CN114657151A

CN114657151A - Porcine pseudorabies virus gE/gI/TK gene deletion vaccine strain and construction method and application thereof

Info

Publication number: CN114657151A
Application number: CN202210191045.6A
Authority: CN
Inventors: 伍建敏; 李中圣; 王凤求; 庞旋飞; 王贵平; 尹兴强
Original assignee: Guangdong Haid Animal Husbandry And Veterinary Research Institute Co ltd
Current assignee: Guangdong Haid Animal Husbandry And Veterinary Research Institute Co ltd
Priority date: 2022-02-25
Filing date: 2022-02-25
Publication date: 2022-06-24
Anticipated expiration: 2042-02-25
Also published as: CN114657151B

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 GD 0304; the preservation number of the gene deletion strain is CCTCC NO: V201955. Compared with the classical Bartha K61 strain, the strain has better virus counteracting protection effect on variant strains, can effectively reduce morbidity and mortality, and has higher serum neutralizing titer. The three-gene deletion strain has higher application and commercial value in the prevention and control of the pseudorabies variant strain, and is a vaccine strain hopefully applied to the current PRV epidemic prevention and control work.

Description

Porcine pseudorabies virus gE/gI/TK gene deletion vaccine strain and construction method and application thereof

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, and a construction method and application thereof.

Background

The pseudorabies virus belongs to alpha herpes virus, can infect various mammals and a few poultry, is a main host of the pseudorabies virus, can cause abortion and stillbirth of pregnant sows, sterility of boars, death of newborn piglets, dyspnea and growth retardation of fattening pigs and other pseudorabies symptoms, and is one of major infectious diseases harming the breeding industry. At present, the prevention and treatment of the pseudorabies mainly depends on vaccines, wherein the pseudorabies is the most classical pseudorabies and the most widely used pseudorabies at home and abroad is K61 passage weak strain separated from Bartha of the scholars Hungarian in 1961, and the strain is called Bartha K61 strain for short. The vaccine based on the strain plays an important role in the prevention and control of the pseudorabies, the purification of the pseudorabies is realized in part of countries and regions, and the pseudorabies virus vaccine used in China at present is mainly derived from the strain. The Bartha K61 strain plays an important role in preventing and controlling the pseudorabies in China, and over 80 percent of breeding pigs immunize the strain since 1990, so that the spreading of the pseudorabies in China is effectively controlled. However, in recent years, variation may occur in the pseudorabies strain circulating in China, so that the Bartha K61 vaccine fails to immunize. From the analysis of gene sequences, the global pseudorabies virus can be divided into two large branches, wherein a gene 1 strain represented by a Bartha strain is mainly distributed in Europe and America, while a domestic strain belongs to a gene 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. It can be seen from the gene analysis data that although the pseudorabies virus still belongs to one serotype, the domestic and foreign pseudorabies strains have been differentiated on the gene sequence, and the domestic strains are further differentiated into new and old branches, which suggests that the domestic strains may have differences in immunogenicity from the foreign strains represented by Bartha, and explains the incomplete protection of the Bartha vaccine in recent years.

On the other hand, in some pig farms, due to the consideration of biological safety control, in order to reduce the contact between personnel and the swinery and avoid the stimulation to the swinery, the use of vaccines is greatly reduced, which also leads to the further increase of the incidence of the pseudorabies in the two years. The two factors present higher requirements and challenges for the development work of the pseudorabies vaccine, on one hand, the vaccine strain is required to provide high-efficiency protection action against the domestic epidemic variant strains, and in order to solve the problem, the domestic epidemic, representative and highly virulent PRV variant wild strains are required to be screened out to serve as vaccine candidate strains; on the other hand, a pseudorabies vaccine which is more stable, safe and reliable, has higher immune efficiency and convenient use and has small stimulation to a swinery is needed, and the construction and screening method of the gene deletion vaccine which is safer and more reliable and the adjustment of the immune dosage form and the immune mode are needed to realize the pseudorabies vaccine.

In summary, the current trend of epidemic and purification of pseudorabies strains in China is that on one hand, a vaccine aiming at an epidemic pseudorabies variant strain is very needed, and on the other hand, higher requirements are provided for screening of vaccine candidate strains and construction of gene deletion vaccines. However, the current strains of the domestic pseudorabies vaccine are mainly the traditional Bartha-K61 strain vaccine, and the safety, the effectiveness and the pertinence of whether regional epidemic strains exist are not fully verified by 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 preparation of vaccines.

The technical scheme adopted by the invention is as follows:

the first aspect of the invention provides a porcine pseudorabies virus gE/gI/TK gene deletion strain, wherein the porcine pseudorabies virus gE/gI/TK gene deletion strain is porcine pseudorabies virus PRV GD0304 with gE, gI and TK gene deletion; (ii) a The preservation number of the porcine pseudorabies PRV GD0304 is CCTCC NO: V201946, and the preservation time is as follows: 2019.8.15, depository: china center for type culture Collection, collection address: wuhan, Wuhan university.

In some embodiments of the invention, the porcine pseudorabies virus gE/gI/TK gene deletion strain is designated as: porcine pseudorabies virus PRV GD0304 gE^-gI^-TK^-The strain has a preservation number of CCTCC NO of V201955, and the preservation time is as follows: 2019.8.15, depository: china center for type culture Collection, collection address: wuhan, Wuhan university.

In a second aspect of the invention, a product for treating or preventing porcine pseudorabies is provided, which comprises the gene deletion strain of the first aspect of the invention or porcine pseudorabies PRV GD 0304.

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 an aluminum salt adjuvant, a protein adjuvant, and a nucleic acid adjuvant that can be applied to 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 drop, intramuscular, intravenous, subcutaneous, or cutaneous injection applications.

In some embodiments of the invention, the porcine pseudorabies virus gE/gI/TK gene deletion strain in the inactivated vaccine is inactivated by an inactivating agent such as formaldehyde or beta-propiolactone.

In the third aspect of the invention, an antibody prepared by using the porcine pseudorabies virus gE/gI/TK gene deletion strain in the first aspect of the invention as an immunogen is provided.

The invention also provides the application of the reagent in the preparation of the diagnostic reagent and the kit for detecting the porcine pseudorabies virus infection. In a third aspect of the invention, the application of the porcine pseudorabies virus gE/gI/TK gene deletion strain or the porcine pseudorabies PRV GD0304 in preparing a medicament for preventing or treating the porcine pseudorabies is provided.

In the fourth aspect of the invention, the application of the porcine pseudorabies virus gE/gI/TK gene deletion strain or the porcine pseudorabies PRV GD0304 in preparing a medicament for diagnosing or detecting the porcine pseudorabies is provided.

The fifth aspect of 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 and pathogenesis model.

In a sixth aspect of the invention, there is provided a method for preparing a vaccine for preventing or treating porcine pseudorabies, comprising the steps of: preparing virus solution of the porcine pseudorabies virus gE/gI/TK gene deletion strain in the first aspect of the invention, and then adding an adjuvant to obtain the porcine pseudorabies virus gE/gI/TK gene deletion strain.

In some embodiments of the invention, the inactivation is performed after the preparation of the virus solution of the porcine pseudorabies virus gE/gI/TK gene deletion strain.

The invention has the beneficial effects that:

the invention screens out a pseudorabies variant strain with stronger toxicity, characteristics and representativeness in the aspects of genotype, serological level and the like, which is named as PRV GD0304 of porcine pseudorabies, and based on the strain, a homologous recombination method is used for constructing the gE of the strain^-/gI^-/TK^-Three-gene deletion strain named porcine pseudorabies virus PRV GD0304 gE^-/gI^-/TK^-As vaccine candidate strains.As can be seen from immune challenge tests of mice and piglets, compared with the classical Bartha K61 strain, the inactivated or live virus vaccine prepared from the pseudorabies variant strain with three-gene deletion has better challenge protection effect on the variant strain, can effectively reduce the morbidity and the mortality, and has higher serum neutralization titer. The three-gene deletion strain has higher application and commercial value in the prevention and control of the pseudorabies variant strain, and is a vaccine strain hopefully applied to the current PRV epidemic prevention and control work.

Drawings

FIG. 1 is a technical route for screening porcine pseudorabies virus epidemic variant strains.

FIG. 2 shows the CPE of Vero cells infected by GD0304 strain (P3).

FIG. 3 is an electron micrograph of PRVGD0304 virions.

FIG. 4 is a phylogenetic tree constructed from pseudorabies gene sequences in the database. FIG. 4A is a tree of evolution of 9 PRV viral genomes constructed using the MP method; FIG. 4B is a tree of gB gene sequences constructed using the NJ method; fig. 4C is an evolutionary tree constructed using the NJ method for the gC gene sequence.

FIG. 5 shows cytopathic effects of PRV GD0304 strain infected Vero cells before, during, and after gene knockout. FIG. 5a is the cytopathic effect of GD0304 wild strain after infecting Vero cells for 36 hours, 200 times; FIG. 5b shows the expression of green fluorescent protein in Vero cells 48 hours after the recombinant viruses replace the TK gene with the green fluorescent protein gene, 200 times; FIG. 5c is a 100-fold representation of red fluorescent protein in Vero cells 48 hours after recombinant viruses infected Vero cells, after replacing viral gE and part of gI genes with red fluorescent protein genes, based on TK gene deletion; FIG. 5d shows the cytopathic effect of the three-gene-deleted strain of pseudorabies GD0304 TK, gE and part gI after the removal of the red fluorescent protein gene, after infecting Vero cells for 72 hours, which is 200 times higher.

FIG. 6 is a gel electrophoresis diagram of the TK and gE genes identified by PCR. FIG. 6a shows PCR detection results of TK gene and gE gene of 6 passage viruses after TK gene deletion, wherein the strains are continuously passaged on Vero cells for 6 times, and the PCR detection results are as follows: passage 1 to passage 6 viruses, PC: positive control (wild virus genome), M: DL2000 Marker; FIG. 6b shows the gE gene detection results of 6 passage viruses after three gene deletion in the Vero cell, wherein the gE gene detection results are 1-6: 1 st to 6 th generation viruses, PC: positive control (wild virus genome), M: DL2000 Marker.

FIG. 7 shows dead mice and their necropsy. FIG. 7A is a trace of abdominal wrapping of a dead mouse; FIG. 7B shows a liver enlargement; FIG. 7C shows spleen atrophy hyperemia; FIG. 7D is a punctate hemorrhage of the kidney; FIG. 7E shows pulmonary congestion; FIG. 7F is meningeal hyperemia.

FIG. 8 shows the pathological changes of different tissues of piglets after challenge in each experimental group. Wherein the virus attacking strain in FIG. 8A is GD0304 strain (10)⁶TCID₅₀Per ml), the vaccine type is three-gene deletion inactivated virus + adjuvant 1; the toxic strain in FIG. 8B is GD0304 strain (10)⁶TCID₅₀Per ml), the vaccine type is three-gene deletion inactivated virus + adjuvant 2; FIG. 8C shows that the toxic strain is GD0304 strain (10)⁶TCID₅₀Per ml), the vaccine type is commercial Bartha strain inactivated vaccine; FIG. 8D shows that the middle challenge strain is GD0304 strain (10)⁶TCID₅₀Per ml), PBS treatment; the piglets in fig. 8E were not treated at all.

Detailed Description

The idea of the invention and the resulting technical effects will be clearly and completely described below in connection with the embodiments, so that the objects, features and effects of the invention can be fully understood. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

Example 1 isolation and identification of epidemic variant strain of porcine pseudorabies Virus (PRV 0304 Strain) and virulence test

The technical route is shown in figure 1.

1. GD0304 strain pseudorabies virus source

In the 4 th month of 2017, PRV suspected epidemic situation occurs in a certain pig farm in the river of Guangdong, tissues such as brain, spleen, lung, pulmonale lymph nodes and the like of dying pigs are collected for laboratory detection, PRV wild virus nucleic acid is positive, and meanwhile, porcine circovirus type 2 nucleic acid and porcine reproductive and respiratory syndrome nucleic acid are positive in disease material; according to investigation, the pig farm is immunized according to a commercial PRV vaccine (Bartha K61 strain) immunization program, and the positive rate of the PRV gI antibody (IDEXX kit) is detected to be 87.7%; by combining diagnostic criteria, it was concluded that PRV vaccines were not effective against this epidemic infection. Epidemiological investigations have found that pig farms in close contact with the pig farm are prevalent with varying degrees of PRV morbidity.

2. Separation, identification and purification of pseudorabies virus

1) PRV rabbit regression

The tissue homogenate supernatant was injected into New Zealand white rabbits and rabbit infection was used to remove or reduce potential other pathogens in the tissue samples. Taking PRV positive brain tissue treatment fluid (centrifuging, sterilizing and diluting by 10 times), inoculating a New Zealand white rabbit (0.5 mL/rabbit) subcutaneously at the neck, and after 16 hours of experiment, generating nervous symptoms and dying 48 hours; the dying rabbits are euthanized, 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), Classical Swine Fever Virus (CSFV), porcine circovirus type 2 (PCV2) and Porcine Parvovirus (PPV) nucleic acid are not detected.

2) PRV separation and purification

PRV is purified by using susceptible cell 'plaque method' test, and potential PRV vaccine virus is removed. Inoculating Vero cells to PRV positive tissue purified by rabbit body (

CCL-81^TM) 6 rounds of 'plaque method' tests are carried out, each plaque is picked to have a single position, the cytopathic shape is typical, and the plaque with higher cytopathic speed is screened in the next round, after 10 virus plaques picked in the final round are re-inoculated with Vero cells, the situation that GD0304 strain (P3) infects Vero cells CPE is shown in figure 2, and 24 hours and 36 hours of infected plaques are respectively observed; respectively carrying out qPCR identification on gD and gE genes of the collected virus, and selecting a strain with the highest lesion speed and highest virus content, namely PRV 0304 strain; after the virus is inoculated into Vero cells, CPE appears rapidly, the cytopathic morphology is typical, and the virus content in cell supernatant fluidHigher; the plasma virus is enlarged and cultured, and is purified by ultracentrifugation, the virus particles are spherical and have the diameter of 100-150nm and a cyst membrane by electron microscope observation, and radial fiber protrusions are arranged outside the cyst membrane, and the virus particle is in a typical PRV virus form (figure 3). The virus purified by the super separation is subjected to NGS second generation sequencing, and finally the 132307bp virus full-length sequence is obtained by splicing (NCBI sequence number: MH 582511). The porcine pseudorabies virus strain is delivered to a preservation organization for preservation, is preserved in China center for type culture Collection (CCTCC, Wuhan university preservation center) and is named as porcine pseudorabies virus PRV GD0304 with the preservation number: CCTCC NO, V201946, the preservation time is as follows: 2019.8.15.

PRV 0304 strains are continuously passaged on Vero cells to 30 generations, and the specificity and the purity of viruses of 10, 20 and 30 generations are checked by referring to the pharmacopoeia of the people's republic of China, and the viruses are specific and have no pollution of bacteria, mildew, mycoplasma and exogenous viruses.

3) PRV GD0304 strain titer determination

The virus titer (TCID50) of PRV GD0304 strain on Vero cells determined by Reed-Muench was: p10 generation 10^-6.2mL, P20 generation 10^-6.57mL, P30 generation 10^-6.6and/mL. The titer of the PRV GD0304 strain low-generation secondary virus on Vero cells is slightly low, and the PRV GD0304 strain low-generation secondary virus is stable after passage for 20 times; compared with other reported PRV epidemic strains, the PRV GD0304 strain has lower TCID50, but has higher infection speed on Vero cells and more obvious cytopathic condition. Compared with Barth K61, the GD0304 strain infected cells can observe obvious cytopathy about 5 hours after inoculation, can observe virus plaque formation about 12 hours, and can cause the infected cells to completely disintegrate and fall off about 24-36 hours under the same 1MOI infection dose. The titer of PRV GD0304 strain is obviously lower than that of PRV epidemic strain reported in China (10)⁸～10⁹TCID50/mL), but the cytopathic rate is faster, which may mean a stronger replication and infection of the virus.

4) Regression test of strain PRV GD0304

PRV GD0304 strain P30 generation Vero cell culture supernatant after plaque purification is 1 × 10⁶The regression test of 5 SPF piglets inoculated with TCID50/mL nasal drops, the inoculated piglets begin to have clinical symptoms of high body temperature 1 day after inoculation (41.4)4 deg.C), 4 days later, the patients began to have symptoms of dyspnea, mania, trembling, and stroke, and all died (100% mortality) in 5 days later, and the control group was normal. After dead piglets are dissected, tissues such as brain, spleen, lung lymph node and the like are taken to carry out PCR detection, amplification and sequencing on PRV gE, gB, gC, gD and TK genes, and the sequence is consistent with that of GD0304 strain. Regression experiments show that GD0304 strain can induce pseudorabies symptoms of susceptible animals, and the strain sequence is stable.

5) PRV GD0304 shelf life test

Periodically, the virus (10 th, 20 th and 30 th generations) stored at-80 ℃ is taken out, 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, after PRV GD0304 viruses of different generations are stored at-80 ℃ for 36 months, the virus titer on Vero cells does not obviously change, which indicates that the strain can be stably stored and meets the storage requirement of vaccine strains.

TABLE 1 Titers of 3 passage viruses on Vero cells at different storage Times (TCID)₅₀)

2. Whole genome sequencing and major virulence gene analysis

Sequencing the DNA of PRV GD0304 strain of porcine pseudorabies virus obtained by separation, identification and purification by using an Illumina platform, and then performing data assembly to finally obtain 1 whole genome sequence. The result shows that the total length of the PRV GD0304 genome is 142307bp, the sequence length is closer to 142334bp representing a domestic traditional pseudorabies strain Ea strain, the sequence length is shorter than 145312bp representing a domestic pseudorabies variant strain JS-2012 strain, the GC content is 73.6%, and 69 open reading frames are coded in total, wherein the open reading frames contain two repetitive sequence regions of IR and TR. Evolutionary analysis is carried out on the GD0304 whole genome and other representative strains, such as Bartha strain representing foreign vaccine strains, Ea strain representing domestic early wild strains and JS-2012 strain representing domestic PRV variant strains, and the analysis result is shown in figure 4, wherein the GD0304 strain is closer to the Ea strain in evolutionary relationship and is relatively far from the JS-2012 strain representing the variant strains. The evolutionary trees of two conserved genes gC and gB are similar to the whole genome evolutionary tree in the whole structure, and show that each strain of PRV can be divided into two obvious branches, European and American strains represented by Bartha strains independently belong to the same branch, and Chinese strains including PRV GD0304 strains belong to the other branch; further analysis on Chinese strains shows that in the conservation gene, the PRV GD0304 strain has a relatively independent branch in domestic strains recently in the evolutionary relationship with epidemic strains such as HB1201, HeN1 and JS2012 separated since 2012 in China, and the Ea strain, Fa strain, SC strain and the like representing the domestic classical epidemic strains form another relatively independent branch, which is different from the analysis result of the whole genome. The sequence analysis results show that the GD0304 strain is closer to the past domestic epidemic strains on the genome, such as the Ea strain, but is closer to the domestic new epidemic variant strain on the conservation gene sequence; this suggests that the GD0304 strain may have a different origin from other domestic epidemic PRV variants (some studies show that the domestic PRV variant may originate from the Bartha strain, but the GD0304 strain is more closely related to the Ea strain from the genome point of view), but genetic recombination may occur between the epidemic strains during transmission. On one hand, the complexity of the domestic PRV epidemic strain is illustrated, the GD0304 strain has very important research value in researching the origin and the evolutionary relation of the domestic epidemic strain, and on the other hand, the uniqueness of the strain in the pseudorabies epidemic variant strain and the potential of the strain serving as a vaccine candidate strain are reflected.

3. Viral virulence test

3.1 virulence in mice

3.1.1 half lethal dose in mice

50 female Balb/c mice, 5-6 weeks old, were randomly divided into 5 groups, 10/group. 1-4 groups of the medicinal herbs are injected subcutaneously into the neck and back of the patient with 0.1mL of the medicinal herbs respectively containing 10²TCID50/mL，10³TCID50/mL，10⁴TCID50/mL，10⁵TCID50/mL PRV GD0304 strain (experimental group) Vero cell culture virus liquid; group 5 neck and back were injected subcutaneously with 0.1mL DMEM medium as a negative control group. The mice were observed and recorded for 2 weeks after challenge with clinical symptoms, death, etc., and diedThe deceased mice were dissected in time to obtain tissues, PCR-detected for PRV gD and gE genes, and part of the tissues was fixed for pathological section.

3-7 days after the toxin attack, 10⁵TCID50/mL panels and 10⁴The TCID50/mL experimental mice have clinical symptoms of listlessness, appetite reduction and the like in different degrees, and can seriously and continuously wrap around the inoculation part, so that hair is lost, skin bleeding is caused, and finally death is caused. 10³The TCID50/mL group began to develop mild clinical symptoms such as listlessness, decreased appetite, etc. on day 4 after challenge, and some mice developed neurological symptoms such as seizing and died. After 7 days, the surviving mice in the groups basically return to normal without obvious clinical symptoms. The negative control mice appeared normal throughout the experimental period. Statistics show that the death time of the mice is mainly concentrated on the 3 rd to 7 th days after the challenge. 10⁵、10⁴、10³、10²The number of deaths of the TCID50/mL mice in the challenge group is 10, 9, 5 and 1 respectively; according to the experimental result, the LD50 of the PRV GD0304 strain to the mouse is calculated as: 10^3.2TCID50/mL, and LD50 of Chinese classical virulent strain SC strain to mouse (10)^5.16TCID50/mL), the virulence of the strain to the mouse is obviously enhanced.

3.1.2 mouse Cross-Immunoprotection test

40 female Balb/c mice, 6-8 weeks old, were randomly divided into 4 groups of 10 mice each, and immunized subcutaneously on the back and neck. 0.1mL of Bartha-K61 strain formaldehyde-inactivated virus containing 104TCID50 is immunized for each group 1, 104TCID50 is immunized for each group 2, and 0.1mL of DMEM culture medium is inoculated for each mouse of the

groups

3 and 4 to serve 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, the 1 st, 2 nd and 3 rd groups respectively use GD0304 Vero strain Vero cell of 10LD50 to culture live virus for subcutaneous challenge in the neck and back, and the clinical symptoms and death condition of the mice are observed for 14 days continuously. The mice in the group 3 begin to have PR symptoms on the 3 rd day after the challenge, the sick mice have the scratching and winding phenomenon, so that local fur falls off and skin is bled, and all serious PR symptoms appear in 4-7 days and die continuously, and the survival rate is 0%; the mice in the group 1 begin to show PR symptoms on the 4 th day of the challenge, and die in 5-8 days, and the survival rate is 20 percent finally; group 2 mice developed PR symptoms partially on day 4, with a small number of deaths on days 5-7, with a final survival rate of 50%; group 4 mice had no apparent symptoms during the observation period.

3.2 virulence in pigs

3.2.1 half lethal dose for piglets

Randomly dividing 20 healthy piglets of 15 days into 5 groups, each group has 5 piglets, and the 1 st to 3 rd groups are inoculated with 2mL 10 mL nasal drops⁶TCID50/mL，10⁵TCID50/mL，10⁴TCID50/mL and 10³TCID50/mL PRV GD0304 strain Vero cell culture virus solution, and group 5 was inoculated with 2mL DMEM medium by nasal drip as a negative control group. Isolated feeding, free feeding, continuous observation for 14 days, recording body temperature and clinical symptoms; and the dead pigs are dissected in time, tissues are taken to carry out PCR detection on PRV gD and gE genes, and part of tissues are fixed for pathological sections. Day 2 after challenge, 10⁶The TCID50/mL piglet group showed a significant body temperature rise, which reached the highest on day 3, and the average body temperature was: 41.4 ℃.

The piglets of the four challenge test groups have clinical symptoms of body temperature rise, appetite reduction, tachypnea, somnolence and the like in different degrees after challenge; 10⁶The TCID50/mL piglets in the group have dyspnea on the 4 th day after the challenge, are in dog sitting posture, are maniac, continuously hit the columna capitata with the head, have severe clinical symptoms such as shaking the whole body and swabbing on the ground in the later period, all die on the 5 th day, and have the lethality of 100%. 10⁵TCID50/mL group, 10⁴TCID50/mL panels and 10³Similar clinical symptoms also occurred in the TCID50/mL group on day 4 after challenge, with death beginning on day 5 and ending 14, 10 days after challenge⁵TCID50/mL group all died, 10⁴TCID50/mL group one end remaining, 10³The remaining 4 piglets of the TCID50/mL group did not die. The negative control group appeared normal throughout the experimental period. Calculating to obtain the median lethal dose of PRV GD0304 strain to piglets as follows: 10^3.2TCID50/mL。

Death and tolerant piglets in a caesarean section challenge test group find that brain tissues of piglets in the challenge group have bleeding and edema phenomena and cerebrospinal fluid increase; edema of the lung, with parenchymal flesh-like lesions; enlargement of the lung portal lymph nodes with white punctate lesions; spleen congestion with infarct foci at the margin; punctate hemorrhage with the size of a needle point which is dispersedly distributed can be seen on the surface of the kidney; inguinal lymph nodes were swollen and congested. The negative control group has no obvious pathological change in the organs of the piglet.

And (3) carrying out PCR detection on the brain tissues of dead piglets, wherein PRV gE and gD genes are positive, and the sequencing result of the amplified fragment is consistent with that of 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 the highest and can reach 3.2x10⁵copies/g。

3.2.2 comparison of neutralizing Capacity of pig immune serum

After 10 parts of Bartha-K61 are randomly selected for immunization, pig serum with gB antibody positive and gE antibody negative is detected by an IDEXX kit, and the neutralizing capacity of the immune serum on wild viruses separated from Bartha-K61 strains, GD0304 strains and other several Guangdong Guangxi regions is respectively tested.

The results show that the average neutralizing antibody titer of Bartha-K61 immune serum against Bartha-K61 strain is 1: 57; the neutralizing antibody titer of the GD0304 strain can reach 1:13 at the highest, the average value is 1:18, and the two have obvious difference (p is less than 0.01); the average neutralizing antibody titers of several other wild viruses were in the range of 1: 20-1: 40. The results of this experiment also indicate that the neutralizing capacity of Bartha-K61 immune serum to GD0304 strain and other several wild strains is reduced, which explains the reason why pseudorabies still occurs in pig farms immunized with Bartha-K61.

Toxicity tests on mice and piglets show that the PRV GD0304 strain newly isolated and identified in the invention is far stronger in toxicity than the classical PRV standard virulent strain (SC strain LD 50: 10)^5.16TCID50/mL) shows that the toxicity of PRV wild virus is obviously changed in the domestic epidemic process, explains the reason of incomplete immunity protection of Bartha-K61 vaccine, and simultaneously shows the potential of the GD0304 strain separated by us on being used as a candidate vaccine strain aiming at the current domestic PRV epidemic strain.

Example 2 construction of three-Gene-deleted Strain

1. Purpose and technical route:

at present, the most widely used Bartha-K61 strain in the pseudorabies vaccine is naturally deleted gE and partial gI genes, other vaccine strains mostly artificially delete gE and gI genes, and partial vaccine strains can also additionally delete TK genes in order to improve safety. Among three frequently deleted genes, the TK gene, namely the gene coding virus thymidine kinase, mainly participates in the salvage synthesis path of nucleotide to maintain and promote virus replication, is one of the main virulence genes of pseudorabies virus, is related to virus latent infection and proliferation in the central nervous system, and the deletion of the TK gene can obviously reduce the infectivity of the pseudorabies virus on nerve cells and the virulence of pigs, but the immunogenicity is not influenced; the gE and gI genes are one of genes for coding envelope proteins of the virus, are one of main virulence genes of the pseudorabies virus, are related to assembly and transportation of the virus in nerve cells, greatly reduce the virulence of the virus after the gE and gI genes are deleted, and are not influenced by immunogenicity.

The GD0304 strain has the potential of serving as an excellent vaccine candidate strain due to the particularity of the epidemic territory, the toxicity and the gene evolution. In order to test the immunity performance and safety of the strain as a vaccine strain, a TK/gE/gI three-gene deletion strain of the GD0304 strain is constructed by 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 Bartha-K61 strain.

In this example, the homology arm amplification primers in table 2 are used to amplify upstream and downstream homologous sequences of GD0304 strain TK gene and gE and gI genes, respectively, and then expression vectors with green fluorescent protein (TK gene recombinant plasmid) and red fluorescent protein (gE and gI gene recombinant plasmid) are digested and ligated, respectively, to construct recombinant plasmids containing homology arms on both sides of each gene and having fluorescent protein selection markers. Screening out GD0304 strain with green fluorescence labeling deleted from TK gene by using recombinant plasmids with green fluorescence protein and homology arms at two sides of TK gene, and removing inserted green fluorescence fragment from recombinant plasmid without fluorescence protein and with homology arms at two sides; next, GD0304 TK base is addedOn the basis of the deletion strain, a recombinant plasmid containing red fluorescent protein, gE and homologous arms at two sides of part of gI gene is used for screening out a TK gene with red fluorescence, a GD0304 three-gene deletion strain with gE and part of gI gene deletion, and finally, the recombinant plasmid only containing homologous arms at two sides is used for removing the inserted red fluorescent gene fragment without fluorescent protein, and finally, the three-gene deletion strain without fluorescent mark is obtained by 3 times of plaque purification, wherein the three-gene deletion strain is called pseudo rabies GD0304 TK^-gE^-gI^-And (4) strain. The fluorescent screening results are shown in FIG. 5, and the strains obtained by final screening are identified by PCR and sequencing, so that all TK and gE genes and part gI genes are completely deleted, and no wild strain pollution is detected.

TABLE 2 amplification primers of homologous arms at two sides of TK 0304 strain of pseudorabies GD0304 and gE, gI

Note: the underlined part of the sequence is the cleavage site.

2. Identification of gene-deleted strains

2.1PCR deletion Gene identification

Three gene deletion strains constructed by amplification of TK and gE specific gene detection primers are completely not amplified, and thus the gE and TK genes are successfully deleted, as shown in FIG. 6. And (3) amplifying the deletion site fragments of the deletion strain by using outer primers of TK, gE and gI genes, and confirming that the deletion site of the target gene is consistent with the designed deletion site through sequencing.

2.2 identification of growth Performance of cells from deleted Strain

TCID50 results for the three-gene deleted strain on Vero cells were 10^-6.29mL, 10 in comparison with the TCID50 of the wild strain^-6.2There was almost no difference in/mL. However, in the cytopathic speed, the three-gene deletion strain is relatively slower than the wild strain, in the TCID50 determination, the time of obvious CPE of the three-gene deletion strain on Vero cells under high dilution is 8-12 hours later than that of the wild strain, and the cell morphology after pathological change is also different, and the wild strain is in Vero cellsThe CPE of (a) initially exhibited a large number of cell fusions, while the three-gene-deleted strain fused relatively less, and thus exhibited a significant cell rounding phenomenon, as shown in fig. 5a and 5d of fig. 5, the three-gene-deleted strain had a concealed cellular CPE morphology similar to the Bartha K61 strain with naturally deleted gE and gI genes, reflecting laterally that the gE and gI genes had been successfully deleted. From the growth characteristics of the virus on cells, the TCID50 of the strain is hardly influenced after three genes are deleted, which indicates that the gene deletion does not influence the replication of the virus on the cells and basically meets the conditions of vaccine production, and from the time when the cells have CPE and the morphology of the CPE, the strain has the sign of weakening the toxicity of the cell after the gene deletion, which is also similar to the Bartha K61 strain; the cell culture level shows that the three-gene deletion strain constructed by the invention has the potential of being used as a vaccine strain.

2.3 cell passage stability

After the constructed three-gene deletion strain is subjected to blind passage on Vero cells for 30 generations, TCID50 of the virus is determined, and the gE and TK genes are amplified through PCR to identify the stability of the deletion strain.

After 30 blind passages, the TCID50 of the three-gene deletion strain is 10^-6.67mL, slightly increased compared to the initial passage, probably due to strain adaptation to the cell. The PCR identification has no TK and gE genes, and proves that the deleted strain can be stably passaged on Vero cells, the gene recombination is avoided, the strain is pure, and the wild virus pollution is avoided.

The three-gene deletion strain is pseudo rabies GD0304 TK^-gE^-gI^-The strain is delivered to China Center for Type Culture Collection (CCTCC) for preservation, named PRV GD0304 gE^-gI^-TK^-The preservation number is: CCTCC NO, V201955, the preservation time is as follows: 2019.8.15.

2.4 drug resistance testing

In the separation stage of GD0304 wild strain, the strain is found to have serious drug resistance to conventional anti-herpes virus drugs acyclovir (acyclovir) and ganciclovir (ganciclovir), and the action mechanisms of the two drugs and the chest expressed by virus TK geneThe adenokinases are related. Therefore, after the three-gene deletion is completed, the drug resistance of the deleted strain is tested. The test method comprises respectively subjecting GD0304 wild strain and GD0304 TK under the same 1MOI infection coefficient^-gE^-gI^-The strain and Bartha K61 strain were inoculated into Vero cells, and then a gradient of diluted antiviral drug was added to the cell supernatant to a final concentration of between 0 and 200mg/mL, and the resistance of the strain was evaluated by observing the CPE of the cells, and the results are shown in Table 3.

TABLE 3 susceptibility of different strains to anti-herpes virus drugs

Note: "+" indicates that the cell had CPE and "-" indicates that the cell had no CPE.

From the test results, under the highest concentration of 200mg/mL, the GD0304 wild strain and the three-gene deletion strain thereof can generate obvious CPE, the occurrence time and the CPE form have no obvious difference with those of a control group without drugs, while the Bartha K61 group used as a comparison has the advantages that the growth of viruses on cells is completely inhibited under the drug concentration of 2mg/mL, after the TK gene is deleted, the three-gene deletion strain still generates strong drug resistance to acyclovir and ganciclovir as the wild strain, the mechanism of the drug resistance generated by the strain is irrelevant to the TK gene, and the drug resistance mechanism of the strain is to be further researched. The value of the GD0304 strain in the research of a drug-resistant mechanism of the herpes virus is reflected, and the three-gene deletion strain is used as a vaccine strain and can possibly play a certain role in protecting the epidemic drug-resistant strain.

2.5 mouse safety test

Are respectively provided with 10^-6.67The three-gene deletion virus culture solution of/mL TCID50, GD0304 wild strain of 10LD50 and cell culture solution control group are used for nasal administration at a dose of 100 mu L and are 6 weeks oldAfter 7 days, the wild virus mice die, and the control group mice and the three-gene deletion group mice survive, which shows that after gene deletion, the virus toxicity is reduced, and the safety of the mice is higher.

2.6 mouse immune challenge test

In order to verify the protective effect of the three-gene deletion strain as a vaccine, an immune challenge test is designed on a mouse. The immunization scheme is that as shown in Table 4, the doses of the three-gene deletion inactivated virus and the supernatant of the live virus are 10⁴TCID₅₀Each mouse was immunized with a dose of greater than 10 after dilution of commercial vaccine groups as specified⁴TCID₅₀. The virus inactivation method comprises the steps of inactivating formaldehyde with the final concentration of 0.2% for 24 hours, and verifying complete inactivation through virus inoculation Vero cells; adjuvant 1 is MONTANIDE from SEPPIC^TMGel 01PR adjuvant, which is prepared according to the adjuvant specification after the inactivated virus supernatant is diluted to the required concentration (the virus supernatant diluent: adjuvant is 9: 1); adjuvant 2 is MONTANIDE from SEPPIC^TMISA201VG adjuvant, which is prepared according to the instructions after the inactivated virus supernatant is diluted to the required concentration (the virus supernatant diluent: adjuvant is 1: 1); the live virus supernatant experimental group is that Vero cell culture virus supernatant is directly used for immunization after being diluted to required immunization dose by DMEM medium; a commercial Bartha K61 live vaccine (sarahrague bio-pharmaceuticals, ltd., pseudorabies live vaccine (Bartha-K61 strain) diluted according to the instruction) was used as a control experiment group, and Vero cell culture supernatant was used as a blank control group, and 3 immunizations were performed by abdominal subcutaneous injection. After the immunization is finished, part of mice are subjected to a wild strain challenge test, the challenge scheme is shown in table 5, and the rest of mice are subjected to a virus neutralization test.

TABLE 4 immune grouping table of three-gene deletion vaccine

TABLE 5 mode of attack and dosage

After the virus is attacked, the death condition of the mouse is continuously observed for 7 days, the death mouse is subjected to autopsy in time, tissues and organs such as brain, liver, spleen, lung, kidney and the like are taken, nucleic acid is extracted, and then 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. The death of the mice after challenge is shown in table 6, the necropsy of the mice is shown in fig. 7, the fluorescent PCR results of the viral genes of the organs of the 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 mice death record after immune challenge

TABLE 7 dead mouse Virus Gene tissue distribution results

TABLE 8 neutralization of different strains by mouse immune sera

From the mouse immune challenge test results, the classical Bartha strain vaccine can weaken the challenge protection capability of the pseudorabies variant strain on the mouse, and can not completely protect the mouse from death, but the three-gene deletion strain prepared based on the GD0304 strain can provide the protection capability superior to the Bartha strain in the inactivated and live virus forms, particularly the inactivated virus and adjuvant group, and the 100% challenge protection rate is achieved; it can also be seen from the serum neutralization titers that the inactivated vaccine prepared based on the pseudorabies GD0304 TK-gE-gI-strain has higher neutralization titers against wild-type GD0304, although in this experiment the live virus immunization group may have lower serum neutralization titers due to lack of adjuvant to stimulate immunization.

2.7 piglet immune challenge test

In order to test the performance of the three-gene deletion strain as a vaccine candidate strain, a piglet immune challenge test is designed on the basis of a mouse test, the 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, and a commercial inactivated vaccine (Haibolai, porcine pseudorabies inactivated vaccine (Bartha K61 strain)) is immunized according to a vaccine specification, the test groups and the design are shown in Table 9, and the live virus group is not set in the immune challenge test due to the limitation of test conditions.

TABLE 9 immunization challenge test groups for piglets

Blood is collected before challenge for serum neutralization titer detection, and the neutralization titer of each experimental group serum for GD0304 strain is shown in Table 10. The clinical symptoms of the experimental pigs, including fever, behavior and feeding change, mortality and the like, were observed within 14 days after challenge, and the results are shown in table 11, and the remaining pigs were killed after 14 days, and the pathological changes of the organs were observed, and are shown in fig. 8. It can be seen that no obvious organ lesion is observed in other experimental groups except the control group D which has obvious hyperemia lesion of main target organs of porcine pseudorabies.

TABLE 10 neutralization titers of piglet sera of different vaccine groups against PRV GD0304 wild strain

Grouping	Class of vaccine	Immunization dose/mL	Number of immunizations	Neutralizing 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 efficacy of inactivated porcine pseudorabies vaccine

Note: the protection rate is the protection rate aiming at the clinical symptoms of the pseudorabies of the piglets.

From the results of the piglet immunity challenge test, the inactivated vaccine prepared by the three-gene deletion strain can stimulate piglets to generate higher serum neutralizing antibodies aiming at wild strains, can also generate better challenge protection effect in a challenge experiment, can protect the piglets from death, and can effectively avoid the clinical symptoms of pseudorabies. The autopsy result also proves that the vaccine group can protect each main organ of the piglets from being attacked by the virus. Meanwhile, the virus challenge result of the commercial Bartha strain vaccine group can also show that 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 pseudorabies clinical symptoms such as body temperature rise, shortness of breath and the like, and although the death of the pigs is not caused at last, the production performance of the pigs is necessarily reduced in the actual breeding process, and the disease response can be aggravated and the death risk can be increased if the pigs are infected by stress or other epidemic diseases.

2.8 summary of immune challenge test

As can be seen from immune challenge tests of mice and piglets, compared with the classical Bartha K61 strain, the inactivated or live virus vaccine prepared from the pseudorabies variant strain with three-gene deletion has better challenge protection effect on the variant strain, can effectively reduce the morbidity and the mortality, and has higher serum neutralization titer. The three-gene deletion strain has higher application and commercial value in the prevention and control of the pseudorabies variant strain, and can be used as an excellent pseudorabies vaccine candidate strain.

The present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.

SEQUENCE LISTING

<110> Guangdong sea Daorhusbandry veterinary research institute Co., Ltd

<120> porcine pseudorabies virus gE/gI/TK gene deletion vaccine strain, and construction method and application thereof

<130>

<160> 8

<170> PatentIn version 3.5

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Claims

1. A porcine pseudorabies virus gE/gI/TK gene deletion strain, wherein the porcine pseudorabies virus gE/gI/TK gene deletion strain is porcine pseudorabies virus PRV GD0304 with gE, gI and TK gene deletion; the preservation number of the porcine pseudorabies PRV GD0304 is CCTCC NO: V201946, and the preservation time is as follows: 2019.8.15, depository: china center for type culture Collection, collection address: wuhan, Wuhan university.

2. The porcine pseudorabies virus gE/gI/TK gene deletion strain according to claim 2, wherein the porcine pseudorabies virus gE/gI/TK gene deletion strain is named: porcine pseudorabies virus PRV GD0304 gE^-gI^-TK^-The preservation number is CCTCC NO: V201955, the preservation time is as follows: 2019.8.15, depository: china center for type culture Collection, collection address: wuhan, Wuhan university.

3. An antibody prepared by using the porcine pseudorabies virus gE/gI/TK gene deletion strain as claimed in claim 1 or 2 as an immunogen.

4. The use of the porcine pseudorabies virus gE/gI/TK gene deletion strain or the porcine pseudorabies PRV GD0304 of claim 1 or 2 in the preparation of a product for diagnosing or detecting porcine pseudorabies virus.

5. Use of the porcine pseudorabies virus gE/gI/TK gene-deleted strain or the porcine pseudorabies PRV GD0304 of claim 1 or 2 in the preparation of a product for preventing or treating porcine pseudorabies; preferably, the product is a vaccine.

6. The use of the porcine pseudorabies virus gE/gI/TK gene deletion strain or the porcine pseudorabies PRV GD0304 of claim 1 or 2 for constructing a porcine pseudorabies virus challenge and pathogenesis model.

7. A product comprising the porcine pseudorabies gE/gI/TK gene-deleted strain of claim 1 or 2 or porcine pseudorabies PRV GD 0304.

8. The product of claim 7, further comprising a pharmaceutically acceptable adjuvant, carrier, protectant, diluent, or excipient; preferably, the adjuvant is Gel 01PR, ISA201VG or aluminum salt adjuvant, protein adjuvant and nucleic acid adjuvant which can be applied to veterinary vaccines.

9. The product according to claim 7, wherein the product is a vaccine, preferably wherein the vaccine is an inactivated vaccine or a live vaccine.

10. A method for preparing a vaccine for preventing or treating porcine pseudorabies comprises the following steps: preparing the virus solution of the porcine pseudorabies virus gE/gI/TK gene deletion strain of claim 1 or 2, and then adding an adjuvant.