtcgtcac gcccacggcc acgaacttcc acctcaacca ctccgggggc 600

ggcgtattct tctcggactc gccggagcgc ttcctgggtt tctaccgcac gctcgacgag 660

gacctcgtgc tgcaccgcat cgagaacgcc aagaacagca tcgacctctc gctgctctcg 720

atggtgccgg tgatcaagca cgccggcgcc gtggagtact ggccgcggat catagacgcg 780

ctgctgcgcg cggccatcga ccgcggcgtg cgcgtgcgcg tgatcatcac cgagtggaag 840

aacgcggacc cgctgtcggt ctcggccgcg cgcagcctcg acgactttgg cgtcggcagc 900

gtggacatgt ccgtgcgcaa gttcgtggta cccggccggg acgacgctgc gaacaacacc 960

aagctgctca tcgtggacga caccttcgcg cacctcacgg tcgccaacct cgacggcacg 1020

cactaccgct accacgcctt cgtgagcgtg aacgccgaga agggcgacat cgtcaaggac 1080

ctgtcagcgg tcttcgagcg ggactggcgc tcggagttct gcaaaccaat aaatggcggt 1140

gggtcgcctc ccgcgccgca ccccaagggc gaccacgtgc tcaaggcggt ggaatggaaa 1200

gacgtggact ccaaagacta cccgcacttc ttcacggaca tgtgcaagtc cacgtgtccg 1260

aaggagatgc agcgccgcgc ggcacaccac ctcaaccttt gggagagcat atcggccggc 1320

actgtctcca ccaagtactc cgacaatgac ttcatcctgg tggtcgacaa cgacatgacc 1380

ttccgcaagc ccgagatggt aaagccgctc atcgaggcga tgaagacgaa cggctggtac 1440

atggcgcagc tcaaggagac ctacatgacc ggcgcgctgg ccaccaacgt ccccggcacc 1500

ggcgaccccg agctcatggt ctaccccggc ggatacgacg tctcgctaga cgcctacatc 1560

atcaacgtcg gcggcatgaa gaagctctac gacgcgatca tcaaggatgg agggctgcgc 1620

agcggcctgc tcaccgaggt gttcacgcta gagaagcggc tctctctggc gcgcgtggtg 1680

ctctccggcg ccgagcaggt ggtctaccct gagtactaca tacaggtgaa gacgcggctc 1740

ggcggcgcgc cctccctgtg gtcgctgctc gccacgtggc tggcgcgcgg cggtgggtcg 1800

ctggcatacc tgctggtgtt cgtgctgctg ctgccgaact cgcggctgct gtggttcatc 1860

gccgggctgc tggtcacggc catcgtgcat catcaccatc accactaaaa gctt 1914

<210> 2

<211> 635

<212> PRT

<213> tandem proteins of full-length gene of Caprae Seu Ovis B2L and truncated gene of F1L (Artificial synthesis)

<400> 2

Met Trp Pro Phe Ser Ser Ile Pro Gly Gly Ala Asp Cys Arg Val Val

1 5 10 15

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

20 25 30

Thr Leu Tyr Cys Phe Thr Ala Ile Ala Glu Ser Ala Lys Lys Phe Leu

35 40 45

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

50 55 60

Asp Val Lys Asp Lys Leu Cys Thr Leu Ala Lys Glu Gly Val Asp Val

65 70 75 80

Thr Leu Leu Val Asp Val Gln Ser Lys Asp Lys Asp Ala Asp Glu Leu

85 90 95

Arg Glu Ala Gly Val Asn Tyr Tyr Lys Val Lys Val Ser Thr Arg Glu

100 105 110

Gly Val Gly Asn Leu Leu Gly Ser Phe Trp Leu Ser Asp Thr Gly His

115 120 125

Trp Tyr Val Gly Ser Ala Ser Leu Thr Gly Gly Ser Val Ser Thr Ile

130 135 140

Lys Asn Leu Gly Leu Tyr Ser Thr Asn Lys His Leu Ala Trp Asp Leu

145 150 155 160

Met Asn Arg Tyr Asn Thr Phe Tyr Ser Met Ile Val Glu Pro Lys Val

165 170 175

Pro Phe Thr Arg Leu Cys Cys Ala Val Val Thr Pro Thr Ala Thr Asn

180 185 190

Phe His Leu Asn His Ser Gly Gly Gly Val Phe Phe Ser Asp Ser Pro

195 200 205

Glu Arg Phe Leu Gly Phe Tyr Arg Thr Leu Asp Glu Asp Leu Val Leu

210 215 220

His Arg Ile Glu Asn Ala Lys Asn Ser Ile Asp Leu Ser Leu Leu Ser

225 230 235 240

Met Val Pro Val Ile Lys His Ala Gly Ala Val Glu Tyr Trp Pro Arg

245 250 255

Ile Ile Asp Ala Leu Leu Arg Ala Ala Ile Asp Arg Gly Val Arg Val

260 265 270

Arg Val Ile Ile Thr Glu Trp Lys Asn Ala Asp Pro Leu Ser Val Ser

275 280 285

Ala Ala Arg Ser Leu Asp Asp Phe Gly Val Gly Ser Val Asp Met Ser

290 295 300

Val Arg Lys Phe Val Val Pro Gly Arg Asp Asp Ala Ala Asn Asn Thr

305 310 315 320

Lys Leu Leu Ile Val Asp Asp Thr Phe Ala His Leu Thr Val Ala Asn

325 330 335

Leu Asp Gly Thr His Tyr Arg Tyr His Ala Phe Val Ser Val Asn Ala

340 345 350

Glu Lys Gly Asp Ile Val Lys Asp Leu Ser Ala Val Phe Glu Arg Asp

355 360 365

Trp Arg Ser Glu Phe Cys Lys Pro Ile Asn Gly Gly Gly Ser Pro Pro

370 375 380

Ala Pro His Pro Lys Gly Asp His Val Leu Lys Ala Val Glu Trp Lys

385 390 395 400

Asp Val Asp Ser Lys Asp Tyr Pro His Phe Phe Thr Asp Met Cys Lys

405 410 415

Ser Thr Cys Pro Lys Glu Met Gln Arg Arg Ala Ala His His Leu Asn

420 425 430

Leu Trp Glu Ser Ile Ser Ala Gly Thr Val Ser Thr Lys Tyr Ser Asp

435 440 445

Asn Asp Phe Ile Leu Val Val Asp Asn Asp Met Thr Phe Arg Lys Pro

450 455 460

Glu Met Val Lys Pro Leu Ile Glu Ala Met Lys Thr Asn Gly Trp Tyr

465 470 475 480

Met Ala Gln Leu Lys Glu Thr Tyr Met Thr Gly Ala Leu Ala Thr Asn

485 490 495

Val Pro Gly Thr Gly Asp Pro Glu Leu Met Val Tyr Pro Gly Gly Tyr

500 505 510

Asp Val Ser Leu Asp Ala Tyr Ile Ile Asn Val Gly Gly Met Lys Lys

515 520 525

Leu Tyr Asp Ala Ile Ile Lys Asp Gly Gly Leu Arg Ser Gly Leu Leu

530 535 540

Thr Glu Val Phe Thr Leu Glu Lys Arg Leu Ser Leu Ala Arg Val Val

545 550 555 560

Leu Ser Gly Ala Glu Gln Val Val Tyr Pro Glu Tyr Tyr Ile Gln Val

565 570 575

Lys Thr Arg Leu Gly Gly Ala Pro Ser Leu Trp Ser Leu Leu Ala Thr

580 585 590

Trp Leu Ala Arg Gly Gly Gly Ser Leu Ala Tyr Leu Leu Val Phe Val

595 600 605

Leu Leu Leu Pro Asn Ser Arg Leu Leu Trp Phe Ile Ala Gly Leu Leu

610 615 620

Val Thr Ala Ile Val His His His His His His

625 630 635

<210> 3

<211> 27

<212> DNA

<213> upstream primer (Artificial synthesis)

<400> 3

cgcgctagca tgtggccgtt ctcctcc 27

<210> 4

<211> 24

<212> DNA

<213> downstream primer (Synthesis)

<400> 4

ccgaagcttt tagtggtgat ggtg 24

<210> 5

<211> 635

<212> PRT

<213> control group 1 (Artificial synthesis)

<400> 5

Met Trp Pro Phe Ser Ser Ile Pro Val Gly Ala Asp Cys Arg Val Val

1 5 10 15

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

20 25 30

Thr Leu Asp Cys Phe Thr Ala Ile Ala Glu Ser Ala Lys Lys Phe Leu

35 40 45

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

50 55 60

Asp Val Lys Asp Lys Leu Cys Thr Leu Ala Lys Glu Gly Val Asp Val

65 70 75 80

Thr Leu Leu Val Asp Val Gln Ser Lys Asp Lys Asp Ala Asp Glu Leu

85 90 95

Arg Glu Ala Gly Val Asn Tyr Tyr Lys Val Lys Val Ser Thr Arg Glu

100 105 110

Gly Val Gly Asn Leu Leu Gly Ser Phe Trp Leu Ser Asp Thr Gly His

115 120 125

Trp Tyr Val Gly Ser Ala Ser Leu Thr Gly Gly Ser Val Ser Thr Ile

130 135 140

Lys Asn Leu Gly Leu Tyr Ser Thr Asn Lys His Leu Ala Trp Asp Leu

145 150 155 160

Met Asn Arg Tyr Asn Thr Phe Tyr Ser Met Ile Val Glu Pro Lys Val

165 170 175

Pro Phe Thr Arg Leu Cys Cys Ala Val Val Thr Pro Thr Ala Thr Asn

180 185 190

Phe His Leu Asn His Ser Gly Gly Gly Val Phe Phe Ser Asp Ser Pro

195 200 205

Glu Arg Phe Leu Gly Phe Tyr Arg Thr Leu Asp Glu Asp Leu Val Leu

210 215 220

His Arg Ile Glu Asn Ala Lys Asn Ser Ile Asp Leu Ser Leu Leu Ser

225 230 235 240

Met Val Pro Val Ile Lys His Ala Gly Ala Val Glu Tyr Trp Pro Arg

245 250 255

Ile Ile Asp Ala Leu Leu Arg Ala Ala Ile Asp Arg Gly Val Arg Val

260 265 270

Arg Val Ile Ile Thr Glu Trp Lys Asn Ala Asp Pro Leu Ser Val Ser

275 280 285

Ala Ala Arg Ser Leu Asp Asp Phe Gly Val Gly Ser Val Asp Met Ser

290 295 300

Val Arg Lys Phe Val Val Pro Gly Arg Asp Asp Ala Ala Asn Asn Thr

305 310 315 320

Lys Leu Leu Ile Val Asp Asp Thr Phe Ala His Leu Thr Val Ala Asn

325 330 335

Leu Asp Gly Thr His Tyr Arg Tyr His Ala Phe Val Ser Val Asn Ala

340 345 350

Glu Lys Gly Asp Ile Val Lys Asp Leu Ser Ala Val Phe Glu Arg Asp

355 360 365

Trp Arg Ser Glu Phe Cys Lys Pro Ile Asn Gly Gly Gly Ser Pro Pro

370 375 380

Ala Pro His Pro Lys Gly Asp His Val Leu Lys Ala Val Glu Trp Lys

385 390 395 400

Asp Val Asp Ser Lys Asp Tyr Pro His Phe Phe Thr Asp Met Cys Lys

405 410 415

Ser Thr Cys Pro Lys Glu Met Gln Arg Arg Ala Ala His His Leu Asn

420 425 430

Leu Trp Glu Ser Ile Ser Ala Gly Thr Val Ser Thr Lys Tyr Ser Asp

435 440 445

Asn Asp Phe Ile Leu Val Val Asp Asn Asp Met Thr Phe Arg Lys Pro

450 455 460

Glu Met Val Lys Pro Leu Ile Glu Ala Met Lys Thr Asn Gly Trp Tyr

465 470 475 480

Met Ala Gln Leu Lys Glu Thr Tyr Met Thr Gly Ala Leu Ala Thr Asn

485 490 495

Val Pro Gly Thr Gly Asp Pro Glu Leu Met Val Tyr Pro Gly Gly Tyr

500 505 510

Asp Val Ser Leu Asp Ala Tyr Ile Ile Asn Val Gly Gly Met Lys Lys

515 520 525

Leu Tyr Asp Ala Ile Ile Lys Asp Gly Gly Leu Arg Ser Gly Leu Leu

530 535 540

Thr Glu Val Phe Thr Leu Glu Lys Arg Leu Ser Leu Ala Arg Val Val

545 550 555 560

Leu Ser Gly Ala Glu Gln Val Val Tyr Pro Glu Tyr Tyr Ile Gln Val

565 570 575

Lys Thr Arg Leu Gly Gly Ala Pro Ser Leu Trp Ser Leu Leu Ala Thr

580 585 590

Trp Leu Ala Arg Gly Gly Gly Ser Leu Ala Tyr Leu Leu Val Phe Val

595 600 605

Leu Leu Leu Pro Asn Ser Arg Leu Leu Trp Phe Ile Ala Gly Leu Leu

610 615 620

Val Thr Ala Ile Val His His His His His His

625 630 635

Claims

1. An orf virus fusion protein rB2LcF L, wherein the amino acid sequence of the orf virus fusion protein rB2LcF L is SEQ ID No:2.

2. a gene encoding the orf virus fusion protein rB2LcF1L of claim 1, the nucleic acid sequence of the gene encoding the orf virus fusion protein rB2LcF L being SEQ ID No. 1.

3. A recombinant noctiluca californica nuclear polyhedrosis virus Bac-ORFV-01 strain which has been delivered 22 days in 2018 to the China general microbiological culture Collection center of the national institute of microbiology, national institute of sciences, national institute of microbiology, national academy of sciences, no. 3, north chen west way No.1, dynasty, and has a deposit number of: CGMCC No.15494; the strain can stably and efficiently express the fusion proteins of the orf virus B2L and the F1L, and the amino acid sequence of the fusion proteins of the orf virus B2L and the F1L is SEQ ID No. 2.

4. The method for preparing the recombinant noctiluca californica nuclear polyhedrosis virus Bac-ORFV-01 strain according to claim 3, which is characterized by comprising the following steps:

step two, constructing a fusion expression vector: PCR amplification is carried out by taking the artificially synthesized gene sequence of SEQ ID No.1 as a template and adopting primer pairs 1F and 1R; wherein the upstream primer 1F sequence is: 5'-CGCGCTAGCA TGTGGCCGTT CTCCTCC-3' (SEQ ID No: 3) having a restriction enzyme introduced at its 5' endNheII site and protective base; the sequence of the downstream primer 1R is as follows: 5'-CCGAAGCTTTTAGTGGTGAT GGTG-3' (SEQ ID No: 4) having a restriction enzyme introduced at its 5' endHind IIIA site and a protective base; amplifying the obtained target DNA band, recovering the target DNA band, and adoptingNheⅠ/Hind IIIDouble enzyme digestion, and is connected with insect baculovirus expression system plasmid pBac5 carrier which is subjected to the same enzyme digestion to obtain the sheep mouth insertedPositive clone pBac5-GB2LcF L of full-length gene of sore virus; transforming DH5 alpha competent cells with the connected plasmids, picking up monoclonal cells to LB liquid medium containing kanamycin, shake culturing overnight at 37 ℃, and extracting plasmids for later use;

step three: construction of recombinant expression of the fusion protein rB2LcF L gene engineering strain of the orf virus: 6-well cell culture plates were taken at 1X 10 ⁶ ml ^-1 Inoculating sf9 cells with good growth state, culturing at 27 ℃ for 2 h, transfecting when the fusion degree of the cells reaches 80%, and uniformly mixing 3 mug pBac5-GB2LcF L and 3 mug linearized Bacmid by using 100 mug serum-free culture medium to obtain solution I; diluting 5 mu L of the fusfollow-up transfection reagent with 100 mu L of serum-free culture medium, slowly dripping the reagent into the solution I, uniformly mixing, and standing for 20min; adding the mixed solution into the cell plate of sf9, incubating at 27 ℃ for 5-7 d, observing the expression condition of a reporter gene cGFP under a fluorescence microscope, and harvesting cell supernatant in a 1 mL centrifuge tube after the diseased cells reach more than 90%, wherein the cell supernatant is marked as P1 generation recombinant baculovirus, and storing at-80 ℃ for a long time; the strain is delivered to China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, which is the national academy of sciences of China, including No. 3, of the national academy of sciences of China, including the Korean area of Beijing, at 22 days 2018, and has a collection number of: CGMCC No.15494.

5. Use of the recombinant california silver streak noctuid nuclear polyhedrosis virus Bac-ORFV-01 strain according to claim 3 for the preparation of a recombinant subunit vaccine of the orf virus.

6. An orf virus recombinant subunit vaccine, characterized in that the vaccine comprises recombinant orf virus B2L and F1L fusion protein rB2LcF L expressed by insect baculovirus; the strain for preparing the seedlings is a baculovirus for recombinant expression of fusion proteins of the sheep sore viruses B2L and F1L, is named as a recombinant alfalfa silver vein noctuid nuclear polyhedrosis virus Bac-ORFV-01 strain, and is delivered to China general microbiological culture Collection center for culture Collection, china institute of microbiological culture Collection, national institute of China, and the national institute of sciences of China, no. 3, the university of microorganisms, and the accession number is: CGMCC No.15494.

7. The recombinant subunit vaccine of the orf virus of claim 6, wherein the alfalfa silver vein moth nuclear polyhedrosis virus Bac-ORFV-01 strain is used as a vaccine production strain, and the recombinant subunit vaccine is prepared by culturing, expressing, separating and purifying target proteins, and adding a biphase oil adjuvant for mixing.

8. A method of preparing a recombinant subunit vaccine for an orf virus comprising the steps of:

(5) Sample collection: centrifugally collecting, and respectively collecting cells and supernatant;

(8) And (3) sterile inspection: according to the annex of the current Chinese animal pharmacopoeia, the bacteria-free growth is needed;

(9) Preparing a vaccine: introducing the biphase oil adjuvant into an oil phase tank, sterilizing at least 121 ℃ for 30 minutes under high pressure, cooling to room temperature for standby, properly diluting and uniformly mixing the purified protein which is qualified by inspection by PBS according to the protein content measurement result, adding the water phase into an emulsifying tank, stirring at 80-100 r/min, slowly adding the oil phase according to the ratio of 1:1 (V/V), and stirring for 20-30 min after the addition; sampling after emulsification, inspecting, and sub-packaging after passing.