Porcine pseudorabies virus strain, construction method of reverse genetic manipulation platform of porcine pseudorabies virus strain, recombinant porcine pseudorabies virus and application

Abstract

The invention discloses a porcine pseudorabies virus strain, a construction method of a reverse genetic manipulation platform of the porcine pseudorabies virus strain, a recombinant porcine pseudorabies virus and application of the recombinant porcine pseudorabies virus, the porcine pseudorabies virus strain is a porcine pseudorabies virus PRV-HL-2021 strain, the preservation number of the porcine pseudorabies virus strain is CGMCC No.46094, and the porcine pseudorabies virus strain is preserved in China General Microbiological Culture Collection Center on July 29, 2024. The invention provides a porcine pseudorabies virus strain, and constructs a college reverse genetic manipulation platform for the porcine pseudorabies virus strain, so that the porcine pseudorabies virus gene can be quickly and accurately modified, the function of the porcine pseudorabies virus gene can be researched, and a new live virus vector is developed for the research of recombinant vaccines.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for constructing a porcine pseudorabies virus strain and a reverse genetic operation platform thereof, a recombinant porcine pseudorabies virus and applications thereof. Background Art

[0002] Pseudorabies in pigs is an acute infectious disease with a high mortality rate caused by pseudorabies virus (PRV). Pseudorabies virus belongs to the Herpesviridae family and the genus Porcine Herpesvirus. Pseudorabies virus can infect a variety of hosts, but pigs are the main natural host, reservoir and transmitter of the virus. Pseudorabies virus in pigs can infect pigs of different ages, but pregnant sows and suckling piglets are the most seriously infected: causing abortion, stillbirth and mummification in pregnant sows; suckling piglets develop neurological symptoms, paralysis, exhaustion and death, with a mortality rate of almost 100%.

[0003] PRV is a member of the alphaherpesviridae family and the genus Varicellavirus. Its genome is a linear double-stranded DNA composed of a unique long segment (UL region), a unique short segment (US region), an internal repeat sequence (IR), and a terminal inverted repeat sequence (TR). The PRV genome contains about 70 to 100 protein-coding genes, among which genes such as gE, gI, gC, TK, and PK are related to PRV virulence and are non-essential genes for PRV proliferation. gE and gI are related to PRV neurotropism and spread in the nervous system, and exert their effects through the formation of heterodimers.

[0004] Since the end of the 20th century, my country's pig farms have started to use pseudorabies gene-deficient vaccines, and the incidence of pseudorabies in pigs has dropped significantly. However, since 2011, many large-scale pig farms that use gE gene-deficient live vaccines for immunization have experienced sows giving birth to weak piglets, stillbirths, abortions, and piglets showing neurological symptoms and death, which are suspected clinical symptoms of pseudorabies and have caused serious economic losses. Relevant researchers have isolated and identified PRV wild virus, analyzed gE gene sequences, and conducted serum neutralization tests on clinical samples from immunized pig farms in many provinces. The preliminary results show that the newly isolated PRV epidemic strain has significantly mutated compared with previous strains, and its pathogenicity to mice and pigs has been significantly enhanced; the diseased piglets have itching symptoms and their tropism for visceral tissues has increased. Therefore, it is very necessary to develop a vaccine against this re-emerging epidemic strain. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide a porcine pseudorabies virus strain and to construct an advanced reverse genetic operation platform for the virus strain, so as to quickly and accurately modify the porcine pseudorabies virus gene and study its function, and then develop a new live virus vector for the research of recombinant vaccines.

[0006] In order to achieve the above-mentioned purpose, the present invention provides a porcine pseudorabies virus strain, which is the porcine pseudorabies virus PRV-HL-2021 strain, whose preservation number is CGMCC No.46094, and it was preserved in the General Microbiology Center of China National Microbiological Culture Collection Administration on July 29, 2024.

[0007] The present invention also provides a method for constructing a reverse genetic operation platform of the porcine pseudorabies virus strain as described above, and the method for constructing the reverse genetic operation platform specifically comprises:

[0008] S100, extracting the genomic DNA of the porcine pseudorabies virus PRV-HL-2021 strain;

[0009] S200, randomly shearing the genomic DNA obtained in step S100 into multiple fragments (each fragment is 25-45 kb in length, preferably 30-45 kb) by physical methods, connecting the sheared fragments to the pCC1FOSTM vector to construct a PRV Fosmid library composed of multiple cosmids, and each cosmid-connected PRV fragment is unique;

[0010] S300, randomly selecting multiple groups of cosmid combinations that can cover the whole genome of the pseudorabies virus PRV-HL-2021 strain from the PRV Fosmid library constructed in step S200;

[0011] S400, constructing a reverse genetics operation platform for porcine pseudorabies virus using the randomly obtained cosmid combination in step S300.

[0012] The present invention also provides a recombinant porcine pseudorabies virus rescued based on the reverse genetic operating system described above.

[0013] Preferably, the recombinant porcine pseudorabies virus is a recombinant porcine pseudorabies virus expressing green fluorescent protein.

[0014] Preferably, the recombinant porcine pseudorabies virus is a gene-deficient recombinant porcine pseudorabies virus.

[0015] Preferably, the deleted gene is selected from at least one of the US9 gene, the gI gene and the gE gene.

[0016] Furthermore, the deleted genes are US9 gene, gI gene and gE gene. That is, the recombinant porcine pseudorabies virus here is a three-gene deleted porcine pseudorabies virus.

[0017] The present invention also provides a use of the recombinant porcine pseudorabies virus as described above as a porcine pseudorabies virus reporter virus or in the preparation of a vaccine or reagent for preventing, treating or diagnosing porcine pseudorabies virus.

[0018] Through the above technical scheme, the present invention constructed a Fosmid library of the pseudorabies virus PRV-HL-2021 genome of porcine pseudorabies virus, from which a combination of cosmids that can cover the complete porcine pseudorabies virus genome and have overlapping regions were screened, and a candidate vaccine strain with a three-gene deletion of pseudorabies virus gE / gI / US9 was constructed using the cosmid combination that met the above screening. There was no difference in titer and growth characteristics between the reporter virus and the parent virus.

[0019] The present invention divides a huge genome into several small fragments to construct a Fosmid library, thereby improving the stability of the library and making the transformation and modification of the pseudorabies virus gene more convenient, with simple operation steps and shortening the library construction cycle.

[0020] The present invention provides an efficient method for rapidly and accurately modifying the pseudorabies virus genome, and has broad application prospects in the field of research and development of live vector vaccines for porcine pseudorabies virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0022] Figure 1 is a carbohydrate gel electrophoresis diagram of the amplified products of the gE and gB genes of the PRV-Bartha-K6 vaccine strain and the PRV-HL-2021 strain in Detection Example 1 of the present invention;

[0023] Figure 2A is the position of the preferred cosmid in Example 2 of the present invention in the whole genome of PRV;

[0024] Figure 2B This is a result diagram of multiple groups of cosmids after transfection in Example 2 of the present invention;

[0025] Figure 3 This is an IFA result diagram of the virus rePRV-PRV-HL-2021 in Detection Example 2 of the present invention; wherein, the first row marked as rPRV refers to the virus rePRV-PRV-HL-2021, the second row marked as PRV refers to the virus PRV-HL-2021, and the third row is a negative control;

[0026] Figure 4 is a sugar gel electrophoresis diagram of the amplified products of gE and gB genes of multiple virus strains rePRV-PRV-HL-2021 rescued in Detection Example 2 of the present invention; wherein, Figure 4In the upper and lower figures, the bands from left to right represent: molecular weight marker, rePRV-PRV-HL-2021 rescued by the cosmid combination grouped as 2 in Table 2, rePRV-PRV-HL-2021 rescued by the cosmid combination grouped as 4 in Table 2, rePRV-PRV-HL-2021 rescued by the cosmid combination grouped as 6 in Table 2, rePRV-PRV-HL-2021 rescued by the cosmid combination grouped as 7 in Table 2, rePRV-PRV-HL-2021 rescued by the cosmid combination grouped as 9 in Table 2, positive control group and negative control group;

[0027] Figure 5 It is the growth curve and virus plaque diagram of the rescued virus rePRV-PRV-HL-2021 and PRV-HL-2021 strain virus in detection example 2 of the present invention; wherein PRV refers to the virus PRV-HL-2021, Res-PRV refers to the virus rePRV-PRV-HL-2021, and Figure 5 A is the growth curve. Figure 5 B is a viral plaque image;

[0028] Figure 6 is a diagram of the construction strategy of the first mutant cosmid in Example 3 of the present invention;

[0029] Figure 7 3 is a graph showing the expression of green fluorescent protein after multiple generations of the rescued virus rPRV-US9-EGFP in Detection Example 3 of the present invention; wherein, from left to right, they are respectively passage 1, passage 3, passage 5, passage 7, and passage 10;

[0030] Figure 8 The growth curve and virus plaque diagram of the rescued virus rPRV-US9-EGFP and rPRV virus in detection example 3 of the present invention; wherein rPRV refers to the virus rePRV-PRV-HL-2021, Res-EGFP refers to the virus rPRV-US9-EGFP, and Figure 8 A is the growth curve. Figure 8 B is a viral plaque image;

[0031] Fig. 9 is a diagram of the construction strategy of the second mutant cosmid in Example 4 of the present invention;

[0032] Fig.10 It is a result diagram of the gene-deficient rescue virus rPRV-delgI / gE / US9 transfection in the detection example 4 of the present invention, and a sugar gel electrophoresis diagram of the amplified products of the gE and gB genes; wherein, Fig.10 A is the transfection result diagram. Fig.10B is the sugar gel electrophoresis diagram of the amplified products of gE and gB genes, rPRV-delgI / gE is the gene-deficient rescue virus rPRV-delgI / gE / US9, PRV is the virus PRV-HL-2021, NC or Mock is the negative control, and the band Marker is the molecular weight marker. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0034] In the present invention, the diseased piglets used are from a diseased pig farm in Heilongjiang. The diseased piglets show the following clinical symptoms: elevated body temperature, mental fatigue, severe emaciation, uncoordinated movement, shivering, convulsions, and the like.

[0035] The mice used were 4-6 weeks old and purchased from Jiangsu Huachuang Xinnuo Co., Ltd.

[0036] Porcine kidney cells (PK15 cells) and African green monkey kidney cells (VERO cells) are both conventional commercial products purchased from Shanghai Beiweimin Technology Co., Ltd. and are stored by culturing in DMEM medium (Gibco) containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Escherichia coli DH10B competent cells are conventional commercial products purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0037] DMEM was purchased from GIBCO; DL2000 DNA Marker and PCR reagents were purchased from Bao Biotechnology (Dalian) Co., Ltd.; DNA extraction kit and gel recovery kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; the cell culture medium was DMEM containing 8% inactivated fetal bovine serum and 100 U (μg) / mL of penicillin and streptomycin; CopyControlFosmid LibraryProduction Kit (CCFOS110), pCC1FOSTMFosmid Vector, EPI300TM-T1R E.coliPlatingStrain, and packaging protein MaxplaxTMLambda Packaging Extracts were all purchased from Epicentre; low melting point agarose was purchased from Invitrogen (UltraPureTM LMP Agarose, 16520-050), all antibiotics and L-arabinose (Sigma, A-3256) were purchased from Sigma; plasmid miniprep kit was purchased from Epigenetics (ZR BACDNA Miniprep Kit, D4049); plasmid extraction kit was purchased from Qiagen (QIAfilter Plasmid MidiKit, 12243); X-tremeGENE HPDNA transfection reagent was purchased from Roche (Cat. No. 06366546001); pEGFP-N1 vector plasmid was purchased from Clontech, and pMD18-T Simple Vector was purchased from TaKaRa; FITC-labeled goat anti-swine IgG fluorescent secondary antibody was purchased from Sigma; antibiotics and L-arabinose (Sigma A-3256) were purchased from Sigma; other reagents used in the experiments were all commercially available imported or domestic analytical grade reagents.

[0038] Example 1. Isolation and identification of pseudorabies virus PRV-HL-2021 strain

[0039] The diseased piglets were autopsied and the brain tissue of the pigs was taken as the diseased material. The diseased material was cut into pieces and ground thoroughly in a mortar. A certain amount of PBS was added and the sample was repeatedly frozen and thawed three times. The sample was centrifuged at 4000r / min for 15min, and the supernatant was taken. After sterilization, the filtrate was obtained by filtering through a 0.22μm filter membrane. The filtrate was stored below -20℃ or immediately inoculated with cells for toxin separation. After the DNA in the filtrate was extracted, PCR analysis was performed.

[0040] The specific virus separation operation process is as follows: VERO cells that have grown into a good monolayer (it should be noted that in actual operation, the VERO cells here can also be replaced by other cells that can be routinely used by those skilled in the art, for example, PK15 cells can be selected), the culture medium is discarded, and then the filtrate after grinding, freezing and thawing, and filtration and sterilization is inoculated into the cultured VERO cell monolayer, adsorbed at 37°C for 1 hour, the adsorbed filtrate is discarded, and then the cell maintenance solution (i.e., the cell culture solution with the content of inactivated fetal bovine serum adjusted to 2%, and the other components unchanged) is added, and the culture is placed in an incubator at 37°C and containing 5% CO2, and observed for 96 hours. If there is CPE, the virus is harvested after freezing and thawing 3 times in a -20°C refrigerator and a 37°C water bath; if there is no CPE, the virus is harvested after continuing blind transmission for 3 generations, and the virus is harvested after freezing and thawing 3 times in a -20°C refrigerator and a 37°C water bath.

[0041] The harvested virus was diluted with pure DMEM in a 10-fold gradient. -1 ~10 -8 Inoculate the suspension of each dilution into a 96-well microplate of VERO cells that have grown into a monolayer. Inoculate 8 wells for each dilution, and set up 8 wells without virus as a control. Culture in a 37°C, 5% CO2 incubator for 5 days. The cell morphology of the control well should be good. Take the diseased well with the highest dilution and collect the supernatant to obtain the first purified virus.

[0042] The virus solution of the highest dilution lesion well (i.e., the first purified virus obtained) was plaque purified. After 48 hours, a single plaque was marked under an inverted fluorescence microscope, and a single plaque was picked in a biosafety cabinet and placed in 1 mL of serum-free DMEM (i.e., pure DMEM), and frozen and thawed three times at -80°C. After gradient dilution, the virus was inoculated into a six-well plate grown into a monolayer of cells (i.e., VERO cells that have grown into a good monolayer) for the next round of plaque purification (it should be noted that, similarly, the virus solution used for the next round of plaque purification here is the virus solution in the highest dilution lesion well after this plaque purification). After four rounds of plaque purification, the viral genomic DNA was extracted, and PCR detection of gB and gE genes was further verified. In the same way, after seven rounds of plaque purification, a pure virus was obtained, named PRV-HL-2021.

[0043] The pure virus solution obtained after seven rounds of plaque purification was diluted 10-fold in a cell culture medium containing 2% newborn calf serum (the same as before, i.e., the inactivated fetal calf serum content was adjusted to 2%, and the other components were unchanged). -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10-8 Eight dilutions were inoculated into 96-well microplates of ST cells that had grown into a good monolayer (i.e., VERO cells that had grown into a good monolayer), and 8 wells were inoculated for each dilution, with 100 μL per well. Each well was supplemented with 100 μL of cell maintenance solution containing 2% newborn calf serum (same as before, i.e., cell culture solution with the content of inactivated fetal calf serum adjusted to 2%, and the other components unchanged). At the same time, 8 wells without inoculation were set up for control, cultured in an incubator containing 37°C and % CO2, and observed for 96 to 120 hours, and the number of wells with cytopathic effect (CPE) was recorded. TCID50 was calculated according to the Reed Muench method.

[0044] Example 2. Establishment of a reverse genetics platform for rapid rescue of recombinant pseudorabies virus PRV-HL-2021 strain

[0045] The pseudorabies virus genome was extracted according to the conventional pig extraction method in the art. Specifically, the virus (ie, PRV-HL-2021 purified in Example 1) was inoculated into PK-15 cells, and when 90% of the cells showed CPE, the cells were collected in a 50 mL centrifuge tube. Use 10 mL of LCM solution to lyse the cells. Add 1 mL of Freon (1,1,2-trifluorotrichloroethane), shake thoroughly, and release viral particles. Centrifuge in an ultracentrifuge for 2 hours, centrifuge at 4 ° C and 26000 r / min, and then add 10 mL of TEN and 500 μL of 10% SDS to release the viral genome. Use an equal volume of balanced phenol solution to extract once, and phenol: chloroform: isopropanol (25:24:1) solution to extract twice. Then use cold anhydrous ethanol to precipitate the DNA, and finally use deionized water to dissolve the precipitate to obtain the genomic DNA of the porcine pseudorabies virus PRV-HL-2021 strain of the present invention.

[0046] The genomic DNA extracted above was randomly sheared by physical method, and the DNA was end-filled according to the instructions of CopyControlFosmidLibrary Production Kit, and low melting point agarose gel pulse electrophoresis was performed, and then the DNA region between 36 and 48 kb (i.e., the target length) was cut and the DNA was recovered. 0.25 μg of the recovered purified DNA was connected to pCC1FOSTM to obtain multiple recombinant plasmids (wherein each DNA fragment reaching the target length was connected to the pCC1FOSTM vector to form the recombinant plasmid corresponding to the DNA fragment), and then packaged and transduced into EPI300 host cells, and the transduction product was evenly coated on an LB plate containing 12.5 μg / mL chloramphenicol and cultured at 37°C overnight. 50 monoclones were randomly selected, the cosmids were extracted, and the end sequencing was performed for identification. The sequence of the correctly sequenced cosmids was spliced ​​to obtain a Fosmid library covering the full length of PRV.

[0047] After terminal sequencing analysis, a total of 200 cosmids with inserted exogenous fragments were obtained. From these 200 clones, 19 cosmids with genome insertion fragment sizes of 30-45kb were screened for subsequent construction of cosmid groups. Random selection and combination were performed from the above-screened 19 cosmids, and finally 12 groups of cosmid groups were randomly selected for rescuing the virus. The randomly selected multiple groups of cosmid groups were all different from each other, and each had its own uniqueness. The two ends of the PRV DNA fragments inserted on multiple cosmids in each group of cosmid groups can overlap with each other (that is, the multiple cosmids in each group of cosmid groups are arranged in sequence according to the order of the DNA inserted thereon in the whole genome gene sequence, and the DNA fragments on two adjacent cosmids partially overlap), and, among the cosmids obtained based on the present invention, a cosmid group consisting of no less than 5 cosmids can cover the entire PRV genome (wherein, although 19 cosmids were screened in the present invention, 12 more suitable cosmids were finally screened for construction of the cosmid group, and the DNA fragments contained therein are specifically shown in Table 1, and their positions in the entire PRV genome are as follows. Figure 2A As shown). The above 12 cosmids were used to construct 12 cosmid groups, and the 12 combinations of cosmids were transfected into Vero cells respectively. The time when typical CPE appeared after transfection was observed respectively, and the toxicity of the rescued virus was detected, and then the best combination of rescued virus was screened out. The best combination results are shown in Table 2 (wherein the letter a in the cosmid combination corresponds to PRV-IF-a in Table 1, and the letter b corresponds to PRV-IF-b in Table 1... and so on. Further, a+c+e+j+k is the cosmid group composed of PRV-IF-a, PRV-IF-c, PRV-IF-e, PRV-IF-j and PRV-IF-k in Table 1, and other cosmid combinations are similar; at the same time, among the 10 cosmid groups shown in Table 1, 8 cosmid groups showed CPE within 48 hours after transfection, and their transfection results are shown in the figure below). Figure 2B As shown). According to the instructions of X-tremeGENE HPDNA transfection reagent, the purified cosmids were transfected into Vero cells in a 10 cm cell culture plate with a density of 90% according to the corresponding cosmid combination. The transfection dose of each cosmid in each cosmid group was 2 μg. After CPE, the cells were repeatedly frozen and thawed, the virus was harvested, and the rescued virus was named rePRV-PRV-HL-2021.

[0048] Table 1

[0049]

[0050] Table 2

[0051] Grouping Clay Combination CPE occurrence time (hpt) 1 a+c+e+j+k 36 2 a+c+f+j+k 24 3 b+d+f+g+h+i 48 4 b+d+h+i+k 36 5 b+d+h+i+j+k 54 6 a+c+e+j+l 36 7 a+c+f+j+l 36 8 b+d+f+g+h+l 54 9 b+d+h+i+l 36 10 b+d+h+i+j+l 36

[0052] Example 3: Construction of recombinant porcine pseudorabies virus expressing green fluorescent protein

[0053] Based on the cosmid combination rescue system constructed in Example 2 (i.e., the rescue of the virus based on the reverse operation platform of the cosmid group of not less than 5 cosmids per group) (specifically, Group 2 in Table 2 is selected here), the EGFP gene is inserted into the cosmid PRV-IF-k (the exogenous DNA fragment inserted into the cosmid is the base fragment at the 100865-end of the whole genome sequence of the PRV-HL-2021 strain in the present invention) after the termination codon of the US9 gene to construct the first mutant cosmid. Specifically, the construction method of the first mutant cosmid here is as follows:

[0054] First, according to the full-length genome sequence of PRV-HL-2021 strain and the EGFP gene sequence, the first primer pair as shown in SEQ ID No: 1 and SEQ ID No: 2 and the second primer pair as shown in SEQ ID No: 3 and SEQ ID No: 4 (primer 1 and primer 2 in Table 3) were designed. Using the Red / ET recombination technology, a recombinant virus expressing EGFP was constructed by two-step recombination. The specific operations are as follows: 1) The expression cassette of the selection marker gene rpsL-neo with 50bp left and right homologous arms at both ends was inserted after the stop codon of the US9 gene of the corresponding cosmid; 2) The expression cassette of the selection marker gene rpsL-neo was replaced with the EGFP gene with the same 50bp left and right homologous arms at both ends; 3) The positive clones were screened on the LB plate containing double resistance to chloramphenicol and streptomycin, and the cosmids were further identified by reverse screening of kanamycin resistance and PCR. The specific construction strategy is as follows Figure 6 shown.

[0055] The cosmids were extracted using the Qiagen medium extraction kit. According to the instructions of the X-tremeGENE HP DNA transfection reagent, the first mutant cosmid constructed above and the other four cosmids (PRV-IF-a, PRV-IF-c, PRV-IF-f, PRV-IF-j in Table 1) were co-transfected into Vero cells in a cell culture plate with a density of 90% and a diameter of 10 cm, and the transfection dose of each cosmid was 2 μg. The positive control group was a cosmid combination transfection formed by the unmodified 5 cosmids (i.e., the first mutant cosmid used the original cosmid PRV-IF-k without the EGFP gene inserted, and the other four cosmids remained unchanged), which could produce CPE; the negative control group only transfected 4 of the cosmids (i.e., the first mutant cosmid was not used, and only the other four cosmids were used for transfection), and CPE could not be produced. After the cells are transfected with the cosmid combination including the first mutant cosmid and the other four cosmids constructed by the present invention and the pathological changes are observed, the mixture of cells and culture supernatant is harvested and repeatedly frozen and thawed to obtain the virus rescued by the cosmid combination based on the first mutant cosmid, and the obtained rescued virus is named rPRV-US9-EGFP.

[0056] Example 4. Construction of gene-deficient recombinant porcine pseudorabies virus (knockout of US9, gI, gE)

[0057] Based on the cosmid combination rescue system constructed in Example 2 (i.e., the rescue of the virus based on the reverse operation platform of the cosmid group with no less than 5 cosmids per group), the US9, gI, and gE genes were knocked out from the cosmid to construct the second mutant cosmid. Specifically, the construction method of the second mutant cosmid here is as follows:

[0058] First, according to the genome sequence, Fosmid sequence and US2 and gI gene sequences of PRV-HL-2021 strain, the third primer pair shown in SEQ ID No: 5 and SEQ ID No: 6 and the first primer pair shown in SEQ ID No: 7 and SEQ ID No: 8 (primer 3 and primer 4 in Table 3) were designed. Using Red / ET recombination technology, a recombinant virus with US9, gI and gE genes knocked out was constructed through two-step recombination. The specific operations are as follows: 1) Insert the rpsL-neo expression cassette of the selection marker gene with 50bp left and right homologous arms at both ends into the corresponding position of the cosmid (PRV-IF-k); 2) Replace the rpsL-neo expression cassette of the selection marker gene with part of the US2 and gI genes with the same 50bp left and right homologous arms at both ends; 3) Screen positive clones on an LB plate containing dual resistance to chloramphenicol and streptomycin, and further identify the cosmid by reverse screening of kanamycin resistance and PCR. The specific construction strategy is as follows. Fig. 9 shown.

[0059] The cosmids were extracted using the Qiagen midi extraction kit. According to the instructions of the X-tremeGENE HP DNA transfection reagent, the above-mentioned modified cosmids (the cosmids with US9, gI, and gE genes knocked out) and the other four cosmids (PRV-IF-a, PRV-IF-c, PRV-IF-f, and PRV-IF-j in Table 1, and similarly, in this embodiment, the cosmid group used here is the group numbered 2 in Table 2) were co-transfected into Vero cells in a cell culture plate with a density of 90% and a diameter of 10 cm, and the transfection dose of each cosmid was 2 μg. The positive control group was transfected with a combination of 5 unmodified cosmids (i.e., PRV-IF-a, PRV-IF-c, PRV-IF-f, PRV-IF-j, and PRV-IF-k), which could produce CPE; the negative control group was transfected with only 4 of the cosmids (i.e., PRV-IF-a, PRV-IF-c, PRV-IF-f, and PRV-IF-j), which could not produce CPE. After the cell pathological changes after transfection with the cosmid combination comprising the second mutant cosmid and the other four cosmids constructed by the present invention, the mixture of cells and the culture supernatant was harvested and repeatedly frozen and thawed to obtain a virus rescued by the cosmid combination based on the second mutant cosmid, and the rescued virus obtained was named rPRV-delgI / gE / US9 (gE, gI and US9 triple gene deletion strain).

[0060] Table 3

[0061]

[0062]

[0063] Test Example 1

[0064] After the filtrate after grinding, freeze-thawing and filtration sterilization in Example 1 was inoculated into Vero cells and blindly propagated for 3 generations, the Vero cells showed typical cytopathic effects, manifested by cell rounding and shedding, while the control cells (i.e., VERO cells that had grown into a good monolayer) still maintained a complete monolayer.

[0065] The virus isolated in Example 1 (i.e., the first purified virus) was cloned and purified for three generations by limiting dilution method (specifically, the pure virus after seven rounds of plaque purification was inoculated and blindly propagated for three generations), and the virus content reached 10 7.5 TCID50 / mL.

[0066] After DNA was extracted from PRV-HL-2021 obtained in Example 1, PCR amplification was performed using the fifth primer pair shown in SEQ ID No: 9 and SEQ ID No: 10 and the sixth primer pair shown in SEQ ID No: 11 and SEQ ID No: 12 (Table 3, primers 5 and 6), and the amplified products were sent to a gene sequencing company for sequencing. The amplification results are shown in FIG. Figure 1 As shown, the amplification results show that two specific bands of 325bp (gB) and 198bp (gE) were amplified. This proves that the PRV-HL-2021 of the present invention contains gE and gB genes and is a currently popular wild strain. Figure 1 A is the existing PRV-Bartha-K61 vaccine strain, Figure 1 B is the porcine pseudorabies virus PRV-HL-2021 strain of the present invention.

[0067] The porcine pseudorabies virus PRV-HL-2021 strain obtained by the present invention was diluted in a gradient manner using TCID50 as a reference to obtain TCID50 values ​​of 10 5 , 10 4 , 10 3 , 10 2 , 10 1 Five dilutions of 50 μg / mL were used, and DMEM was used as a negative control. There were six groups of experiments in total. Five mice aged 4 to 6 weeks were taken from each group of experiments. Each mouse was intraperitoneally inoculated with 0.2 mL of virus isolation culture. After inoculation, the mice's mental state, diet, and clinical manifestations were observed. The results are shown in Table 4. After the isolated virus was inoculated into mice, the mice died, indicating that the isolated virus was highly toxic.

[0068] In summary, the present invention isolated and obtained the porcine pseudorabies virus PRV-HL-2021 strain, and confirmed that it was highly toxic through virus isolation, cell culture, toxicity determination, virus morphological observation, PCR identification, sequence analysis, animal regression test and other methods. It was further confirmed that the porcine pseudorabies virus PRV-HL-2021 strain isolated and obtained by the present invention had a high toxicity, and the animal regression test showed that it was a highly toxic strain.

[0069] Table 4

[0070]

[0071] Test Example 2

[0072] The sequencing results of the cosmids extracted and terminal sequenced in Example 2 showed that the 200 cosmids covered the entire PRV genome, but there were relatively more cosmids containing UL region fragments, indicating that the constructed Fosmid library had a certain bias.

[0073] The virus rePRV-PRV-HL-2021 rescued in Example 2 was repeatedly frozen and thawed, and the supernatant was inoculated into PK-15 cells, which produced typical CPE, indicating that the virus was successfully rescued.

[0074] The virus rePRV-PRV-HL-2021 rescued in Example 2 was inoculated into PK-15 cells at a density of 90%, and an IFA experiment was performed 48 hours later using an anti-PRV polyclonal antibody. The specific operation is as follows: discard the culture medium, wash the cells twice with PBS, and then fix the cells with cold anhydrous ethanol. After 30 minutes, add mouse anti-PRV antibody, and after 2 hours at room temperature, wash the cells 5 times with PBS, and add 100-fold diluted FITC-labeled goat anti-mouse IgG, and incubate at room temperature in the dark for 1 hour, wash the cells 5 times with PBS, 5 minutes each time, and observe and take pictures under an inverted fluorescence microscope. Figure 3 As shown, the IFA results showed that both the rescued virus and the parental virus could react with PRV-positive sera.

[0075] The virus rePRV-PRV-HL-2021 rescued in Example 2 was repeatedly frozen and thawed at -80°C / 37°C, and then continuously propagated on PK-15 cells for 10 generations. The DNA of the 10th generation virus of each strain was extracted, and the gB and gE fragments were amplified using the fifth and sixth primer pairs as described above, and the amplified products were sequenced and analyzed. Figure 4 As shown, each strain can detect PRV gE and gB genes.

[0076] The virus rePRV-PRV-HL-2021 rescued in Example 2 was inoculated into PK-15 cells grown to a monolayer at a multiplicity of infection (MOI) of 10, and the virus was harvested at 0h, 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 48h, 60h and 72h after infection. The virus titers harvested at different time points were determined by IFA, and the growth curve of the virus was plotted. Figure 5 As shown, there is no obvious difference in the growth curve of the rescued virus rePRV-PRV-HL-2021 and the PRV-HL-2021 strain virus.

[0077] Test Example 3

[0078] The transfection results of Example 3 show that the rescue virus rPRV-US9-EGFP expressing green fluorescent protein constructed in Example 3 can produce CPE. After the rescue virus rPRV-US9-EGFP was propagated for 10 generations, the EGFP gene was still stably expressed. Figure 7 shown.

[0079] The virus rPRV-US9-EGFP rescued in Example 3 was inoculated into PK-15 cells grown to a monolayer at a multiplicity of infection (MOI) of 10, and the virus was harvested at 0h, 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 48h, 60h and 72h after infection. The virus titers harvested at different time points were determined by IFA, and the growth curve of the virus was plotted. Figure 8 As shown, there is no obvious difference in the growth curves of the rescued virus rPRV-US9-EGFP and the PRV-HL-2021 strain virus, proving that the growth characteristics of the two are consistent.

[0080] Test Example 4

[0081] It can be seen from the transfection results of Example 4 that the gene-deficient rescue virus rPRV-delgI / gE / US9 constructed in Example 4 can produce CPE, the positive control group can also produce CPE, and the negative control group cannot produce CPE. Fig.10 As shown in A.

[0082] The virus rPRV-delgI / gE / US9 rescued in Example 4 was repeatedly frozen and thawed at 80°C / 37°C, and then continuously propagated on PK-15 cells for 10 generations. The DNA of the 20th generation virus of each strain was extracted, and the gB and gE fragments were amplified using the fifth and sixth primer pairs as described above, and the amplified fragments were sequenced and analyzed. It was able to effectively amplify gB, but not gE. The results are shown in Figure 2. Fig.10 As shown in B, it can detect the gB gene but cannot detect the gE gene.

[0083] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0084] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

[0085] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A porcine pseudorabies virus strain, characterized in that: The porcine pseudorabies virus strain is the porcine pseudorabies virus PRV-HL-2021 strain, whose preservation number is CGMCC No.46094, and it was deposited in the General Microbiology Center of China National Culture Collection Administration on July 29, 2024.

2. A method for constructing a reverse genetic operation platform for porcine pseudorabies virus strains as claimed in claim 1, characterized in that: The construction method of the reverse genetic operation platform comprises: S100, extracting the genomic DNA of the porcine pseudorabies virus PRV-HL-2021 strain; S200, constructing a corresponding PRV fosmid library consisting of a plurality of cosmids according to the genomic DNA obtained in step S100; S300, selecting multiple groups of cosmids that can cover the entire genome of the porcine pseudorabies virus PRV-HL-2021 strain from the PRV fosmid library constructed in step S200; S400, using the cosmid group obtained in step S300 to construct a reverse genetic operation platform for the pseudorabies virus strain.

3. The construction method according to claim 2, characterized in that: The length of the exogenous DNA fragment inserted into the cosmid in step S200 is 25-45 kb; The vector used for the cosmid in step S200 is the pCC1FOSTM vector.

4. A recombinant porcine pseudorabies virus rescued by the reverse genetic operating system according to claim 2 or 3.

5. The recombinant porcine pseudorabies virus according to claim 4, characterized in that: The recombinant porcine pseudorabies virus is a recombinant porcine pseudorabies virus expressing green fluorescent protein.

6. The recombinant porcine pseudorabies virus according to claim 4, characterized in that: The recombinant porcine pseudorabies virus is a gene-deficient recombinant porcine pseudorabies virus.

7. The recombinant porcine pseudorabies virus according to claim 6, characterized in that: The deleted gene is selected from at least one of the US9 gene, the gI gene and the gE gene.

8. Use of the recombinant porcine pseudorabies virus according to any one of claims 4 to 7 as a porcine pseudorabies virus reporter virus or in the preparation of a vaccine or reagent for the prevention, treatment or diagnosis of porcine pseudorabies virus.

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