ORF130 gene deleted strain of orf virus as well as preparation method and application of ORF130 gene deleted strain

Abstract

The invention belongs to the field of preparation of veterinary vaccines, and particularly relates to an orf virus ORF130 gene deleted vaccine strain as well as a preparation method and application thereof, and the gene deleted vaccine strain is prepared by inhibiting ORF130 gene expression in orf virus and deleting or conditionally knocking out an ORF130 gene. The highest TCID50 of the obtained gene deletion strain ORFV-HB-TS09F65 ORF130 can reach 10 <-5.12 > / 0.1 mL, and the toxicity of the gene deletion strain ORFV-HB-TS09F65 ORF130 is obviously weakened; compared with an artificial passage attenuated orf vaccine strain, the orf vaccine strain has no difference in safety; meanwhile, in the vaccine immunization process, the specific marker in the region can be used as an effective target for distinguishing vaccine immunization from wild virus infection, so that the gene deletion strain is suitable for being used as a candidate strain of the orf gene deletion vaccine.

Description

Technical Field

[0001] The invention belongs to the field of veterinary vaccine preparation, and specifically relates to a sheep sore virus ORF130 gene deleted vaccine strain and a preparation method and application thereof. Background Art

[0002] Orfvirus, also known as Contagious ecthyma (CE) or Contagious pustular dermatitis (CPD), is an acute, contact and epithelial zoonosis of goats, sheep and humans caused by infection with Orfvirus (ORFV), a member of the parapoxvirus. It is characterized by the formation of erythema, papules, pustules, ulcers and thick warty crusts on the skin and mucous membranes of the lips, hooves, udders and vulva of infected sheep. ORFV belongs to the genus Parapoxvirus and is classified as Poxviridae and Chordata Poxvirinae. The virus mainly includes two types, namely, sheep-type virus (S type) that infects sheep and goat-type virus (G type) that infects goats. Both originated in Europe and Asia in the 19th century, respectively. In recent years, contagious pustular disease has occurred in sheep flocks in sheep-raising countries and regions around the world, and is considered to be a global epidemic. Existing epidemiological survey and research data show that the incidence of the disease is about 50%, while the incidence in sensitive sheep flocks can reach 90%. Therefore, once the disease occurs, it will cause significant economic losses to sheep farmers and seriously endanger the healthy development of the mutton sheep industry. What is more serious is that the disease can infect breeders through wounds. It is a zoonotic infectious disease with relatively serious harm. At present, there is no effective preventive or therapeutic drug for sheep canker sores, and vaccination is the only way to prevent the occurrence and spread of the disease. Traditional sheep canker sores attenuated vaccines have been developed in China, but they have not been mass-produced due to process and other issues. In addition, the existing sheep canker sores vaccines cannot distinguish between vaccine immunity and virulent infection, and cannot trace the immune effect of the vaccine. At the same time, traditional attenuated vaccines also have the potential risk of virulence reversion.

[0003] In the previous research, the research group of the inventor used the MDBK cell line to adapt and isolate the Hubei strain of sheep oral ulcer disease positive material, and cultured the OrfVirus HB-TS09 strain to the 17th generation, and then inoculated the cell virus into newborn calf testicular primary cells for culture attenuation, and obtained the sheep infectious pustular virus attenuated vaccine strain, named the sheep infectious pustular virus cell-passaged attenuated OrfVirus HB-TS09F65 strain, classified and named the sheep infectious pustular virus cell-passaged attenuated, and sent it to the China Center for Type Culture Collection for preservation on March 19, 2014, with the preservation number CCTCC NO: V201406, and the preservation address is Wuhan University.

[0004] The ORFV ORF130 gene is located in the terminal variable region of the ORFV genome. The function of this gene is currently unknown and no researchers have studied it. Based on the above vaccine strain research, the present invention uses a genetic engineering method to delete the ORF130 gene in the vaccine strain, resulting in a significantly reduced virulence of the strain. Summary of the invention

[0005] The purpose of the present invention is to delete the ORFV ORF130 gene by a gene recombination method, so as to provide a safe, low-toxic and long-lasting immune vaccine candidate strain for the research and development of sheep oral ulcer gene deletion vaccine.

[0006] The primary purpose of the present invention is to provide an application of preparing an attenuated orf virus strain by inhibiting the expression of ORF130 gene, deleting or conditionally knocking out the ORF130 gene in orf virus, wherein the ORF130 gene sequence is shown in SEQ ID NO.1, and the orf virus is an attenuated strain of OrfVirus HB-TS09F65 caused by cell passage of orf virus, which was deposited in China Center for Type Culture Collection on March 19, 2014, with a deposit number of CCTCC NO.V201406.

[0007] The second object of the present invention is to provide an application of preparing an attenuated oropharyngeal canker sore vaccine by inhibiting the expression of the ORF130 gene, deleting or conditionally knocking out the ORF130 gene in oropharyngeal canker sore virus, wherein the ORF130 gene sequence is shown in SEQ ID NO.1, and the oropharyngeal canker sore virus is the OrfVirus HB-TS09F65 strain attenuated by cell passage of the sheep contagious pustular virus, which was deposited in the China Center for Type Culture Collection on March 19, 2014, with a deposit number of CCTCC NO.V201406.

[0008] The third object of the present invention is to provide an attenuated oropharyngeal canker virus strain, wherein the attenuated oropharyngeal canker virus strain includes an oropharyngeal canker virus strain with ORF130 gene expression inhibition, deletion or conditional knockout, and the oropharyngeal canker virus is an OrfVirus HB-TS09F65 strain attenuated by cell passage of sheep infectious pustular virus, which was deposited in the China Center for Type Culture Collection on March 19, 2014, with a deposit number of CCTCC NO.V201406.

[0009] Preferably, the attenuated oropharyngeal canker virus strain is an oropharyngeal canker virus strain with conditional knockout of ORF130 gene, and the oropharyngeal canker virus is the OrfVirus HB-TS09F65 strain attenuated by cell passage of ovine contagious pustular virus, which was deposited in China Center for Type Culture Collection on March 19, 2014, with the deposit number of CCTCC NO.V201406.

[0010] The fourth object of the present invention is to provide a method for preparing the attenuated sheep oral sore virus strain, wherein the method is to conditionally knock out ORF130 of the sheep infectious pustular virus cell-passaged attenuated OrfVirus HB-TS09F65 strain by genetic engineering means, and the ORF130 gene sequence is shown in SEQ ID NO.1. The sheep infectious pustular virus cell-passaged attenuated OrfVirus HB-TS09F65 strain was deposited in the China Center for Type Culture Collection on March 19, 2014, and the deposit number is CCTCC NO: V201406.

[0011] Preferably, the method comprises the following steps:

[0012] (1) Design and synthesize a gene-deleted recombinant plasmid with the reporter gene EGFP: pUC-130L-EGFP-130R;

[0013] (2) The designed and synthesized gene-deficient recombinant plasmid: pUC-130L-EGFP-130R was transfected into BT cells inoculated with OrfVirus HB-TS09F65 virus by liposome transfection;

[0014] (3) Through fluorescent plaque combined with fluorescent single cell screening, a monoclonal gene-deficient virus with a green fluorescent reporter gene was screened. After multiple rounds of screening and PCR identification, a recombinant virus strain lacking the ORF130 gene was obtained: ORFV-HB-TS09F65△ORF130.

[0015] The fifth object of the present invention is to provide the use of the attenuated aphthous stomatitis virus strain in the preparation of an attenuated aphthous stomatitis virus vaccine.

[0016] The sixth object of the present invention is to provide an attenuated oropharyngeal sore virus vaccine, wherein the attenuated oropharyngeal sore virus vaccine comprises the attenuated oropharyngeal sore virus strain.

[0017] The beneficial effects of the present invention are as follows: the ORF130 gene is deleted in the attenuated sheep oral sore vaccine strain Orf VirusHB-TS09F65 strain attenuated by laboratory passage, and the TCID of the obtained ORF130 gene-deficient strain ORFV-HB-TS09F65△ORF130 is 50 The maximum value can reach 10 -5.83 / 0.1mL, the attenuated canker sore gene-deficient strain has significantly weakened virulence compared with its parental strong strain; the attenuated canker sore gene-deficient strain has significantly reduced virulence compared with its parental strong strain, and its virulence has also decreased compared with its parental vaccine strain, proving that the ORF130 gene is a virulence gene. There is no difference in safety between this strain and the canker sore vaccine strain weakened by artificial passage; after immunizing rabbits with this attenuated strain, tracking its antibody changes is consistent with the vaccine strain. This deletion strain introduces the reporter gene EGFP in the ORF130 deficiency region, which can be visualized during virus screening and purification. At the same time, during the vaccine immunization process, the specific markers in this region can be used as an effective target to distinguish vaccine immunity from wild virus infection. Therefore, this gene-deficient strain is suitable as a candidate strain for canker sore gene-deficient vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the construction of ORFV-HB-TS09F65ΔORF130 gene deletion strain

[0019] Figure 2 Recombinant plasmid transfection results

[0020] A: Cells with green fluorescence observed by fluorescence microscope; B: Photo of fluorescence and cells observed by fluorescence microscope; C: CPE observed by fluorescence microscope

[0021] Figure 3 Fluorescent plaque screening of recombinant viruses

[0022] A, B, C: first round of screening; D, E, F: second round of screening; G, H, I: third round of screening; A, D, G: green fluorescence produced after the recombinant virus infects the cells; B, E, H fluorescence and cell superposition photos; C, F, I: CPE produced by the recombinant virus

[0023] Figure 4 Fluorescence single cell screening of recombinant toxins

[0024] A, B, C: first round of screening; D, E, F: second round of screening; G, H, I: third round of screening; A, D, G: green fluorescence produced after the recombinant virus infects the cells; B, E, H fluorescence and cell superposition photos; C, F, I: CPE produced by the recombinant virus

[0025] Figure 5 Subculture and expansion of gene-deficient recombinant virus

[0026] A, B, C: 96-well plate culture; D, E, F: 24-well plate culture; G, H, I: 12-well plate culture; J, K, L: 12-well plate culture; M, N, O: T75 cell flask culture

[0027] Figure 6PCR identification results of gene deletion recombinant virus

[0028] 1: DL5000 DNA Marker; 2: ORFV-HB-TS09 parental virus; 3: ORFV-HB-TS09△ORF130 gene deletion virus

[0029] Figure 7 Growth curves of ORFV-HB-TS09ΔvIL-10 gene-deficient virus and its parental virus

[0030] Figure 8 Electrophoresis of recombinant virus PCR products Notes: M. DNA molecule marker; 1. Gene-deficient virus, generation 5; 2. Gene-deficient virus, generation 10; 3. Gene-deficient virus, generation 10

[0031] Fig. 9 Detection of antibody levels in rabbits inoculated with gene-deficient virus and its parental virus DETAILED DESCRIPTION

[0032] The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description of the specific embodiments. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the technical solutions and detailed forms and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0033] It should be noted that, in the following examples, unless otherwise specified, the methods used are all conventional methods; the reagents used are all conventional reagents and can be purchased from the market.

[0034] It should be noted that the ORFV-HB-TS09 strain virus described in the present invention was isolated from a clinical case of oral ulcer in goats in Hubei Province, and is also known as OrfVirusHB-TS09.

[0035] It should be noted that the ORFV-HB-TS09F15 virulent strain described in the present invention is obtained by passage of the ORFV-HB-TS09 strain to the 15th generation.

[0036] It should be noted that the ORFV-HB-TS09F65 parent virus described in the present invention is a vaccine strain obtained by passage of the ORFV-HB-TS09 strain to the 65th generation, and it was deposited in the China Center for Type Culture Collection on March 19, 2014, with the deposit number CCTCC NO: V201406, and an invention patent ZL201410160855.0 has been applied for.

[0037] In the early stage of the experiment of the present invention, the research group of the inventor deleted the gene ORF130 in the strong toxin of ORFV-HB-TS09F15, and the virulence of the strain was significantly reduced, confirming that the gene ORF130 was a virulence gene. In order to further obtain a vaccine strain with lower virulence and better safety, the existing preserved vaccine strains were iteratively upgraded. The research group of the inventor deleted the gene ORF130 in the vaccine strain ORFV-HB-TS09F65 (i.e., the parental toxin) preserved in the early stage, and conducted the experiment described in the present invention in detail. The ORFV-HB-TS09F65△ORF130 described in the following examples is a strain in which the gene ORF130 in the parental toxin of ORFV-HB-TS09F65 is deleted.

[0038] The term "vaccine" refers to a biological preparation that can provide a protective response in an animal, wherein the vaccine has been delivered and cannot cause serious disease. The sheep sore virus vaccine of the present invention further optionally comprises one or more adjuvants, excipients, carriers and diluents. The adjuvant can be any suitable adjuvant, chemical immunoadjuvants such as aluminum hydroxide, Freund's adjuvant, mineral oil, Span, etc.; microbial immunoadjuvants such as mycobacteria, lipopolysaccharide, muramyl dipeptide, cell peptide, fat-soluble wax D, short rod-shaped bacteria; plant immunoadjuvants are mostly polysaccharides extracted from plants or large fungi, such as tuckahoe polysaccharide, safflower polysaccharide, Chinese herbal medicine, etc. And biochemical immunoadjuvants such as thymosin, transfer factor, interleukin, etc. Preferred adjuvants can be nano adjuvants, biological adjuvants, interleukins, interferons, etc.

[0039] The Cre / Loxp system consists of two parts: Cre recombinase and Loxp site. Cre recombinase is a specific recombinase discovered from P1 phage. It is a 38kDa protein that can recognize specific DNA sequences, namely LoxP sites. It mediates specific recombination between two LoxPs, causing the gene sequence between the LoxP sites to be deleted or recombined. The LoxP site also comes from P1 phage and is composed of two 13bp inverted repeat sequences and an 8bp spacer sequence. The spacer sequence determines the direction and is a specific recombination site for the Cre recombinase. Depending on the location and direction of the loxP site, recombination can generally have three results:

[0040] ① Synchronous deletion: If the loxP sites are on the same DNA chain and in the same direction, the sequence between the loxP sites will be deleted (not retained) as a circular DNA fragment.

[0041] ② Reverse flipping: construct two opposite Loxp sites on the same chromosome. Under the action of Cre recombinase, the target gene between the Loxp sites flips in the opposite direction.

[0042] ③ Translocation on different chromosomes: A LoxP site is constructed on each chromosome. Under the action of the Cre group enzyme, the genes behind the LoxP site are recombined, large fragments of genes on different chromosomes are exchanged with each other, and chromosome translocation occurs.

[0043] Example 1 Construction and screening of the ORF130 gene deletion strain of the oropharyngeal canker sore virus

[0044] 1.1 Construction of a green fluorescent marker gene deletion virus using the Cre-Loxp recombinase system

[0045] The specific method and construction diagram of deleting the ORF130 gene and introducing the EGFP reporter gene in the present invention are as follows: Figure 1 Based on the attenuated strain of ORFV-HB-TS09F65, the LoxP sequence with the same orientation was constructed into the gene expression frame of the transfer vector using the homologous recombination method, which was used for the excision of the EGFP fluorescent tag after the subsequent vaccine strain was determined.

[0046] 1.2 Construction of ORFV-HB-TS09ΔORF130 gene deletion strain

[0047] In the present invention, we simultaneously utilize homologous recombination technology and Cre-LoxP system to construct an ORFV gene deletion strain (ORFV-HB-TS09F65ΔORF130) lacking the green fluorescent marker of the ORF130 gene.

[0048] According to the full sequence of ORFV-HB-TS09F65 vaccine strain, 1500 bp were selected upstream and downstream of ORF130 gene to design primers to amplify homology arms (see Table 1). Figure 1 Design and synthesize pUC57-130L-EGFP-130R transfer vector plasmid.

[0049] Table 1 Primer sequences

[0050]

[0051] 1.3 Screening of gene-deleted recombinant virus of ORFV-HB-TS09F65ΔORF130 strain

[0052] 1.3.1 Cell transfection

[0053] Select BT cells in good condition and inoculate them in a 6-well plate. When the cell density reaches about 70%, inoculate the monolayer BT cells with the attenuated ORFV-HB-TS09F65 strain vaccine at an MOI of 0.1. Incubate in a 37°C, 5% CO2 cell culture incubator for 1 hour, then add DMEM high-glucose culture medium and incubate in a 37°C, 5% CO2 cell culture incubator for 6 hours.

[0054] According to the instructions for use of the ThermoFisher Scientifi Lipo 3000 liposome transfection kit, 125 μL Opti-MEM culture medium, 2 μg pUC57-130L-EGFP-130R transfer vector plasmid, and 4 μL P3000 reagent were added to a 1.5 mL sterile EP tube, and mixed with a pipette; then take another sterile EP tube, add 125 μL Opti-MEM culture medium, and then add 4 μL Lipo3000, and gently mix with a pipette, incubate at room temperature for 5 minutes, and then mix the two parts of the incubated liquid, and gently mix with a pipette, incubate at room temperature for 20 minutes, and then add 250 μL of the mixture to one well of the above-mentioned 6-well plate. Incubate at 37 ° C for 6 hours, supplement with a medium containing 10% serum; at 24 hours, 48 ​​hours, and 72 hours of transfection, use an inverted fluorescence microscope to observe fluorescence. Use a marker pen to mark the strong fluorescent area on the back of the six-well plate, and discard the culture medium of the six-well plate. Add 2 μL of trypsin digestion solution to each area of ​​the marked area to digest the cells in the marked area. When all the cells in the marked area are digested, add 2 μL of DMEM culture medium to the marked digestion area, and collect the digested cells in this area together with the liquid in 50 μL of serum-free DMEM culture medium and store at -80°C for later use.

[0055] 1.3.2 Green fluorescent plaque screening

[0056] Use BT cells to lay 6-well plates in advance. When the cell density reaches about 80%, discard the cell culture medium, wash the 6-well plates with PBS 2-3 times, add a small amount of serum-free medium to each well of the 6-well plate, dilute the green fluorescent virus harvested in 1.3.1 10 times and inoculate the 6-well plate. One well is not inoculated with virus as a control. After standing in a 37℃5% CO2 incubator for 1h, take out the 6-well plate and add 2% FBS cell culture medium. Put it in a 37℃5% CO2 incubator again and observe whether fluorescence appears every 12h. After 24h of fluorescent cells appearing (to make the fluorescent cells grow and concentrate), discard the liquid culture medium, mix 1.5% agarose and 4% FBS2×DMEM in a ratio of 1:1, add 6-well plates, add 2mL to each well, and place in a 37℃ incubator after the agarose solidifies. After 72h, use a fluorescence microscope to obtain the green fluorescent aggregation area, collect and store at -80℃. Repeat the screening in this way.

[0057] 1.3.3 Fluorescence single cell screening

[0058] Use BT cells to spread 96-well plates in advance, wait until the cell density reaches about 80%, discard the original cell culture medium, wash the 96-well plates 3 times with PBS, add 100 μL serum-free DMEM to the control wells, dilute the fluorescent plaque venom collected in 1.3.2 10-fold, and add 10 -1, 10 -2 , 10 -3 Three dilutions were added to each well of a 96-well plate, with 50 μL of the virus inoculated. After standing in a 37°C 5% CO2 incubator for 1 hour, the 6-well plate was taken out and supplemented with 50 μL of cell culture medium containing 2% FBS. It was placed in a 37°C 5% CO2 incubator again, and fluorescence was observed every 12 hours to see if there was any fluorescence, and the wells with more concentrated fluorescence were marked. 72 hours after inoculation, the fluorescent wells with the largest dilution and the highest overlap between cytopathic effect and green fluorescence were selected, the culture medium was aspirated, 100 ul of trypsin was added to each well, and the cell digestion was observed under a fluorescence microscope. When the cells became round, digestion was stopped. 100 μL of DMEM was added and gently blown, and all digested cells were blown into single cells as much as possible. The digested single cells were placed in a 35 mm dish, and fluorescent cells were picked up with a pipette under a fluorescence microscope. The selected fluorescent single cells were placed in an EP tube containing 100 μL of DMEM culture medium, and frozen and thawed 3 times, and stored at -80°C. The screening was repeated in this way.

[0059] 1.3.4 Expansion and subculture of gene-deficient virus

[0060] Inoculate the fluorescent single cell toxins selected in 1.3.3 into the BT cells cultured in the 96-well plate, and perform subculture, and observe whether the green fluorescence increases with the increase in the number of subcultures, and observe whether the green fluorescence aggregation area coincides with the cell CPE. If the green fluorescence of the BT cells does not coincide with the CPE, it means that the single cell toxin selected in 1.3.3 is not pure enough, and 1.3.3 is repeated for screening. Finally, the green fluorescence of the single cell green fluorescent toxin is increased with the increase in the number of subcultures on BT, and the area where the green fluorescence appears should coincide with the CPE of the BT cells caused by ORFV. Collect such culture wells and expand the culture to 24-well plates. As with the 96-well plates, observe whether the green fluorescence coincides with the CPE, collect the overlapping wells, and expand the culture to 12-well plates, 6-well plates, and T75 cell bottles. The viruses of each generation that are screened and expanded are stored at -80°C for standby use.

[0061] 1.4 Identification of gene-deleted recombinant viruses

[0062] According to the gene sequence of the pUC57-130L-EGFP-130R recombinant plasmid, upstream and downstream primers located at the front and rear ends of the EGFP green fluorescent gene were designed, and the primer sequences are shown in Table 2.

[0063] Table 2 Primer sequences

[0064]

[0065] TaKaRa's MiniBEST Viral RNA / DNA Extraction Kit Ver.5.0 was used to extract DNA from the ORFV-HB-TS09ΔvIL-10 gene-deficient strain and the parental strain ORFV-HB-TS09F65 vaccine strain, and used them as templates for PCR reactions. The ORFV-HB-TS09F65ΔORF130 gene-deficient strain was identified.

[0066] 1.5 Results

[0067] 1.5.1 Transfection results of recombinant plasmid pUC57-130L-EGFP-130R

[0068] The gene recombinant plasmid pUC57-130L-EGFP-130R was transfected into BT cells inoculated with ORFV-HB-TS09F65 vaccine virus. After 48 hours of culture, green fluorescence could be observed under an inverted fluorescence microscope. After 72 hours, the green fluorescence of BT cells gradually increased, and ORFV-induced cytopathic effect (CPE) could be observed in the fluorescent BT cells. Figure 2 However, the number of BT cells with green fluorescence and lesions was small, indicating that most of the lesions were caused by infection with the vaccine parent virus. The virus-transfected cells were collected blindly, frozen and thawed three times, and stored at -80℃ for later use.

[0069] 1.5.2 Screening and purification of ORFV-HB-TS09F65ΔORF130 recombinant virus

[0070] 1.5.2.1 Fluorescent plaque screening results

[0071] The virus obtained after transfection was diluted 10 times and inoculated into 6-well BT cells for fluorescent plaque screening. Green fluorescence generally appeared in the infected cells in about 24 hours. At 72 hours, the 6-well plate was placed under a fluorescence microscope, and the areas where the green fluorescence was concentrated and the green fluorescence emission area could completely cover the cell CEP were marked for plaque selection. A total of three rounds of screening were carried out, and the fluorescent monoclonal plaque virus selected in each round was passaged for five generations before the next round of screening. As the number of screenings increased, the green fluorescent spots that could be screened became stronger and stronger, and the strong fluorescence area basically coincided with the CPE that appeared on the cells. Figure 3 shown.

[0072] 1.5.2.2 Results of fluorescent single-cell recombinant virus screening

[0073] Single cell fluorescence screening was also performed for 3 rounds, and the fluorescent single cells selected in each round were passaged for 5 generations. Under an inverted fluorescence microscope, the diseased cells in the white light field and the luminescent area in the green fluorescence field almost completely overlapped ( Figure 4). It was preliminarily proved that the gene-deficient virus screened had reached a certain purity.

[0074] 1.5.2.3 Expansion and subculture of gene-deficient recombinant virus

[0075] The single-cell fluorescent gene-deficient monoclonal recombinant virus screened in 1.5.2.2 was first subcultured in a 96-well plate, and the monoclonal recombinant virus whose fluorescence gradually increased with subculture and whose fluorescent luminescence site completely overlapped with the CPE of the virus-inoculated cells was selected for subculture ( Figure 5 :A, B, C). The monoclonal recombinant virus passaged in 96-well plates was collected and frozen and thawed three times, and then inoculated into 24-well plates for passage. The fluorescence-producing site completely overlapped with the CPE produced by the cells, and the cell area where CPE was produced produced fluorescence ( Figure 5 :D, E, F), and a total of 5 generations. The pure monoclonal gene-deficient recombinant virus cultured in 24-well plates was further cultured and expanded to 12-well plates ( Figure 5 :G, H, I); After 5 generations of monoclonal gene deletion and recombination in 24-well plates, the cytotoxic cells whose fluorescence increased with the passage and completely overlapped with the cell CPE were selected and expanded to 6-well plates ( Figure 5 :J, K, L), and finally expanded to T75 cell flask culture ( Figure 5 :M, N, O). Then gradually expand the culture according to demand. The gene-deficient recombinant virus obtained by expanded culture: ORFV-HB-TS09ΔORF130, from the observation of the passage process, the fluorescence intensity of the recombinant virus gradually increased with the increase of the number of primary generations, and completely overlapped with the CPE produced by the cells (such as Figure 5 ). It can be seen that the constructed and screened ORFV-HB-TS09ΔORF130 recombinant virus is relatively pure.

[0076] 1.6 Identification of ORFV-HB-TS09ΔORF130 recombinant virus

[0077] The genomic DNA of the deletion strain was identified by PCR using the primers designed in 1.4 at both ends of the EGFP gene, such as Figure 6 As shown, a band of 835bp can be amplified in the deletion strain, and a band of 669bp can be amplified in the parental strain, which is consistent with the expected band size. After the band gel was recovered and purified, it was sent to the company for sequencing. The sequencing results showed that the deletion strain amplified the complete EGFP gene (724bp), and the parental strain amplified the complete ORF130 gene, which proved that the gene deletion strain successfully deleted the ORF130vIL-10 gene and had completely inserted the reporter gene EGFP. This result is consistent with the green fluorescence observed during the deletion strain screening process.

[0078] Example 2 Biological characteristics of ORFV-HB-TS09F65△ORF130 gene deletion strain and its safety and immune evaluation

[0079] 2.1 Proliferation characteristics of ORFV-HB-TS09F65△ORF130 gene deletion strain

[0080] Cell preparation: 1 day in advance, culture BT cells in 60 mm cell dishes. When the cell confluence reaches 80%, it can be used for virus inoculation; culture cell dishes should be no less than 20 to prepare TCID 50 Adequate sampling was performed for virus growth curve determination.

[0081] Virus inoculation: ORFV-HB-TS09F65△ORF130 deletion virus, ORFV-HB-TS09F65 parental virus and ORFV-HB-TS09F15 strong virus were inoculated into well-growing BT cells at an inoculation dose of MOI=0.1. ORFV-HB-TS09F65△ORF130 deletion virus and ORFV-HB-TS09F65 parental virus were inoculated into 12 cell culture dishes, incubated in a 37°C, 5% CO2 cell culture incubator for 1 hour, and then supplemented with 2% FBS DMEM cell culture medium and placed in a 37°C, 5% CO2 incubator for culture.

[0082] Virus collection: After the ORFV-HB-TS09F65△ORF130 deletion virus, ORFV-HB-TS09F65 parental virus and ORFV-HB-TS09F15 strong virus are inoculated and cultured, the time is set, and random samples (one culture dish) are taken at 6h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, and 96h to collect the virus, and records are kept. The collected virus liquid is repeatedly frozen and thawed in a -80℃ refrigerator for 3 times for standby use. In addition, 3 culture dishes inoculated with ORFV-HB-TS09F65△ORF130 deletion virus and ORFV-HB-TS09F65 parental virus are randomly selected, and the cultured virus is observed 48h after inoculation. The virus is collected after more than 90% of the cells show cytopathic effect (CPE), and is repeatedly frozen and thawed in a -80℃ refrigerator for 3 times to prepare for the later virus TCID 50 Determination

[0083] TCID 50 Determine the virus titer and draw the virus growth curve: After the virus collected in the previous step was frozen and thawed three times, the TCID of the virus collected at each time point was determined. 50 The growth curve of the virus was drawn to compare the intracellular replication level of ORFV-HB-TS09F65△ORF130 and the parent virus. The cytotoxicity of more than 90% of the collected cells showing cytopathic effect (CPE) was determined as TCID 50 , comparing their value-added characteristics.

[0084] 2.2 Genetic stability of ORFV-HB-TS09F65△ORF130 gene deletion strain

[0085] The ORFV-HB-TS09F65△ORF130 gene-deficient virus after screening and purification was continuously passaged on BT cells for 15 generations, and the reporter gene EGFP was determined by PCR to detect the genetic stability of the ORFV-HB-TS09F65△ORF130 gene-deficient virus during virus passage (primers see Table 3).

[0086] Table 3 EGFP PCR primers

[0087]

[0088] 2.3 Safety evaluation of ORFV-HB-TS09F65△ORF130 gene deletion strain

[0089] 2.3.1 Mice

[0090] A total of 112 healthy three-week-old BALB / c male mice were randomly divided into 4 groups, including 3 groups of 35 mice each as experimental groups and 1 group of 7 mice as control groups. The TCID of the purified and expanded ORFV-HB-TS09F65△ORF130 gene deletion, ORFV-HB-TS09 F65 parental virus and ORFV-HB-TS09F15 strong strain was determined. 50 Value (requires TCID50 ≥ 10 -5.00 ), according to the TCID 50 , which was converted into PFU value (plaque forming unit). The three experimental groups were inoculated by intraperitoneal injection, one group was inoculated with ORFV-HB-TS09F65△ORF130 gene deletion virus, another group was inoculated with ORFV-HB-TS09F65 parental virus, and the third group was inoculated with ORFV-HB-TS09F15 strong virus; each group was inoculated with 10 3 , 10 4 , 10 5 , 10 6 , 10 7 PFU inoculation dose, 5 dose groups were inoculated, and 7 mice were inoculated in each dose group (see Table 4). 7 mice were in the control group, and 0.5 ml DMEM was injected intraperitoneally in each mouse (see Table 4). Each group and each dose group was marked, and the incidence, death and recovery of orchitis in male mice were observed and recorded daily.

[0091] Table 4 Mouse safety test

[0092]

[0093] 2.3.2 Suckling mice

[0094] 112 healthy 2-3 day old BALB / c suckling mice with mothers were selected and randomly divided into 4 groups, 3 experimental groups with 35 mice in each group and 1 control group with 7 mice. The 2 experimental groups were inoculated by intracranial injection, one group was inoculated with ORFV-HB-TS09F65△ORF130 gene deletion virus, another group was inoculated with ORFV-HB-TS09F65 parental virus, and the third group was inoculated with ORFV-HB-TS09F15 strong virus; each group was inoculated with 10 3 , 10 4 , 10 5 , 10 6 , 10 7 PFU was divided into 4 dose groups, and 7 suckling mice were inoculated in each dose group (see Table 5). The control group had 7 mice, and each was intraperitoneally injected with 0.02 ml DMEM (see Table 5). Those who died within 24 hours after inoculation were treated as lethal by inoculation and supplemented according to the group. The cases of onset and death after 24 hours were observed daily and recorded.

[0095] Table 5 Safety test on suckling mice

[0096]

[0097] 2.3 Evaluation of the immune effect of rabbits immunized with ORFV-HB-TS09F65△ORF130 gene deletion strain

[0098] Twenty-four New Zealand rabbits aged 5-6 months were randomly divided into four groups, including 7 rabbits in the ORFV-HB-TS09F65 parental virus group, 7 rabbits in the ORFV-HB-TS09F65△ORF130 gene deletion virus group, 7 rabbits in the ORFV-HB-TS09F15 virulent virus group, and 3 rabbits in the control group. The rabbits were immunized by intralip streak inoculation and intradermal inoculation of the inner thigh. The immunization dose was 10 5 TCID 50 / rabbit, and boost immunization with the same dose 2 weeks after immunization. Observe daily, record clinical symptoms of test rabbits, collect blood once a week, separate serum, and track changes in antibody levels of immunized rabbits.

[0099] 2.4 Results

[0100] 2.4.1 TCID of ORFV-HBTS09F65ΔORF130 gene deletion virus and its parent virus 50 Determination

[0101] The TCID of parental virulent ORFV-HB-TS09F65 strain was determined using the Reed-Muench method 50 For 10 -5.91 / 0.1mL, virulent ORFV-HB-TS09F15 strain TCID 50 For 10-5.85 / 0.1mL ORFV-HBTS09F65ΔORF130 gene-deleted recombinant strain TCID 50 For 10 -5.83 / 0.1mL. Compared with the parental virus, the virus titer of ORFV-HBTS09ΔORF130 gene deletion virus decreased slightly, but the decrease was very small. This indicates that after the ORF130 gene was deleted, the proliferation ability of ORFV-HBTS09F65ΔORF130 gene deletion virus on BT cells did not decrease.

[0102] 2.4.2 Virus growth curve

[0103] The horizontal axis is the virus infection time, and the TCID of virus samples collected at different time points is measured. 50 The one-step growth curves of the ORFV-HB-TS09ΔORF130 gene-deficient recombinant strain, the parental strain of the ORFV-HB-TS09F65 strain, and the strong strain of ORFV-HB-TS09F15 were drawn with the values ​​as the vertical axis ( Figure 7 ). As can be seen from the growth curve, the growth and proliferation of ORFV-HB-TS09F65ΔORF130 gene deletion virus and its parental virus are basically similar, except that the virus titer of ORFV-HB-TS09F65ΔORF130 gene deletion virus at each time point is lower than that of the parental virus, which proves that the replication ability of ORFV virus decreases after deleting the virulence gene ORF130, but the decrease is small, and some time points overlap.

[0104] 2.4.3 Virus genetic stability determination

[0105] The recombinant virus ORFV-HB-TS09F65ΔORF130 was continuously passaged on BT cells, and the virus liquid of F5, F10, and F15 was taken to extract the DNA as a template. The primers in Table 3 were used for PCR amplification, and the expected size bands were amplified. The sequencing results were correct ( Figure 8 ). This proves that the genetic traits of the ORFV-HB-TS09ΔORF130 gene deletion virus are stable.

[0106] 2.4.4 Safety evaluation of gene-deficient strains

[0107] 2.4.4.1 Death status and clinical symptoms of BALB / c male mice:

[0108] Male rat safety test group 10 7 All BALB / c male mice inoculated with ORFV-HB-TS09F65 strong virus, ORFV-HB-TS09F65 parental virus, and ORFV-HB-TS09F65ΔORF130 gene deletion virus developed orchitis around 7 days after inoculation, and all died on 14 days.6 In the PFU dose group, all the ORFV-HB-TS09F15 strong virus showed orchitis, and the number of male mice inoculated with ORFV-HB-TS09ΔORF130 gene deletion virus in each group with orchitis was less than that of its parental virus. Statistical data were used to calculate the ID of ORFV-HB-TS09F15 strong virus, ORFV-HB-TS09F65 parental virus and ORFV-HB-TS09F65ΔORF130 gene deletion virus on BALB / c male mice. 50 , strong poison is 10 3.400 PFU, parental toxin is 10 4.549 PFU, ORFV-HB-TS09F65ΔORF130 gene deletion virus is 10 4.833 PFU. The pathogenicity of ORFV-HB-TS09F65ΔORF130 gene deletion virus was significantly reduced compared with its parental strong virus, which proved that the ORF130 gene is a virulence gene. The pathogenicity of ORFV-HB-TS09F65ΔORF130 gene deletion virus to male mice was lower than that of ORFV-HB-TS09F65 parental virus, which proved that the pathogenicity of ORFV-HB-TS09 vaccine virus was further reduced after deleting the ORF130 gene. After male mice were inoculated with ORFV-HB-TS09F65ΔORF130 gene deletion virus and its parental virus, 10 3 No disease or death occurred when the PFU inoculation dose was reached, so the safety of the gene-deficient virus is equivalent to that of the vaccine strain parent virus; the virulence of the gene-deficient virus is significantly reduced compared with the strong parent virus.

[0109] 2.4.4.2 Results of the safety test on suckling mice

[0110] In the suckling mouse safety test group, 10 6 All suckling mice inoculated with ORFV-HB-TS09F65 virus and ORFV-HB-TS09F65ΔORF130 gene deletion virus died within 7 days of inoculation; 5 In the PFU inoculation dose group, 4 mice died in the parental toxin group during the 14th day of observation, and 5 mice died in the deletion toxin group; 4 In the PFU inoculation dose group, 2 mice died in the parental poison group and 3 mice died in the deletion poison group; 3 There was no death in the PFU inoculation dose group, either the parental virus or the deletion virus. Statistical data were used to calculate the LD of ORFV-HB-TS09F15 strong virus, ORFV-HB-TS09F65 vaccine virus, and ORFV-HB-TS09ΔORF130 gene deletion virus against suckling mice 50 , strong poison is 10 3.25 PFU, parental toxin is 10 4.36 PFU, gene deletion virus is 104.64 PFU. This proves that the lethality of ORFV-HB-TS09F65ΔORF130 gene deletion virus to suckling mice is significantly lower than that of its parental strong virus, and slightly lower than that of its parental vaccine virus. The suckling mouse safety test also proves that the ORFV ORF130 gene is a virulence gene. After suckling mice were inoculated with ORFV-HB-TS09F65ΔORF130 gene deletion virus and its parental virus, 10 3 No deaths occurred at the PFU inoculation dose, so the safety of the gene-deficient virus is comparable to that of the vaccine strain parental virus. The virulence of the gene-deficient virus is significantly reduced compared with its parental strong virus. The gene-deficient virus can be used as a candidate strain for the sheep oral canker sore gene-deficient vaccine.

[0111] The animal experiments in 2.4.4.1 and 2.4.4.2 further proved that the ORF130 gene is a virulence gene of ORFV.

[0112] 2.4.5 Evaluation of the immune effect of gene-deficient strains

[0113] The antibody level of the immunized rabbits was detected using the indirect ELISA method established in the laboratory. Fig. 9 As shown in the figure, antibodies can be detected in the immunized group 7 days after immunization, and the antibody level increases rapidly after booster immunization on the 14th day, reaching a peak between 21 and 30 days. The antibody level began to decline after 60 days. The antibody level changes of ORFV-HB-TS09F65ΔORF130 gene deletion virus and its parent vaccine virus and parent strong virus are basically the same. This proves that the ORFV-HB-TS09F65ΔORF130 deletion virus can maintain the original immunogenicity of the vaccine strain after the virulence gene ORF130 is deleted.

[0114] In summary, the present invention deletes the ORF130 gene in the attenuated sheep oral canker sore vaccine strain OrfVirus HB-TS09F65 strain attenuated by laboratory passage, and the TCID of the obtained ORF130 gene-deficient strain ORFV-HB-TS09F65△ORF130 is 50 The maximum value can reach 10 -5.83 / 0.1mL, the attenuated canker sore gene-deficient strain has significantly weakened virulence compared with its parental strong strain; the attenuated canker sore gene-deficient strain has also decreased virulence compared with its parental vaccine strain, proving that the ORF130 gene is a virulence gene. There is no difference in safety compared with the canker sore vaccine strain attenuated by artificial passage; after immunizing rabbits with this attenuated strain, tracking the changes in its antibodies is consistent with the vaccine strain. The deletion strain introduces the reporter gene EGFP in the ORF130 deficiency region, which can be visualized during virus screening and purification. At the same time, during the vaccine immunization process, the specific markers in this region can be used as an effective target to distinguish vaccine immunity from wild virus infection. Therefore, this gene-deficient strain is suitable as a candidate strain for canker sore gene-deficient vaccine.

Claims

1. The use of preparing an attenuated oropharyngeal canker sore virus strain by inhibiting the expression of ORF130 gene, deleting or conditionally knocking out ORF130 gene in oropharyngeal canker sore virus, characterized in that: The ORF130 gene sequence is shown in SEQ ID NO.1, and the sheep oral sore virus is the sheep infectious pustular virus cell-passaged attenuated OrfVirus HB-TS09F65 strain, which was deposited in the China Center for Type Culture Collection on March 19, 2014, with a deposit number of CCTCC NO.V201406.

2. The use of an attenuated oropharyngeal canker sore virus vaccine prepared by inhibiting the expression of ORF130 gene, deleting or conditionally knocking out the ORF130 gene in the oropharyngeal canker sore virus, characterized in that: The ORF130 gene sequence is shown in SEQ ID NO.1, and the sheep oral sore virus is the sheep infectious pustular virus cell-passaged attenuated OrfVirus HB-TS09F65 strain, which was deposited in the China Center for Type Culture Collection on March 19, 2014, with a deposit number of CCTCC NO.V201406.

3. An attenuated sheep sore virus strain, characterized in that The attenuated oropharyngeal canker virus strain includes an oropharyngeal canker virus strain with ORF130 gene expression inhibition, deletion or conditional knockout. The oropharyngeal canker virus is an OrfVirus HB-TS09F65 strain attenuated by cell passage of sheep infectious pustular virus, which was deposited in the China Center for Type Culture Collection on March 19, 2014, with a deposit number of CCTCC NO.V201406.

4. The attenuated aphthous stomatitis virus strain according to claim 3, characterized in that The attenuated oropharyngeal canker virus strain is an oropharyngeal canker virus strain with conditional knockout of ORF130 gene, and the oropharyngeal canker virus is an OrfVirus HB-TS09F65 strain attenuated by cell passage of sheep infectious pustular virus, which was deposited in China Center for Type Culture Collection on March 19, 2014, with a deposit number of CCTCC NO.V201406.

5. A method for preparing the attenuated aphthous stomatitis virus strain according to claim 3, characterized in that: The method is to conditionally knock out ORF130 of the attenuated OrfVirus HB-TS09F65 strain caused by cell passage of sheep infectious pustular virus by genetic engineering means. The ORF130 gene sequence is shown in SEQ ID NO.

1. The attenuated OrfVirus HB-TS09F65 strain caused by cell passage of sheep infectious pustular virus was deposited in the China Center for Type Culture Collection on March 19, 2014, and the deposit number is CCTCC NO.V201406.

6. The preparation method according to claim 5, characterized in that: The method comprises the following steps: (1) designing and synthesizing a gene-deleted recombinant plasmid with the reporter gene being EGFP: pUC-130L-EGFP-130R; (2) The designed and synthesized gene-deficient recombinant plasmid: pUC-130L-EGFP-130R was transfected into BT cells inoculated with ORFV HB-TS09F65 strain virus by liposome transfection; (3) Through fluorescent plaque combined with fluorescent single cell screening, a monoclonal gene-deficient virus with a green fluorescent reporter gene was screened. After multiple rounds of screening and PCR identification, a recombinant virus strain lacking the ORF130 gene was obtained: ORFV-HB-TS09F65△ORF130.

7. Use of the attenuated aphthous stomatitis virus strain according to claim 3 or 4 in the preparation of an attenuated aphthous stomatitis virus vaccine.

8. An attenuated vaccine for oropharyngeal canker sore virus, characterized in that: The attenuated oropharyngeal sore virus vaccine comprises the attenuated oropharyngeal sore virus strain according to claim 3 or 4.

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