CN114657151B - Porcine pseudorabies virus gE/gI/TK gene deletion vaccine strain, construction method and application thereof - Google Patents
Porcine pseudorabies virus gE/gI/TK gene deletion vaccine strain, construction method and application thereof Download PDFInfo
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- CN114657151B CN114657151B CN202210191045.6A CN202210191045A CN114657151B CN 114657151 B CN114657151 B CN 114657151B CN 202210191045 A CN202210191045 A CN 202210191045A CN 114657151 B CN114657151 B CN 114657151B
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Abstract
The invention discloses a porcine pseudorabies virus gE/gI/TK gene deletion vaccine strain, a construction method and application thereof, wherein the deletion strain is obtained by deleting gE, gI and TK genes through a genetic engineering method on the basis of porcine pseudorabies virus PRV GD0304; the preservation number of the gene deletion strain is CCTCC NO: V201955. Compared with classical Bartha K61 strain, the strain has better toxicity attack protection effect on variant strains, can effectively reduce morbidity and mortality, and has higher serum neutralization titer. The three-gene deletion strain has higher application and commercial value in prevention and control of pseudorabies variation strains, and is a vaccine strain hopefully applied to current PRV epidemic prevention and control work.
Description
Technical Field
The invention belongs to the field of viruses, and particularly relates to a porcine pseudorabies virus gE/gI/TK gene deletion vaccine strain, a construction method and application thereof.
Background
Pseudorabies virus belongs to alpha herpes virus, can infect various mammals and few birds, pigs are main hosts of the pseudorabies virus, and the pseudorabies virus can cause abortion and stillbirth of pregnant sows, sterility of boars, death of newborn piglets, dyspnea of fattening pigs, growth arrest and other pseudorabies symptoms, and is one of serious infectious diseases endangering the breeding industry. At present, the prevention and treatment of pseudorabies mainly depends on vaccines, wherein the most classical and the most widely used at home and abroad are K61 passage attenuated strains separated from Bartha of a hungarian in 1961, and the K61 strain is called Bartha for short. The vaccine based on the strain plays a very important role in prevention and control of pseudorabies, and part of countries and regions realize purification of pseudorabies, and the currently used pseudorabies vaccine in China is mainly derived from the strain. The Bartha K61 strain plays an important role in prevention and control of pseudorabies in China, and more than 80% of farmed pigs are immunized with the strain in 1990, so that the spread of pseudorabies in China is effectively controlled. However, in recent years, the domestically popular pseudorabies strain may have variation, resulting in failure of the Bartha K61 vaccine. From the aspect of gene sequence analysis, the global pseudorabies virus can be divided into two large branches, wherein a gene type 1 strain represented by Bartha strain is mainly distributed in Europe and America, and a domestic strain belongs to a gene type 2 and is positioned on the other large branch; domestic strains can be further divided into classical strain branches represented by Ea strains and variant strain branches appearing in recent years. From the gene analysis data, it can be seen that although pseudorabies virus belongs to one serotype, the pseudorabies strains at home and abroad have been differentiated in gene sequence, and the domestic strains are further differentiated into new and old branches, so that the immunogenicity of the domestic strains is possibly different from that of foreign strains represented by Bartha, and the situation that the vaccine protection of the Bartha strain is incomplete in recent years is explained.
On the other hand, in part of pig farms, in order to reduce the contact between personnel and pig groups and avoid the stimulation to pig groups, the use of vaccines is greatly reduced, which also results in further increase of the incidence of pseudorabies in these two years. The factors in the two aspects both provide higher requirements and challenges for the development work of pseudorabies vaccines, on one hand, vaccine strains are required to provide efficient protection against domestic epidemic variant strains, and in order to solve the problem, domestic epidemic PRV variant wild strains with representativeness and stronger virulence are required to be screened out as vaccine candidate strains; on the other hand, a more stable, safe and reliable pseudorabies vaccine with higher immune efficiency and convenient use and small stimulation to a pig group is needed, and the method is realized by a safer and more reliable gene deletion vaccine construction and screening method and adjustment of immune dosage forms and immune modes.
In summary, the current trend of epidemic and purification of pseudorabies strains in China is that vaccines aiming at epidemic pseudorabies variant strains are very needed on one hand, and on the other hand, higher requirements are put forward on screening vaccine candidate strains and constructing gene deletion vaccines. The strain of the current domestic pseudorabies vaccine is mainly the traditional Bartha-K61 strain vaccine, and the safety and effectiveness of the new vaccine strain and the pertinence of whether the strain has a regional epidemic strain are not fully verified in the market.
Disclosure of Invention
The invention aims to provide a porcine pseudorabies virus gE/gI/TK gene deletion strain based on porcine pseudorabies virus PRV GD0304 and application thereof in preparing vaccines.
The technical scheme adopted by the invention is as follows:
the invention provides a porcine pseudorabies virus gE/gI/TK gene deletion strain, which is porcine pseudorabies virus PRV GD0304 with gE, gI and TK genes deleted; the method comprises the steps of carrying out a first treatment on the surface of the The preservation number of the PRV 0304 is CCTCC NO: V201946, and the preservation time is as follows: 2019.8.15, accession number: china center for type culture collection, preservation address: chinese university of Wuhan and Wuhan.
In some embodiments of the invention, the porcine pseudorabies virus gE/gI/TK gene deleted strain is designated: PRV GD0304 gE - gI - TK - The plant has a preservation number of CCTCC NO: V201955, and the preservation time is as follows: 2019.8.15, accession number: china center for type culture collection, preservation address: chinese university of Wuhan and Wuhan.
In a second aspect of the invention, there is provided a product for the treatment or prophylaxis of porcine pseudorabies comprising the gene-deleted strain of the first aspect of the invention or porcine pseudorabies PRV GD0304.
In some embodiments of the invention, the product further comprises a pharmaceutically acceptable adjuvant, carrier, diluent or excipient.
In some embodiments of the invention, the adjuvant is Gel 01PR, ISA201VG, or aluminum salt adjuvants, protein adjuvants, and nucleic acid adjuvants that can be used in veterinary vaccines.
In some embodiments of the invention, the vaccine is an inactivated vaccine or a live vaccine.
In some embodiments of the invention, the vaccine is provided in a physiologically administrable form and is suitable for oral, nasal drip, intramuscular, intravenous, subcutaneous or dermal injection applications.
In some embodiments of the invention, the porcine pseudorabies virus gE/gI/TK gene-deleted strain in the inactivated vaccine is inactivated by an inactivating agent such as formaldehyde or beta-propiolactone.
In a third aspect of the invention, there is provided an antibody prepared by using the porcine pseudorabies virus gE/gI/TK gene-deleted strain of the first aspect of the invention as an immunogen.
The invention also provides application of the kit in preparation of a diagnostic reagent for detecting porcine pseudorabies virus infection. In a third aspect, the invention provides an application of the porcine pseudorabies virus gE/gI/TK gene deletion strain or porcine pseudorabies PRV GD0304 in preparation of a medicament for preventing or treating porcine pseudorabies.
In a fourth aspect, the invention provides an application of the porcine pseudorabies virus gE/gI/TK gene deletion strain or porcine pseudorabies PRV GD0304 in preparation of a medicament for diagnosing or detecting porcine pseudorabies.
In a fifth aspect, the invention provides an application of the porcine pseudorabies virus gE/gI/TK gene deletion strain or the porcine pseudorabies PRV GD0304 in a porcine pseudorabies virus challenge disease model.
In a sixth aspect of the invention, there is provided a method of preparing a vaccine for preventing or treating porcine pseudorabies, comprising the steps of: preparing the porcine pseudorabies virus gE/gI/TK gene deletion strain virus liquid according to the first aspect of the invention, and then adding an adjuvant.
In some embodiments of the invention, the porcine pseudorabies virus gE/gI/TK gene deleted strain virus solution is inactivated after preparation.
The beneficial effects of the invention are as follows:
the invention screens out a pseudorabies mutant strain with strong toxicity, characteristics in genotype, serological level and the like and representativeness, is named as porcine pseudorabies PRV GD0304, and constructs gE of the strain by using a homologous recombination method based on the strain - /gI - /TK - Three-gene deletion strain named PRV GD0304 gE - /gI - /TK - As vaccine candidate strains. The immune toxicity attack tests of mice and piglets show that the inactivated or live virus vaccine prepared from the three-gene deletion pseudorabies mutant strain has better toxicity attack protection effect on the mutant strain compared with the classical Bartha K61 strain, can effectively reduce morbidity and mortality, and has higher serum neutralization titer. The three-gene deletion strain has higher application and commercial value in prevention and control of pseudorabies variation strains, and is a vaccine strain hopefully applied to current PRV epidemic prevention and control work.
Drawings
FIG. 1 is a technical route for screening epidemic variant strains of porcine pseudorabies virus.
FIG. 2 shows the case of GD0304 strain (P3) infected with Vero cell CPE.
FIG. 3 is an electron microscope image of PRVGD0304 viral particles.
FIG. 4 is a phylogenetic tree constructed from pseudorabies gene sequences in a database. FIG. 4A is a phylogenetic tree of 9 PRV viral genomes constructed using MP method; FIG. 4B is a phylogenetic tree constructed by using the NJ method for gB gene sequences; FIG. 4C is a phylogenetic tree constructed by using the NJ method for gC gene sequences.
FIG. 5 shows cytopathic effects of strain GD0304 pseudorabies virus infected Vero cells before, during and after gene knockout. FIG. 5a shows cytopathic effect of GD0304 wild strains at 200-fold after 36 hours of infection of Vero cells; FIG. 5b shows the expression of green fluorescent protein in Vero cells after 48 hours of infection by recombinant virus after replacement of the viral TK gene with the green fluorescent protein gene, 200-fold; FIG. 5c shows the expression of red fluorescent protein in cells 48 hours after infection of Vero cells with recombinant viruses after replacement of the viral gE gene and part of the gI gene by red fluorescent protein gene based on TK gene deletion, 100 times; FIG. 5d shows cytopathic effects of pseudorabies GD0304 TK, gE and partial gI deletion strain after removal of the red fluorescent protein gene for 72 hours in Vero cells, 200-fold.
FIG. 6 shows gel electrophoresis of PCR identified TK and gE genes. FIG. 6a shows PCR detection results of TK gene and gE gene of 6-generation virus after 6 times of continuous passage of strain on Vero cell after TK gene deletion, 1-6: 1 st to 6 th generation viruses, PC: positive control (wild virus genome), M: DL2000 Marker; FIG. 6b shows the gE gene test results of 6-generation virus after 6 serial passages of the strain on Vero cells after three gene deletions, 1-6: 1 st to 6 th generation viruses, PC: positive control (wild virus genome), M: DL2000 Marker.
Figure 7 shows a dead mouse and its profile. FIG. 7A is an abdominal grab trace of a dead mouse; FIG. 7B is a hepatomegaly; FIG. 7C is spleen atrophy hyperemia; fig. 7D is a renal punctate hemorrhage; FIG. 7E is pulmonary congestion; fig. 7F is meningeal hyperemia.
Fig. 8 shows pathological changes of different tissues of piglets after toxin challenge in each experimental group. Wherein the challenge strain in FIG. 8A is GD0304 strain (10 6 TCID 50 The vaccine is three-gene deletion inactivated virus and adjuvant 1; the challenge strain in FIG. 8B was GD0304 strain (10 6 TCID 50 The vaccine is three-gene deletion inactivated virus and adjuvant 2; the challenge strain in FIG. 8C was GD0304 strain (10 6 TCID 50 The type of the vaccine is commercial Bartha strain inactivated vaccine; FIG. 8D shows that the middle challenge strain is GD0304 strain (10 6 TCID 50 /ml), PBS treatment; the piglets in fig. 8E were not subjected to any treatment.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
Example 1 isolation and identification and virulence test of porcine pseudorabies virus epidemic variant strain (PRV 0304 strain)
The technical route is shown in figure 1.
1. GD0304 strain pseudorabies virus source
In 2017, 4 months, PRV suspected epidemic situation occurs in a pig farm of Guangdong river origin, tissues such as brain, spleen, lung, pulmonary lymph node and the like of dying pigs are collected for laboratory detection, PRV wild virus nucleic acid is positive, and porcine circovirus type 2 nucleic acid and porcine reproductive and respiratory syndrome nucleic acid positive are detected in disease materials; according to investigation, the pig farm is immunized according to a commercial PRV vaccine (Bartha K61 strain) immunization program, and the PRV gI antibody (IDEXX kit) positive rate is detected to be 87.7%; combining with various diagnostic indicators, it was concluded that PRV vaccines were not effective against this epidemic infection. Epidemiological surveys have found that pig farms in close contact with the pig farm are ubiquitous with varying degrees of PRV morbidity.
2. Isolation and identification and purification of pseudorabies virus
1) PRV rabbit regression
The tissue homogenate supernatant is injected into New Zealand white rabbits, and the rabbit infection is used for removing or reducing potential other pathogens in the tissue sample. Taking PRV positive brain tissue treatment fluid (centrifuging, sterilizing and diluting 10 times), subcutaneously inoculating New Zealand white rabbits (0.5 mL/rabbit) on the neck, and enabling the experimental rabbits to have neurological symptoms and dying for 48 hours after 16 hours; euthanasia is carried out on dying rabbits, brain groups are taken, PRV gE nucleic acid strong positive (Ct is less than or equal to 18) is detected by real-time fluorescence PCR (qPCR), and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), swine fever virus (CSFV), porcine circovirus type 2 (PCV 2) and Porcine Parvovirus (PPV) nucleic acids are not detected.
2) PRV separation and purification
PRV was purified using the susceptible cell "plaque assay" assay to remove potential PRV vaccine toxins. Inoculating the PRV positive tissue purified by rabbit body into Vero cellCCL-81 TM ) 6 rounds of 'plaque method' tests are carried out, each time, a single plaque is picked, the plaque with typical cytopathic morphology and relatively high pathological change speed is subjected to the next round of screening, after the Vero cells are re-inoculated to 10 virus plaques picked by the final round, the situation that the Vero cells are infected by GD0304 strain (P3) is shown in FIG. 2, and the plaques after 24 hours and 36 hours are respectively infected; qPCR identification is carried out on gD and gE genes for collecting viruses respectively, and the most rapid lesion speed and the highest virus content are selectedIs named PRV 0304 strain; after the virus is inoculated into Vero cells, CPE appears faster, the cytopathic morphology is typical, and the virus content in cell supernatant is higher; the virus particles are spherical, have a diameter of 100-150nm, are provided with a capsule membrane, and have radial fibers outside the capsule membrane, and are typical PRV virus morphology (figure 3) after the virus particles are subjected to amplification culture and purification by ultracentrifugation. The super-isolated and purified virus was subjected to NGS second generation sequencing, and finally spliced to obtain a 132307bp full-length virus sequence (NCBI sequence number: MH 582511). And the porcine pseudorabies virus strain is sent to a preservation institution for preservation, and is preserved in China Center for Type Culture Collection (CCTCC), named porcine pseudorabies virus PRV GD0304, and the preservation number is: CCTCC NO: V201946, the preservation time is: 2019.8.15.
the PRV 0304 plants are continuously passaged to 30 generations on Vero cells, and the 10, 20 and 30 generation viruses are subjected to specificity and purity test by referring to the pharmacopoeia of the animal of the people's republic of China, and the viruses are specific and have no bacterial, mould, mycoplasma and exogenous virus pollution.
3) PRV GD0304 Strain titer determination
The virus titer (TCID 50) of PRV GD0304 strain on Vero cells was determined using Reed-Muench as follows: p10 generation 10 -6.2 /mL, generation P20 10 -6.57 /mL, generation P30 10 -6.6 /mL. The PRV GD0304 strain low-generation secondary virus has slightly lower titer on Vero cells and is stable after 20 passages; compared with other PRV epidemic strains, the TCID50 of the PRV GD0304 strain is lower, but the infection speed of Vero cells is faster, and cytopathic effect is more obvious. Compared with Barth K61, at the same 1MOI infection dose, the GD0304 strain infected cells can observe obvious cytopathy about 5 hours after inoculation, and can observe the formation of virus plaque about 12 hours, and the infected cells can be completely disintegrated and fall off about 24-36 hours. The titer of PRV GD0304 strain is significantly lower than that of the domestic reported PRV epidemic strain (10) 8 ～10 9 TCID 50/mL), but the cytopathic rate is faster, potentially implying greater replication and infection capacity of the virus.
4) PRV GD0304 strain regression test
The culture supernatant of PRV GD0304 strain P30 generation Vero cell after plaque purification is 1×10 6 The TCID50/mL concentration nasal drip is used for inoculating 5 SPF piglets, clinical symptoms (41.4 ℃) of body temperature rise start to appear after 1 day of inoculation, nerve symptoms such as dyspnea, mania, systemic tremor, water-drawing exercise and the like start to appear after 4 days, and all the piglets die successively on the 5 th day (100% mortality rate), and the control group is normal. And after the dead piglet is dissected, taking tissues such as brain, spleen, lung, pulmonary lymph node and the like, carrying out PCR detection on PRV gE, gB, gC, gD and TK genes, and carrying out amplification sequencing, wherein the sequence is consistent with GD0304 strain. Regression experiments prove that the GD0304 strain can induce the susceptible animals to generate pseudorabies symptoms, and the strain sequence is stable.
5) PRV GD0304 shelf life test
The virus (10 th, 20 th and 30 th generation) stored at-80 ℃ is taken out periodically, and Vero cells without exogenous virus infection are inoculated. PRV virus titers were measured after 0, 12, 24, 36 months of storage, respectively. The results are shown in Table 1, and after the PRV GD0304 viruses of different generations are stored at-80 ℃ for 36 months, the virus titer on Vero cells is not changed obviously, which indicates that the strain can be stored stably and meets the storage requirement of vaccine strains.
TABLE 1 titre of 3 passages of subviruses on Vero cells at different shelf-lives (TCID 50 )
2. Whole genome sequencing and major virulence gene analysis
And (3) sequencing the porcine pseudorabies virus PRV GD0304 strain DNA obtained by separation, identification and purification through an Illumina platform, and then carrying out data assembly to finally obtain 1 whole genome sequence. The result shows that the total length of PRV GD0304 genome is 142307bp, the sequence length is closer to 142334bp representing domestic traditional pseudorabies strain Ea strain, and is shorter than 145312bp representing JS-2012 strain of domestic pseudorabies variation strain, the GC content is 73.6%, 69 open reading frames are encoded in total, and the sequence contains two repetitive sequence regions of IR and TR. The entire genome of GD0304 was subjected to evolution analysis with the entire genome of several other representative strains, such as Bartha strain representing foreign vaccine strain, ea strain representing domestic earlier wild strain, and JS-2012 strain representing domestic PRV variant strain, the analysis results being shown in fig. 4, wherein GD0304 strain has a relatively close evolutionary relationship with Ea strain and is relatively distant from JS-2012 strain representing variant strain. The evolutionary trees of the conserved genes gC and gB are similar to the whole genome evolutionary tree in the whole structure, and each strain of PRV can be divided into two obvious branches, european and American strains represented by Bartha strain independently belong to the same branch, and Chinese strains including PRV GD0304 strain belong to the other branch; further analysis of Chinese strains shows that PRV GD0304 strain has a recent evolution relationship with epidemic strains such as HB1201, heN1, JS2012 and the like separated from the strain in 2012 of China on the conservation gene, one relatively independent branch is formed in the strain in China, and Ea strain, fa strain, SC strain and the like representing the classical epidemic strain in China form another relatively independent branch, which is different from the analysis result of the whole genome. The sequence analysis result shows that GD0304 strain is more similar to the past domestic epidemic strains such as Ea strain on genome, but is more similar to the domestic new epidemic variant strain on the conservation gene sequence; this suggests that strain GD0304 may have a different origin than other domestic epidemic PRV variants (some studies have shown that domestic PRV variants may originate from Bartha strain, but that strain GD0304 is more closely related to Ea strain from the genome), but that genetic recombination may occur between epidemic strains during transmission. The complexity of the PRV epidemic strain in China is described, the GD0304 strain has very important research value in researching the origin and evolution relation of the PRV epidemic strain in China, and the uniqueness of the strain in the pseudorabies epidemic variation strain and the potential of the strain serving as a vaccine candidate strain are also embodied.
3. Virus toxicity test
3.1 virulence against mice
3.1.1 half lethal dose in mice
Female Balb/c mice of 5-6 weeks of age were randomly divided into 5 groups, 10/group. 1-4 groups each contained 10 in each case by subcutaneous injection into the neck and back of the patient 0.1mL 2 TCID50/mL，10 3 TCID50/mL，10 4 TCID50/mL，10 5 PRV GD0304 strain (experimental group) Vero cell culture virus liquid with TCID of 50/mL; group 5 nape of the neckA portion was subcutaneously injected with 0.1mL of DMEM medium as a negative control group. And continuously observing and recording clinical symptoms, death conditions and the like of the mice in 2 weeks after the virus attack, timely dissecting and taking tissues of the dead mice, carrying out PCR detection on PRV gD and gE genes, and fixing part of tissues for pathological sections.
Day 3-7, 10 after detoxification 5 TCID50/mL group and 10 4 The TCID50/mL group experimental mice have clinical symptoms of listlessness, appetite reduction and the like with different degrees, and the mice can also have continuous grasping and winding grafting parts, so that hair is shed, skin is bleeding and finally dies. 10 3 The TCID50/mL group started to develop mild clinical symptoms such as listlessness, appetite decrease and the like on day 4 after toxin challenge, and partial mice developed neurological symptoms such as grab winding and the like and died. After 7 days, the surviving mice in each group are basically recovered to normal, and no obvious clinical symptoms appear. The negative control mice appeared normal throughout the experimental period. The statistics show that the death time of mice is mainly concentrated on days 3-7 after toxin attack. 10 5 、10 4 、10 3 、10 2 The death number of the TCID50/mL challenge group mice is 10, 9, 5 and 1 respectively; according to the experimental result, the LD50 of PRV GD0304 strain to mice is calculated as follows: 10 3.2 TCID50/mL, LD50 against mice with classical virulent strain SC strain of China (10) 5.16 TCID 50/mL), the virulence of the strain to mice is obviously enhanced.
3.1.2 mice Cross immunoprotection test
40 female Balb/c mice of 6-8 weeks of age were randomly divided into 4 groups of 10 animals each, and immunized subcutaneously on the nuchal. Group 1 was immunized with 0.1mL of each of the Bartha-K61 strain formaldehyde inactivated virus containing 104TCID50, group 2 was immunized with each of the GD0304 strain formaldehyde inactivated virus containing 104TCID50, and groups 3 and 4 were inoculated with 0.1mL of DMEM medium as a negative control group and a blank control group, respectively; the same dose and method are used for boosting once every other week, after 3 times of immunization, groups 1, 2 and 3 respectively use 10LD50 GD0304 strain Vero cells to culture live virus cervical back subcutaneous challenge, and the clinical symptoms and death condition of the mice are observed for 14 days. The group 3 mice begin to have PR symptoms on the 3 rd day after the virus attack, the sick mice have the scratch phenomenon, so that local hair is shed, skin is bleeding, and serious PR symptoms are all generated within 4-7 days and die successively, and the survival rate is 0%; group 1 mice began to develop PR symptoms on day 4 of challenge and succumb to death on days 5-8, with a final survival rate of 20%; group 2 mice developed PR symptoms in part on day 4, developed small deaths on days 5-7, and a final survival of 50%; group 4 mice had no apparent symptoms during the observation period.
3.2 virulence against pigs
3.2.1 half lethal dose for piglets
Randomly dividing 20 healthy piglets of 15 days into 5 groups, each group of 5 piglets, and inoculating 2mL of the healthy piglets 10 by nasal drip respectively in the 1 st to 3 rd groups 6 TCID50/mL，10 5 TCID50/mL，10 4 TCID50/mL and 10 3 PRV GD0304 strain Vero cell culture virus liquid with TCID of 50/mL, and group 5 nasal drip inoculation of 2mL DMEM culture medium as a negative control group. Isolated feeding, free feeding, continuous observation for 14 days, recording of body temperature and clinical symptoms; and the dead pigs are cut in time, the PRV gD and gE genes are detected by PCR through the tissues, and part of tissues are fixed for pathological sections. Day 2, 10 after detoxification 6 The piglets in the TCID50/mL group have obvious temperature rise, reach the highest temperature on the 3 rd day, and have average temperature of: 41.4 ℃.
The four test groups of piglets with toxicity attack show clinical symptoms such as raised body temperature, anorexia, shortness of breath, somnolence and the like with different degrees after toxicity attack; 10 6 The TCID50/mL group piglets have dyspnea on the 4 th day after toxicity attack, are in a sitting posture, are manic, are continuously hit against a column with heads, have serious clinical symptoms such as whole body tremble, land pouring and water drawing in the later period, and die completely on the 5 th day, and have the mortality rate of 100%.10 5 TCID50/mL group, 10 4 TCID50/mL group and 10 3 The TCID50/mL group also showed similar clinical symptoms successively on day 4 after challenge, death began on day 5, and finally on day 14, 10 after challenge 5 TCID50/mL group all died, 10 4 TCID50/mL group remaining head, 10 3 The remaining 4 piglets of the TCID50/mL group did not die. The negative control group appeared normal throughout the experimental period. The half-lethal dose of PRV GD0304 strain to piglets is calculated as follows: 10 3.2 TCID50/mL。
The death of the virus-attack test group and the bleeding of brain tissues of piglets in the virus-attack group are found by piglets which are resistant to the death, the edema phenomenon is caused, and the cerebrospinal fluid is increased; lung edema, the appearance of substantial sarcoidosis; enlargement of the lymph nodes of the lung door, which appears as white punctate lesions; spleen congestion, infarct foci at the edge; the surface of the kidney can be seen to be in diffuse distribution with point bleeding with the size of the needle point; inguinal lymphadenectasis and congestion. The negative control group has no obvious pathological changes of the organs of the tissues of the piglets.
And (3) carrying out PCR detection on brain tissues of dead piglets, wherein PRV gE and gD genes are positive, and the sequencing result of the amplified fragment is consistent with GD0304 strain. The fluorescent PCR method is adopted for each pathological organ, the constructed pMD18-gD recombinant plasmid is used as a standard plasmid, a standard curve is drawn after gradient dilution, the virus copy number in each tissue is calculated, and the calculation result shows that the virus content in the brain of the dead piglet is highest and can reach 3.2x10 5 copies/g。
3.2.2 comparative test of neutralizing Capacity of pig immune serum
After 10 parts of Bartha-K61 are randomly selected for immunization, detecting pig serum positive for gB antibody and negative for gE antibody by an IDEXX kit, and respectively testing the neutralizing capacity of immune serum to wild viruses separated from Bartha-K61 strain, GD0304 strain and other Guangdong Guangxi regions.
The results show that the average neutralizing antibody titer of the Bartha-K61 immune serum to the Bartha-K61 strain is 1:57; the highest neutralizing antibody titer of GD0304 strain reaches 1:13, and the average is 1:18, and the two have obvious difference (p is less than 0.01); average neutralizing antibody titers for several other strains were 1: 20-1:40. The test result also shows that the Bartha-K61 immune serum has reduced neutralizing capacity for GD0304 strain and other wild strains, and the reason that pseudorabies still occurs in pig farms immunized with Bartha-K61 is explained.
The toxicity test on mice and piglets shows that the PRV GD0304 strain newly separated and identified in the invention is far more virulent than the classical PRV standard virulent strain (SC strain LD 50:10) 5.16 TCID 50/mL), the PRV wild toxin has obvious change in the domestic epidemic process, the cause of incomplete immunoprotection of the Bartha-K61 vaccine is explained, and meanwhile, the method is describedPotential of our isolated GD0304 strain as a candidate vaccine strain against the now domestic PRV epidemic strain.
EXAMPLE 2 construction of three Gene-deleted Strain
1. Purpose and technical route:
the most widely used Bartha-K61 strain in the current pseudorabies vaccine is naturally deleted gE and partial gI genes, other vaccine strains are mostly artificially deleted gE and gI genes, and partial vaccine strains can additionally delete TK genes for improving the safety. Among the three frequently deleted genes, the TK gene, namely the gene encoding the viral thymidine kinase, is mainly involved in the salvage synthesis way of nucleotides to maintain and promote virus replication, is one of the main virulence genes of pseudorabies virus, is related to latent infection of viruses and proliferation in the central nervous system, and the deletion of the TK gene can obviously reduce the infectivity of pseudorabies virus on nerve cells and the virulence of pigs, but the immunogenicity is not affected; the gE and gI genes are one of genes for encoding envelope proteins of viruses, are one of main virulence genes of pseudorabies viruses, are related to assembly and transportation of viruses in nerve cells, greatly reduce the virulence of the viruses after the gE and gI genes are deleted, and have no influence on immunogenicity.
The GD0304 strain has potential as an excellent vaccine candidate strain due to the specificity of the GD0304 strain in epidemic regions, virulence and genetic evolution. In order to test the immunity and safety of the strain as a vaccine strain, a TK/gE/gI three-gene deletion strain of GD0304 strain is constructed by adopting a method of artificial homologous recombination. Wherein the TK gene and the gE gene are completely deleted, and the gI gene is the first half part of the deletion gene imitating the Bartha-K61 strain.
In the embodiment, homologous arm amplification primers in table 2 are firstly used for respectively amplifying the GD0304 strain TK gene and upstream and downstream homologous sequences of gE and gI genes, and respectively enzyme-cutting and connecting the homologous sequences into expression vectors with green fluorescent protein (TK gene recombinant plasmid) and red fluorescent protein (gE and gI gene recombinant plasmid) to construct recombinant plasmids containing homologous arms on two sides of each gene and provided with fluorescent protein screening markers. Recombinant plasmids with green fluorescent protein and TK gene two-side homology arms are utilized to screen out GD0304 strain with green fluorescent marked TK gene deletion, howeverThen removing the inserted green fluorescent fragment by using a recombinant plasmid which does not contain fluorescent protein and only has homologous arms at two sides; the next step is to screen out the TK gene with red fluorescence and the GD0304 three-gene deletion strain with gE and partial gI gene deletion by utilizing the recombinant plasmid containing the red fluorescent protein and homologous arms on two sides of gE and partial gI genes on the basis of the GD0304 TK gene deletion strain, finally, remove the inserted red fluorescent gene fragment by utilizing the recombinant plasmid without fluorescent protein and with homologous arms on two sides, finally, purify by 3 times of plaque to obtain the three-gene deletion strain without fluorescent markers, which is named as pseudorabies GD0304 TK by the applicant - gE - gI - A strain. The fluorescence screening result is shown in figure 5, and the strains obtained by final screening are identified by PCR and sequencing, all TK, gE genes and part of gI genes are completely deleted, and wild strain pollution is not detected.
TABLE 2 amplification primers for two homologous arms of TK, gE and gI strain GD0304 of pseudorabies
Note that: the underlined sequence is the cleavage site.
2. Identification of Gene-deleted strains
2.1 identification of PCR deletion Gene
The three-gene deletion strain constructed by the TK and gE specific gene detection primer amplification is completely free of amplification of the TK and gE genes, which indicates that the gE and TK genes are successfully deleted, as shown in FIG. 6. The deletion site fragments of the deletion strain are amplified by using the outer primers of TK, gE and gI genes, and the deletion site of the target gene is confirmed to be consistent with the designed deletion site by sequencing.
2.2 identification of cell growth Properties of deletion strains
TCID50 of the three-gene-deleted strain on Vero cells was found to be 10 -6.29 Per mL, tcid50=10 with the wild strain -6.2 There was little difference in/mL. However, the three-gene deleted strain is relatively slower than the wild strain in cytopathic speed, and in TCID50 assay, the three-gene deleted strain is at high dilutionThe strain has obvious CPE on Vero cells 8-12 hours later than the wild strain, and the cell morphology after pathological changes is also different, the CPE of the wild strain on Vero cells shows a large amount of cell fusion phenomenon at the initial stage, and the fusion of the three-gene deletion strain is relatively less, so that obvious cell circle shrinkage phenomenon is shown, as shown in fig. 5a and 5d of fig. 5, the cell CPE morphology hidden by the three-gene deletion strain is similar to that of the Bartha K61 strain with the gE and gI genes naturally deleted, and the gE and gI genes are reflected to be deleted successfully from the side. From the growth characteristics of the virus on cells, the TCID50 of the strain is hardly affected after three genes are deleted, which shows that the deletion of the genes does not affect the replication of the virus on cells, the condition of vaccine production is basically satisfied, and from the time of appearance of CPE on cells and the morphology of CPE, the strain has weakened signs on the virulence of the cells after the deletion of the genes, which is similar to that of Bartha K61 strain; the three-gene deletion strain constructed by the invention has potential as a vaccine strain on the cell culture level.
2.3 cell passage stability
After 30 generations of blind transmission of the constructed three-gene deletion strain on Vero cells, the TCID50 of the virus is determined, and the gE and TK genes are amplified by PCR to identify the stability of the deletion strain.
After 30 generations of blind transmission, the TCID50 of the three-gene deleted strain was 10 -6.67 The increase in/mL compared to the initial generation may be due to strain adaptation to the cell. PCR identification has no TK and gE genes, and proves that the deleted strain can be stably passaged on Vero cells, has no gene recombination, and has pure strain and no wild toxin pollution.
Three-gene deletion strain pseudorabies GD0304 TK - gE - gI - The strain is sent to China center for type culture collection (China center for type culture collection, CCTCC) for preservation, and is named PRV GD0304 gE of porcine pseudorabies virus - gI - TK - The preservation number is: CCTCC NO: V201955, the preservation time is: 2019.8.15.
2.4 drug resistance test
The strain is found to have the effect of resisting the conventional anti-herpesvirus drug A in the separation stage of GD0304 wild strainAcyclovir (acyclovir) and ganciclovir (ganciclovir) are severely resistant, and the mechanism of action of these two drugs is associated with thymic kinase expressed by the viral TK gene. Therefore, after the three genes are deleted, the drug resistance of the deleted strain is tested. The test method is to respectively test GD0304 wild strain and GD0304 TK under the same 1MOI infection coefficient - gE - gI - Strains and Bartha K61 strains are inoculated into Vero cells, then antiviral drugs which are diluted in a gradient mode are added into cell supernatant to reach the final concentration of 0-200mg/mL, and the drug resistance of the strains is evaluated by observing the condition of cell CPE, and the results are shown in Table 3.
TABLE 3 susceptibility of different strains to anti-herpesvirus drugs
Note that: "+" indicates that the cells are CPE and "-" indicates that the cells are CPE free.
From the test results, at the highest concentration of 200mg/mL, the GD0304 wild strain and the three-gene deletion strain can generate obvious CPE, the appearance time and the CPE form have no obvious difference from those of a control group without adding the drug, the growth of the virus on cells is completely inhibited by the Bartha K61 group which is compared with the control group without adding the drug at the drug concentration of 2mg/mL, after the TK gene is deleted, the three-gene deletion strain still has strong drug resistance to acyclovir and ganciclovir as the wild strain, and the mechanism of the drug resistance generation of the strain is independent of the TK gene, and the drug resistance mechanism of the strain needs to be further studied. The value of GD0304 strain in the research of herpesvirus drug resistance mechanism is reflected, and meanwhile, the three-gene deletion strain is used as a vaccine strain, and a certain protection effect on epidemic drug-resistant strains is possible.
2.5 safety test of mice
Respectively at 10 -6.67 /mL TCID50, GD0304 wild strain of 10LD50, and cell culture fluid control group, the mice of 6 weeks old were stunted by nasal drip at a dose of 100 μl, and after 7 days, the wild mice all died, and the control group and the three gene deletion mice all survived, indicating that after gene deletion, the virus virulence was reduced, and higher safety was achieved on the mice.
2.6 mice immune toxicity test
In order to verify the protective effect of the three-gene deleted strain as a vaccine, an immune challenge test was designed on mice. The immunization protocol is shown in Table 4, and the immunization dose of the three-gene deletion inactivated virus and the live virus supernatant is 10 4 TCID 50 After dilution according to instructions, the immunization dose of each mouse is more than 10 4 TCID 50 . The virus inactivation method is formaldehyde with the final concentration of 0.2%, the virus is inactivated for 24 hours, and the virus is completely inactivated through the verification of virus-inoculated Vero cells; adjuvant 1 is Montanide of SEPPIC company TM Gel 01PR adjuvant, after diluting the inactivated virus supernatant to the desired concentration, formulated according to the adjuvant instructions (virus supernatant dilution: adjuvant=9:1); adjuvant 2 is Montanide of SEPPIC company TM ISA201VG adjuvant, after diluting the inactivated virus supernatant to a desired concentration, the preparation was performed according to the instructions (virus supernatant diluent: adjuvant=1:1); the live virus supernatant experimental group is that the virus supernatant of the Vero cell culture is directly used for immunization after being diluted to the required immunization dose by a DMEM culture medium; commercial Bartha K61 live vaccine (Szechwan Hailin biopharmaceutical Co., ltd., pseudorabies live vaccine (Bartha-K61 strain), diluted according to the instruction of use) was used as a control experiment group, and the supernatant of Vero cell culture was used as a blank control group, and 3 immunizations were performed by abdominal subcutaneous injection. After the immunization is completed, part of mice are subjected to a wild strain virus challenge test, the virus challenge scheme is shown in table 5, and the rest mice are sampled for virus neutralization test.
Table 4 three gene deleted vaccine immunization grouping table
TABLE 5 toxicity counteracting modes and dosages
After the virus is tapped, the death condition of the mice is observed for 7 days continuously, the dead mice are examined in time, tissues and organs such as brain, liver, spleen, lung, kidney and the like are taken, and the distribution condition of the gD gene and the gE gene of the virus in each tissue is detected by using a real-time fluorescence PCR method after nucleic acid is extracted. The death of the challenged mice is shown in Table 6, the sectional examination of the dead mice is shown in FIG. 7, the fluorescent PCR results of the viral genes of the organs of the dead mice are shown in Table 7, and the neutralization test results of the serum viruses of the mice are shown in Table 8.
TABLE 6 record of death of mice after immune challenge
TABLE 7 results of distribution of viral Gene tissue in dead mice
TABLE 8 neutralization of different strains by mouse immune serum
From the results of the mice immune challenge test, the classical Bartha strain vaccine has reduced challenge protection capability on mice aiming at pseudorabies variation strains, can not completely protect the mice from death, but the three-gene deletion strain prepared based on GD0304 strain can provide better protection capability than Bartha strain under the inactivated and live virus forms, especially the inactivated virus and adjuvant group, and achieves 100% challenge protection rate; the neutralizing titer of the inactivated vaccine prepared based on the pseudorabies GD0304 TK-gE-gI strain can be seen to have higher neutralizing titer against wild virus GD0304, but the live virus immune group in the test probably has lower neutralizing titer of the serum because of lack of adjuvant to stimulate immunity.
2.7 immunization and toxicity attack test for piglet
In order to test the performance of the three-gene deletion strain serving as a vaccine candidate strain in the invention, a piglet immune challenge test is designed on the basis of a mouse test, a preparation method of the three-gene deletion inactivated vaccine is the same as that of groups B1 and B2 of the mouse immune challenge test, commercial inactivated vaccines (Haibles, porcine pseudorabies inactivated vaccine (Bartha K61 strain)) are immunized according to a vaccine specification, test groups and designs are shown in Table 9, and a live virus group is not arranged in the immune challenge test because of test condition limitation.
Table 9 group of immunization and toxicity attack tests for piglets
Blood was collected before challenge for serum neutralization titer detection, and neutralization titers of the serum of each experimental group against GD0304 strain are shown in table 10. Clinical symptoms of the experimental pigs, including fever, action and feeding changes, mortality and the like, are observed within 14 days after the toxicity attack, the results are shown in table 11, the rest pigs are killed after 14 days, and the pathological changes of all organs are observed, wherein the pathological changes of all organs are shown in figure 8. It can be seen that no obvious organ lesions were observed in the experimental groups except the control group D, which had more obvious hyperemic lesions in the main target organs of porcine pseudorabies.
TABLE 10 neutralization titers of piglet sera from different vaccine groups against PRV GD0304 wild strain
| Grouping | Vaccine species | Immunization dose/mL | Number of immunizations | Neutralization potency |
| A | Three gene deletion inactivated virus + adjuvant 1 | 2 | 2 | 29.9 |
| B | Three gene deletion inactivated virus + adjuvant 2 | 2 | 2 | 25.8 |
| C | Commercial Bartha strain inactivated vaccine | 2 | 2 | 11.2 |
| D | PBS | 2 | 2 | ＜2 |
Table 11 comparison of the immune Effect of porcine pseudorabies inactivated vaccine
Note that: the protection rate here is the protection rate against the occurrence of typical porcine pseudorabies clinical symptoms in piglets.
According to the results of the immunity challenge test of the piglets, the inactivated vaccine prepared from the three-gene deletion strain can stimulate the piglets to generate higher serum neutralizing antibodies aiming at wild strains, can generate better challenge protection effect in the challenge test, can protect the piglets from death, and can effectively avoid clinical symptoms of pseudorabies of the piglets. The results of the section test also prove that the vaccine group can protect all main organs of the piglets from virus attack. Meanwhile, as can be seen from the toxicity attacking results of the commercial Bartha strain vaccine group, the protection effect of the classical Bartha K61 strain on the variant strain is weakened, and 2 piglets in the commercial vaccine group have obvious clinical pseudorabies symptoms such as body temperature rise, shortness of breath and the like, and although the pigs do not die at last, the production performance of the pigs is inevitably reduced in the actual breeding process, and the disease reaction is possibly aggravated if the pigs are subjected to stress or other epidemic diseases and the death risk is increased.
2.8 summary of immune toxicity attack tests
The immune toxicity attack tests of mice and piglets show that the inactivated or live virus vaccine prepared from the three-gene deletion pseudorabies mutant strain has better toxicity attack protection effect on the mutant strain compared with the classical Bartha K61 strain, can effectively reduce morbidity and mortality, and has higher serum neutralization titer. The three-gene deletion strain has higher application and commercial value in prevention and control of pseudorabies variant strains, and can be used as an excellent pseudorabies vaccine candidate strain.
The present invention has been described in detail in the above embodiments, but the present invention is not limited to the above examples, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present invention. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.
SEQUENCE LISTING
<110> Guangdong sea big animal husbandry veterinary research institute Co., ltd
<120> porcine pseudorabies virus gE/gI/TK gene deletion vaccine strain, construction method and application thereof
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Claims (12)
1. A porcine pseudorabies virus gE/gI/TK gene deletion strain, wherein the porcine pseudorabies virus gE/gI/TK gene deletion strain is a porcine pseudorabies virus PRV GD0304 with gE, gI and TK genes deleted; the preservation number of the PRV GD0304 is CCTCC NO: V201946, and the preservation time is as follows: 2019.8.15, accession number: china center for type culture collection, preservation address: chinese university of Wuhan; the porcine pseudorabies virus gE/gI/TK gene deletion strain is named as follows: PRV GD0304 gE - gI - TK - The preservation number is CCTCC NO: V201955, and the preservation time is: 2019.8.15, accession number: china center for type culture collection, preservation address: chinese university of Wuhan and Wuhan.
2. An antibody prepared by taking the porcine pseudorabies virus gE/gI/TK gene deletion strain as an immunogen.
3. The use of the porcine pseudorabies virus gE/gI/TK gene deleted strain of claim 1 in the preparation of a product for diagnosing or detecting porcine pseudorabies virus.
4. The use of the porcine pseudorabies virus gE/gI/TK gene deleted strain of claim 1 in the preparation of a product for preventing porcine pseudorabies.
5. The use according to claim 4, wherein the product is a vaccine.
6. The use of the porcine pseudorabies virus gE/gI/TK gene deleted strain of claim 1 in constructing a porcine pseudorabies virus challenge disease model.
7. A medicament comprising the porcine pseudorabies virus gE/gI/TK gene deleted strain of claim 1.
8. The medicament of claim 7, further comprising a pharmaceutically acceptable adjuvant, carrier, protectant, diluent or excipient.
9. The medicament according to claim 8, wherein the adjuvant is Gel 01PR, ISA201VG or aluminum salt adjuvant, protein adjuvant and nucleic acid adjuvant applicable to veterinary vaccines.
10. The medicament of claim 7, wherein the medicament is a vaccine.
11. The medicament according to claim 10, characterized in that the vaccine is an inactivated vaccine or a live vaccine.
12. A method of preparing a vaccine for preventing porcine pseudorabies comprising the steps of: preparing the porcine pseudorabies virus gE/gI/TK gene deletion strain virus liquid according to claim 1, and then adding an adjuvant.
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