CN115386556A - Genetically engineered vaccine for recombinant pseudorabies virus of genes P30 and P54 of African swine fever virus expressed in series and application of genetically engineered vaccine - Google Patents

Genetically engineered vaccine for recombinant pseudorabies virus of genes P30 and P54 of African swine fever virus expressed in series and application of genetically engineered vaccine Download PDF

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CN115386556A
CN115386556A CN202210482517.3A CN202210482517A CN115386556A CN 115386556 A CN115386556 A CN 115386556A CN 202210482517 A CN202210482517 A CN 202210482517A CN 115386556 A CN115386556 A CN 115386556A
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pseudorabies virus
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童武
周艳君
童光志
包玮玲
高飞
郑浩
李国新
于海
单同领
姜一峰
虞凌雪
刘长龙
李丽薇
孔宁
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Shanghai Veterinary Research Institute CAAS
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Abstract

The invention provides a gene engineering vaccine for recombining an African swine fever virus P30 and P54 genes in a tandem expression manner and a pseudorabies virus gene and application thereof, and the research is based on a PRV JS-2012-delta gI/gE strain and utilizes a homologous recombination method to insert a gene sequence of main antigen proteins (P30 and P54 proteins) of the African swine fever virus which is popular in China at present into a vector PRV JS-2012-delta gI/gE vaccine strain so as to construct a recombined pseudorabies virus gene deletion vaccine strain rPRV JS-2012-delta gI/gE + P3054 containing the African swine fever virus P30 and P54 gene sequences. Through PCR and indirect immunofluorescence verification, the inserted genes P30 and P54 of the African swine fever virus can be stably and efficiently expressed in the vector virus PRV JS-2012-delta gI/gE. The biological properties further suggest: the recombinant virus rPRV JS-2012- Δ gI/gE + P3054 is basically similar to the parent virus PRV JS-2012- Δ gI/gE.

Description

Genetically engineered vaccine for recombinant pseudorabies virus of genes P30 and P54 of African swine fever virus expressed in series and application of genetically engineered vaccine
Technical Field
The invention belongs to the technical field of genetic engineering vaccines, and particularly relates to a recombinant pseudorabies virus rPRV and an African swine fever vaccine prepared by using the rPRV, and more particularly relates to construction of a gene recombinant pseudorabies virus genetic engineering vaccine PRV JS-2012-delta gE/gI + P3054 for serially expressing African swine fever virus P30 and P54, and specific application thereof.
Background
African Swine Fever (ASF) is an acute, virulent, and contact infectious disease caused by African Swine Fever Virus (ASFV). The disease is reported to be developed for the first time in 8 months in 2018 in China, then the disease is rapidly spread, in a short time, ASF epidemic situations occur in 32 provinces, cities and autonomous regions in China, and the most disastrous economic loss is caused to the pig industry in China. The ASF epidemic situation reduces the stock quantity and the stock quantity of domestic pigs by 20-50%, and the effective supply of pork in China is seriously weakened in many pig farms because the ASF epidemic situation completely covers, so that the prices of domestic pigs and pork are doubled. The popularity of ASF in our country has attracted considerable attention all over the world, both in the world's first-pig and pork-consuming countries. The control of the ASF epidemic situation is the first work task of veterinarians in China, and the pig farm is used as the main battlefield of veterinary science and technology workers, so that an efficient ASF prevention and control technology and an effective ASF prevention and control measure are urgently needed.
ASFV is an enveloped, linear double-stranded DNA virus, the only member of the genus African swine fever virus of the family African swine fever Virus. The virus particle has a complex structure and is divided into five layers from inside to outside: a nucleoid (nucleoid) containing viral genome, an inner core shell (core shell), an inner membrane (inner envelope), a capsid (capsid), and an outer envelope (outer envelope). The ASFV genome has a total length of 170-194kbp, consists of a central conserved region and two end variable regions, and encodes 151-167 Open Reading Frames (ORFs). Because of the variable number of repeats that a multigene family (MGF) encoded by a viral gene can acquire or lose in ORFs and short tandem repeats (short tandems repeats) present in or between viral genes, there are differences in genome length of different viral isolates. At present, the quantity and function of the ASFV genome-encoded proteins are not completely clear.
ASFV-susceptible mammals are limited to pigs, a variety of pigs of different sizes, including domestic pigs, wild pigs, and wart pigs. Apart from pigs, ASFV can infect ticks, and ticks play an important role in the transmission of ASFV. Soft ticks and wart pigs are natural storage hosts of ASFV, maintaining the virus in nature for a long time. The virus survives for years in the soft tick and can be vertically transmitted to offspring. The wart pig has certain resistance to ASFV, does not produce clinical symptoms after infection, but can produce viremia for a long time, and is an important carrier and an infection source of the virus. Warts pigs do not transmit ASFV horizontally or vertically and must be mediated by ticks. However, in the domestic and wild boar groups, ASFV presents a high degree of contact transmission, which is achieved by direct contact between pigs or contact with infected pig excreta and its contaminants, infected pig meat and blood products and residues. The susceptibility of domestic pigs and wild pigs to ASFV is similar, the infection latency is usually 4-19 days, and the infection with different virulent strains presents different clinical symptoms: the most acute and acute cases are caused by virulent strain infection, and the death rate reaches 100 percent; the mesogenic strains cause sub-acute cases, and the death rate is 30-70%. The chronic type is caused by the infection of low virulent strains, the disease course can last for 5 to 12 months, the internal hemorrhage of the pig is not caused, the symptoms such as skin necrosis and ulcer, joint inflammation and swelling, lymph node hyperplasia and the like are mainly shown, and the growth is slow and even the emaciation is realized. The chronic pig is infectious during the persistent infection process, and can infect the contacted susceptible animals. Pigs with latent infection are also infectious and may be detoxified for hours before clinical symptoms appear. The rehabilitated pigs may also be toxic for long periods and may transmit the virus. ASF has great harm to the pig industry, is listed as an animal epidemic disease which is legally reported by the world animal health Organization (OIE), and is listed as an animal infectious disease in China. Currently, there are no effective vaccines and drugs available for ASF for prophylaxis and therapy.
Vaccines are a common technique and efficient means of controlling viral and bacterial infectious diseases. Various human infectious diseases and animal epidemic diseases are effectively controlled or even eradicated through the use of vaccines. However, due to the complex biological characteristics of ASFV, there is still no clear recognition in many aspects such as virus invasion and immune protection response induction, and in the course of ASF vaccine research in the last half century, different research groups have utilized various technical methods, but have not developed a safe and effective ASF vaccine. Although inactivated vaccines induce the production of antibodies against ASFV, immunized pigs are not resistant to challenge with virulent strains. Even if the modern adjuvant is used for preparing the ASF inactivated vaccine, the ASF inactivated vaccine still has no protective effect on the virus attack of the homologous virulent strain.
Through long-term research, a plurality of ASFV virulence genes, such as K196R, B119L, EP402R, DP148R, DP71L, DP96R, MGF360/505 and the like, are identified. With the continuous development of molecular biology, the genetic operation of viral genomes is easier, and the construction of ASFV gene deletion attenuated strains by a genetic engineering means is more convenient. However, the attenuated strains still have doubts about safety, which are mainly expressed in three aspects: one is the problem of side reactions. Some attenuated strains have damage to hosts after inoculation, and are clinically manifested as fever, arthritis and chronic damage; secondly, there is a risk of virus scattering in the inoculation of the attenuated strain. Some of the attenuated strains produce low levels of viremia after inoculation and can be excreted in vitro to infect other susceptible animals; thirdly, the virulence returns strongly. Although ASFV is a DNA virus, it has a hypervariable region in its genome and is susceptible to mutation. Meanwhile, the low virulent strain needs to be cultured by primary cells, which is not beneficial to industrialized production, so the low virulent strain is not preferable for the African swine fever vaccine at present.
DNA vaccines and virus live vector vaccines show better safety, but the immune efficiency thereof still needs to be improved. Among them, subunit vaccines can produce neutralizing antibodies, but after homologous strong virus challenge, only delay the occurrence of clinical symptoms, but cannot resist virus infection. DNA vaccines lack some good viral antigens. The live virus vector vaccine can induce specific antibody immunity and cell immune response, but can play a role in resisting ASFV after immunizing pigs, and needs to be discussed.
With the vigorous development of DNA recombination technology, reverse genetic manipulation, gene editing and other technologies, the research direction of vaccines is gradually transiting to genetic engineering vaccines, and recombinant live vector vaccines are more and more attracting attention. The vectors currently used for developing viral vaccines involved in the study of live viral vector vaccines mainly include poxviruses, herpesviruses, adenoviruses, and the like. Adenovirus is replication-defective virus, while poxvirus has large genome and more genome expression products, and foreign proteins expressed by the two are difficult to stimulate the body to generate stronger immune response. The genome of the herpesvirus is large, a plurality of replication nonessential regions can be inserted into a plurality of exogenous genes, and the live vector vaccine constructed by the herpesvirus can induce specific mucosal immunity. At present, herpesviruses involved in veterinary live vector vaccine research mainly include Pseudorabies virus (PRV) and Duck plague virus (DEV). The pig is not a host of the duck plague virus, and the immunoprotection effect of the recombinant duck plague virus on ASFV infection after immunizing a pig body is uncertain.
Because the PRV genome is large, PRV has many non-essential genes and non-coding regions, and the insertion of foreign gene sequences in these regions does not affect the replication of PRV itself, which also makes PRV live vector vaccines a hot spot for virus genetic engineering vaccine research. Meanwhile, the comparison of domestic applied patents shows that: zhejiang Hippocampus Biotech Co., ltd constructs a recombinant pseudorabies virus (PRV-BAC-P72-B602L-CD 2V-delta TK-delta gG strain) expressing African swine fever virus P72, B602L and CD2V genes, namely deleting the TK gene and the gG gene of the PRV virus, inserting the P72 and B602L genes of the African swine fever virus at the TK gene deletion position, and inserting the CD2V gene of the African swine fever virus at the gG gene deletion position. The recombinant virus has defects, firstly, the deletion of TK gene leads the virus virulence to be greatly reduced for PRV, the virus virulence and the immunity effect are in positive correlation for vaccine, the virus virulence is too weak to enter the organism and then is replicated and limited, so that the excited immunogenicity is not strong, and the carrier vaccine mainly depends on the recombinant virus to replicate after entering the organism, so that the inserted exogenous gene is expressed along with the replication of the virus to generate the immune protection effect, if the recombinant virus does not replicate in the organism or the replication level is low, the protective effect on the exogenous gene is definitely not good. It is known that PRV is extremely lethal to mice, onlyThe TK gene is knocked out before it is safe for mice, while pigs are much more tolerant to PRV, and the virus safe for pigs may still be highly lethal to mice (Wu Tong, guoxin Li, chao Liang, et al.a live, attentuated viruses strain JS-2012deleted for gE/gii protects infected with bone virus and inspecting strain, additive Research 130 (110-117), gazec cloud, pseudorabies virus variant JS-2012 and gene deleted strain JS-2012- Δ gii/gE infected mice tissue distribution and experiments, university of young state, givea, while the virus safe for mice is almost completely pathogenic to pigs, in which they can produce specific antibodies after immunization of mice, but because the recombinant virus is too virulent to the pig, 2016 is still unknown for the foreign proteins. And secondly, the gE gene of the recombinant virus is not deleted, and the gE gene is not deleted for PRV, so that PRVgE antibody of a pig farm is positive, PRV purification of the pig farm cannot be carried out, and the recombinant virus is not suitable for clinical application in China. Thirdly, the PRV viral vector used by the recombinant virus is a pseudorabies virus strain (PRV HL), the strain is not yet developed for vaccine at present, and the condition for developing and using the PRV viral vector as a live vector vaccine is not provided. The Jiangxi agriculture university constructs a recombinant pseudorabies virus (PRV TK-/gI) expressing genes of African swine fever virus CD2V and P72 or P30 and P54 - /gE - - CD2v + /p72 + Strain or PRV TK-/gI - /gE - -P54 + /P30 + Strain), namely deleting TK gene and gI/gE gene of PRV virus, inserting CD2V or P54 gene of African swine fever virus at the TK gene deletion position, and inserting P72 or P30 gene of African swine fever virus at the gI/gE gene deletion position. Firstly, zhejiang Hailong Biotechnology limited company has weak toxicity to the constructed recombinant virus, which is not beneficial to the expression of foreign protein in the pig body, and can understand whether to provide protection effect for immune pig group. Secondly, part of the foreign gene of the recombinant virus is inserted at the deletion of gI/gE gene, while it is known that the gI/gE gene of PRV is unstable and is susceptible to deletion mutation with passage of the virus (Chao Liang, wu Tong, hao Zheng, fei Liu, jiqiiangWu, guoxin Li, en-min Zhou, guangzhi)Tong, A high-temperature passaging infected Pseudorabies viruses vaccine salts against which PRV variant is expressed 109-115. In this case, the investigator inserted the foreign gene into this position, apparently not the correct choice (Tong Guang, zhenghao, tong Wu, lixin, zhou Jun, shanghai, gao, jiangyan, yu Ling, recombinant Pseudorabies virus strain expressing classical swine fever virus E2 protein and its preparation method and application, yu jin, china, ZL 810627238.5, 02/08/2022). Third, there is no information on the development of vaccines for the same PRV viral vector strains used for the recombinant viruses.
The PRV virus live vector selected in the patent is 2016 Tongwu virus, and the like, utilizes a homologous recombination technology, and takes a variant strain JS-2012 as a framework, so as to successfully construct a pseudorabies double-gene deletion virus JS-2012-delta gE/gI (Tongliu, tongwu, zhenhao, liufei, liangqiao, zhouyanjun, union collar, hai, jiangyi, gaoshan, pseudorabies virus gene deletion attenuated strain and a preparation method and application thereof, 09.07.2018, ju, ZL10002656.7. Wu Tong, guoxin Li, chao Liang, fei Liu, qing Tian, yanyun Cao, lin Li, xuche Zheng, hao Zheng, guangzhi tong.a live, attentuated pseudovitates virus strain JS-2012deleted for gE/gI protects against bone clinical and empirical tissues antibiotic Research 130 (2016) 110-117), studies have shown that this two-gene deletion strain has good safety and efficacy for piglets, and this vaccine is currently waiting for new veterinary drug certificates in the nuclear (clinical trial approval no: 2015051, 2017069, 20190012). In the early stage, the porcine pseudorabies virus double-gene deletion attenuated vaccine strain (JS-2012-delta gE/gI strain) is also used as a recombinant virus rPRV JS-2012-delta gI/gE + E2 for expressing the classical swine fever virus E2 protein by using a vector. The recombinant virus can be used for vaccinating pigs, and does not cause any side effect or tissue damage. The immune pig can completely resist the attack of CSFV virulent virus (shimen) by a single inoculation. Even for piglets with PRV maternal antibodies, rPRV JS-2012-delta gI/gE + E2 immunization can achieve good immune protection effect, and completely protect the piglets against virulent attacks (childhood, zhenghao, tongwu, lizhongxin, zhouyujinjun, shanghai, gaifeng, jiangyiping, yuzhuang, recombinant pseudocanine virus strain expressing classical swine fever virus E2 protein and preparation method and application thereof, no. 02/08 of 2022, china, ZL201810627238.5.Wu Tong, hao Zhuang, guo-xin Li, fei Liu, qing Tien, yanyun Cao, FV Li, xuche Zhuhen Zheng, hao Zhuang, guangzhhi Tong. The early-stage research finds that although the rPRV JS-2012-delta gI/gE + E2 can play a good immune protection effect after immunizing piglets, the E2 antibody level of CSFV is not ideal after immunizing piglets by the rPRV JS-2012-delta gI/gE + E2, in the research, when an exogenous gene of African swine fever virus is selected, a certain codon optimization is carried out on the sequence, and the immune piglets generate specific antibodies aiming at P30 and P54 obviously increased after immunizing the recombinant virus according to the animal test result, and the E2 antibody level is obviously better than that after immunizing the rPRV JS-2012-delta gI/gE + P3054 according to the induced antibody level.
Disclosure of Invention
In order to construct recombinant attenuated strains for the preparation of live vector vaccines for the treatment and prevention of African swine fever, we have conducted intensive studies on pseudorabies virus (PRV) as a live vector for African swine fever virus. In earlier researches, a gene-deleted attenuated vaccine strain of a porcine Pseudorabies virus (PRV) is constructed as a recombinant virus rPRV JS-2012-delta gI/gE + E2 for expressing a Classical Swine Fever Virus (CSFV) E2 protein by using a vector. The recombinant virus can be used for vaccinating pigs, and does not cause any side effect or tissue damage. The immune pig can completely resist the attack of CSFV virulent virus (shimen) by a single inoculation. Even for piglets with PRV maternal antibodies, the rPRV JS-2012-delta gI/gE + E2 immunity can obtain good immune protection effect, and the piglets are completely protected against virulent attack. This is far superior to the immune effect against CSFV produced by recombinant viruses expressing E2 protein using Newcastle disease virus, adenovirus and poxvirus as vectors. These results show that the PRV gene deletion attenuated vaccine strain can effectively express and present foreign protein in the pig body, induce strong immune response and is a high-efficiency live vaccine virus vector for pigs. According to the previous research, although a good immune protection effect can be achieved after the piglets are immunized by the rPRV JS-2012-delta gI/gE + E2, the E2 antibody level of CSFV is not ideal, in the research, when the exogenous gene of African swine fever virus is selected, the sequence of the exogenous gene is optimized by certain codons, and from the animal test result, after the immunization of the recombinant virus, the specific antibodies of the immunized piglets against P30 and P54 are obviously increased, and from the induced antibody level, the P30 and P54 antibody levels after the immunization of the PRV JS-2012-delta gE/gI + P3054 are obviously better than the E2 antibody level after the immunization of the rPRV JS-2012-delta gI/gE + E2.
On the basis, the invention provides a recombinant pseudorabies virus genetic engineering vaccine for serially expressing genes P30 and P54 of African swine fever virus, which is characterized in that the recombinant pseudorabies virus genetic engineering vaccine expresses exogenous genes P30 and P54 of the African swine fever virus and lacks genes gI and gE in the pseudorabies virus;
further, the recombinant pseudorabies virus genetic engineering vaccine is pseudorabies virus PRV JS-2012-delta gE/gI + P3054; the recombinant pseudorabies virus genetic engineering vaccine (pseudorabies virus PRV JS-2012-delta gE/gI + P3054) is preserved in China center for type culture Collection (CCTCC for short) at 16 months 4 in 2022, and is addressed to eight-path Wuhan university Collection 299 # in Wuhan district, wuhan City, hubei province, and the preservation number is CCTCC No. V202230.
Further, in another aspect of the present invention, there is provided a method for preparing a recombinant pseudorabies virus genetic engineering vaccine, comprising the following steps:
(5) Constructing a recombinant vector, designing primers to amplify homologous recombination left and right recombinant arms, carrying out enzyme digestion on a CMV promoter-EGFP gene fragment and an SV40 polyA signal sequence fragment, and sequentially assembling the left recombinant arm-CMV promoter-EGFP gene-SV 40 polyA signal sequence-right recombinant arm to obtain the recombinant transfer vector.
(6) Extracting total DNA of a pseudorabies virus JS-2012-delta gI/gE strain, and cotransfecting the total DNA and the recombinant vector in the step (1) to obtain a recombinant pseudorabies virus containing an EGFP fluorescent label;
(7) Constructing a recombinant vector carrying P30 and P54, wherein the recombinant vector replaces the EGFP gene in the recombinant vector in the step (1) by utilizing a P3054 protein;
(8) Extracting the total DNA of the recombinant pseudorabies virus obtained in the step (2), and cotransfecting the total DNA and the recombinant vector carrying P3054 in the step (3) to obtain the recombinant pseudorabies virus not containing EGFP fluorescent markers, wherein the recombinant pseudorabies virus is the recombinant pseudorabies virus genetic engineering vaccine PRV JS-2012-delta gE/gI + P3054.
In another aspect of the invention, there is also provided a vaccine composition comprising the recombinant pseudorabies virus genetically engineered vaccine PRV JS-2012- Δ gE/gI + P3054 strain in admixture with an adjuvant or a pharmaceutically acceptable carrier.
The vaccine composition is suitable for nasal drop and injection inoculation.
In another aspect of the invention, the invention also provides an application of the recombinant pseudorabies virus genetic engineering vaccine PRV JS-2012-delta gE/gI + P3054 strain in preparing a vaccine for preventing or treating pseudorabies and African swine fever.
The recombinant pseudorabies virus genetic engineering vaccine JS-2012-delta gE/gI + P3054 strain is inoculated to a 15-day-old piglet, no clinical symptoms appear, and meanwhile, immune pigs can generate specific antibodies aiming at African swine fever virus P30 and P54 proteins.
Drawings
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a diagram showing the results of PCR identification of insertion of rPRV JS-2012- Δ gE/gI + P3054 virus into P30 and P54 genes, wherein "M" refers to DNA Marker DL2000, "1" rPRV JS-2012- Δ gE/gI + P3054 virus, "2" parent virus JS-2012- Δ gI/gE, "3" positive control.
FIG. 2 is a diagram showing the results of PCR identification of the insertion sites of rPRV JS-2012- Δ gE/gI + P3054 virus, wherein "M" means DNA Marker DL5000, "1" rPRV JS-2012- Δ gE/gI + P3054 virus, "2" parental virus JS-2012- Δ gI/gE strain, and "3" means positive control.
FIG. 3 one-step growth curves of rPRV JS-2012- Δ gE/gI + P3054 virus and parental virus.
FIG. 4 plaque morphology observations of rPRV JS-2012- Δ gE/gI + P3054 virus and parental virus.
FIG. 5 is a PCR identification result chart of different generations of inserted P30 and P54 genes of rPRV JS-2012- Δ gE/gI + P3054 virus, wherein "M" refers to DNA Marker DL2000, "1-20" rPRV JS-2012- Δ gE/gI + P3054 virus generation 1-20, "21" positive control, and "22" refers to negative control.
FIG. 6 is a diagram showing the results of PCR identification of different generations of passaged disease insertion sites of rPRV JS-2012- Δ gE/gI + P3054 virus, wherein "M" refers to DNA Marker DL5000, "1-20" rPRV JS-2012- Δ gE/gI + P3054 virus generation 1-20 passaged virus, "21" parent virus PRV JS-2012- Δ gE/gI strain (positive control), "22" refers to negative control, and "M" refers to DNA Marker DL2000. FIG. 7 result of IFA experiment using P30 polyclonal antibody for rPRV JS-2012- Δ gE/gI + P3054 different generations of viruses
FIG. 8 results of experiments with P54 mAb as IFA for rPRV JS-2012- Δ gE/gI + P3054 different progeny viruses
FIG. 9 sequencing results of the P30 and P54 genes of rPRV JS-2012- Δ gE/gI + P3054 different generation viruses
FIG. 10 body temperature profile of rPRV JS-2012- Δ gE/gI + P3054 Virus after vaccination of suckling piglets
FIG. 11 Ocular anatomic lesions following rPRV JS-2012- Δ gE/gI + P3054 Virus inoculation on suckling piglets
FIG. 12 dynamic changes in P30 protein antibodies following rPRV JS-2012- Δ gE/gI + P3054 Virus immunization of suckling piglets
FIG. 13 dynamic changes in P54 protein antibodies following rPRV JS-2012- Δ gE/gI + P3054 Virus immunization of suckling piglets
Detailed Description
In the following examples, the experimental procedures without specifying the specific conditions were generally carried out by the methods described in "molecular biology laboratory Manual of Fine text" (edited by F.M. Osber, R.E. Kingston, J.G. Sedman, et al, mashimi, shujiong, beijing: scientific Press, 2004).
Viral strains, cells and vectors
PRV JS-2012-delta gI/gE virus, BHK-21 cell and Vero cell are all preserved in pig infectious disease research laboratory of Shanghai veterinary research institute of Chinese academy of agricultural sciences. Plasmid pBIE-GFP was previously constructed by the swine infectious disease research laboratory of Shanghai veterinary institute, national academy of agricultural sciences. Plasmid and strain: pEGFP-N1, pEGFP-C3 and pBluescript SK (+) were purchased from Shanghai Basidione Biotech Ltd.
MEM and DMEN, available from Invitrogen; scaI, smaI, sacI, nheI, aseI, xbaI, ecorV, xhoI, aflII, and T4 DNA ligase, products of NEB corporation; fugene HD transfection reagent, product of promega corporation; a kit for rapid recovery of small amounts of DNA fragments, omega; 2 XGC Buffer II, dNTP Mix (2.5 mmol/L each), LA-Taq, DL-15000, DL-2000DNA Marker from Takara; other chemical reagents are imported or domestic analytical reagents; e.coli DH 5. Alpha. Competence; purchased from beijing tiangen.
Example 1 construction of recombinant Pseudorabies Virus Gene engineering vaccine JS-2012- Δ gE/gI + P3054 Strain
Primer design
According to the whole genome sequence of PRV JS-2012 strain, multiple primers (shown in table 1) are designed by Oligo 6.0 software, JS + EGFP-LF/JS + EGFP-LR and JS + EGFP-RF/JS + EGFP-RR are used for amplifying homologous recombination left arm and right arm, sacI enzyme digestion sites and aseI enzyme digestion sites are respectively added at the front end and the rear end of the left recombination arm, ecoRV enzyme digestion sites and AflII enzyme digestion sites are added at the front end of the right recombination arm, and XhoI enzyme digestion sites are added at the rear end. P30 identification up/P30 identification down, P54 identification up/P54 identification down and JS gG identification up/JS gG identification down are respectively used for amplifying the size of the exogenous gene and the size of the insertion position.
TABLE 1 primer sequences
Figure BDA0003628400750000081
Construction of PRV JS-2012-delta gI/gE virus containing EGFP green fluorescent protein
Two fragments of 119652 to 121157 bases and 121168 to 122680 bases in PRV JS-2012 genome are amplified by using primers JS + EGFP-LF/JS + EGFP-LR and JS + EGFP-RF/JS + EGFP-RR by a PCR method and are used as a left recombination arm and a right recombination arm of homologous recombination. Meanwhile, a CMV promoter-EGFP gene fragment is cut out from a pEGFP-C3 plasmid through endonuclease digestion, an SV40 polyA signal sequence fragment is cut out from a pEGFP-N1 plasmid, and a left recombination arm-CMV promoter-EGFP gene-SV 40 polyA signal sequence-right recombination arm are sequentially assembled in a pBluescript SK (+) vector, so that the recombinant transfer vector pBG-GFP is obtained.
Infecting BHK-21 cells with PRV JS-2012-delta gI/gE, collecting diseased cells after 70% -80% of cells are diseased, and extracting the total DNA of PRV JS-2012-delta gI/gE from the infected cells by a phenol-chloroform method. And (3) co-transfecting PRV JS-2012-delta gI/gE total DNA and pBG-GFP to BHK-21 cells, performing plaque purification on the collected recombinant viruses, and after 4 rounds of plaque purification, inoculating the obtained viruses to vero cells to generate plaques, wherein the plaques can express EGFP. The successful construction of the PRV JS-2012-delta gI/gE virus containing the EGFP green fluorescent protein is prompted, and the virus is named as rPRV JS-2012-delta gI/gE + EGFP.
Replacement of EGFP marker gene containing EGFP green fluorescent protein PRV JS-2012-delta gI/gE virus
The EGFP gene of the pBG-GFP plasmid of the recombinant vector is cut off by NheI and XbaI double enzyme digestion, the remaining large fragment is recovered, the P3054 gene and the recovered large fragment are connected into a new recombinant plasmid, and the new recombinant plasmid is named as pBG-P3054.
And extracting the total DNA of the PRV JS-2012-delta gI/gE virus containing the EGFP green fluorescent protein by the method. The extracted total DNA and pBG-P3054 are co-transfected into BHK-21 cells, and PRV JS-2012-delta gI/gE recombinant viruses which do not contain EGFP green fluorescent markers and contain P3054 genes are rescued. The primary construction of the recombinant virus is successfully observed by a fluorescence microscope.
Extracting DNA of PRV JS-2012-delta gI/gE virus of the tandem expression P3054 gene, and performing PCR identification by using a P30 identification up/P54 identification down primer. The PCR reaction system is 20 μ L:10 × LA-Taq Buffer II 2ul; dNTP Mix (2.5 mmol/L each) 2ul; p30 identifies up 0.5ul (10 pmol/ul); p54 identifies down 0.5ul (10 pmol/ul); LA-Taq 0.5ul；ddH 2 O12.5 ul; viral DNA 2ul. The reaction conditions are all that is pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, extension at 72 ℃ for 2min, and circulation for 35 times; finally, extension was carried out at 72 ℃ for 10min. The PCR product was electrophoresed in 1% agarose and visualized in a gel imager. The PCR result shows that the sizes of the foreign gene sequences of the sizes of the fragments amplified by the P30 identification up/P54 identification down primers are consistent (figure 1).
And performing PCR identification by using JS gG identification up/JS gG identification down primers. The PCR reaction system is 20 μ L:2 XGC Buffer II10ul; dNTP Mix (2.5 mmol/L each) 2ul; JS gG identifies up 0.5ul (10 pmol/ul); JS gG identifies down 0.5ul (10 pmol/ul); LA-Taq 0.5ul; ddH 2 O4.5 ul; viral DNA 2ul. The reaction conditions are all that is pre-denaturation at 94 ℃ for 5min; denaturation at 94 ℃ for 30s, annealing at 64 ℃ for 30s, extension at 72 ℃ for 4min, and circulation for 35 times; finally, extension was carried out at 72 ℃ for 10min. The PCR product was electrophoresed in 1% agarose and visualized in a gel imager. The PCR result shows that JS gG identification up/JS gG identification down primer amplification is positive, and the size of the fragment is larger than that of parent virus PRV JS-2012-delta gI/gE, which is consistent with the expected result (figure 2). The results show that the PRV JS-2012-delta gI/gE virus for serially expressing P30 and P54 genes is successfully constructed, and the virus is named as rPRV JS-2012-delta gE/gI + P3054.
Example 2 identification of PRV JS-2012- Δ gI/gE Virus expressing P30, P54 genes in tandem and determination of Virus Titers
The PRV JS-2012-delta gI/gE virus of the tandem expression P30 and P54 genes is inoculated to Vero cells, and the virus is harvested when 80 percent of the cells have CPE. The titer of the virus was determined by 96-well tissue culture plate method, reference (Invitrogen et al, animal virology, science publishers, 1997). After 10-fold serial dilution of the virus with DMEM containing 2% fbs, the diluted virus was inoculated into Vero monolayers on 96-well cell culture plates. 8 wells were inoculated per dilution, 8-well control (inoculated with 2% FBS-containing DMEM) was set, the cells were incubated at 37 ℃ in a 5% carbon dioxide incubator, the number of wells in which CPE appeared was recorded every day until 4 days after inoculation, and TCID was calculated according to the Reed-Muench method 50 . The result shows that the titer of the rPRV JS-2012-delta gE/gI + P3054 virus on Vero cells is 10 8.5 TCID 50 /ml。
Example 3 analysis of biological Properties of PRV JS-2012- Δ gI/gE viruses expressing P30, P54 genes in tandem the PRV JS-2012- Δ gI/gE viruses expressing P30, P54 genes in tandem and their parent viruses were inoculated at a titer of 1MOI into Vero cells, the cell supernatants were harvested at different time points (4, 8, 12, 16, 20, 24, 28, 32, 36 h) after the inoculation, respectively, and the TCID of the viruses were performed, respectively 50 And (4) measuring, and drawing a curve according to the measurement result. The results showed that the growth trends of the rPRV JS-2012- Δ gE/gI + P3054 virus and the parent virus JS-2012- Δ gI/gE were substantially consistent (see FIG. 3).
The PRV JS-2012-delta gI/gE virus and the parent virus which serially express the P30 and P54 genes are both expressed by 10,1,0.1TCID 50 Infection of each well of 6-well plates with/mL virus, adsorption of 5% CO2 at 37 ℃ for 2h, addition of equal volumes of agarose and 2 x MEM, respectively, and mixing well until 2mL of mixed solution is added per well when the temperature is appropriate; standing at 4 deg.C until the coating layer is completely solidified, then reversing it, standing at 37 deg.C, 5% CO2 culturing for 4d; typical plaque formation is seen at appropriate viral infection levels after formaldehyde fixation and crystal violet staining. The plaque morphology formed by the rPRV JS-2012- Δ gE/gI + P3054 virus on Vero cells is not obviously different from that of the parent virus JS-2012- Δ gI/gE (see FIG. 4).
Genetic stability verification of PRV JS-2012-delta gI/gE virus for tandem expression of P30 and P54 genes
The PRV JS-2012-delta gI/gE virus with genes of P30 and P54 expressed in series is blind passed on Vero cells for 20 generations, and the genomes of each generation of virus are respectively extracted for exogenous gene amplification and insertion identification. F1, F5, F10, F15 and F20 virus generations are inoculated with Vero cells to extract the genome of each virus generation, and P30 identification up/P54 identification down and JS gG identification up/JS gG identification down primers are used for respectively carrying out exogenous gene amplification and insertion identification. The results show that: each progeny virus amplified the foreign gene (FIG. 5) and the size of the insertion site was consistent with the expected results (FIG. 6). The IFA results show: the foreign genes P30 and P54 in the F1, F5, F10, F15 and F20 generation viruses are all expressed with high efficiency (FIG. 7 and FIG. 8). The sequencing result shows that: no mutation was found in the P30 and P54 genes in the F1, F5, F10, F15 and F20 viruses (FIG. 9).
Safety and effectiveness analysis of PRV JS-2012-delta gI/gE virus for tandem expression of P30 and P54 genes
In order to determine the safety and effectiveness of the rPRV JS-2012-delta gE/gI + P3054 on piglets, PRV JS-2012-delta gI/gE viruses expressing P30 and P54 genes in tandem are divided into 10 5.0 TCID 50/head, immunizing 5 piglets of 15 days old respectively, and setting 5 piglets of the same day old as a blank control. Body temperature measurement is carried out every day within 10 days after immunization, clinical symptoms of the test pigs are observed every day, all the test pigs are killed after 35 days, and pathological changes of all tissues are observed. At the same time, the protein antibodies of African swine fever virus P30 and P54 are detected by blood collection at 7, 14, 21, 28 and 35 days after immunization. The experimental results show that: after the recombinant virus rPRV JS-2012- Δ gE/gI + P3054 is inoculated to the piglets, no adverse clinical reaction occurs (see Table 2), the body temperature of the piglets is similar to that of a blank control group, no fever occurs (figure 10), the ocular pathological changes of all organs are similar to that of the blank control group, no obvious ocular pathological changes occur (figure 11), and therefore the recombinant virus rPRV JS-2012- Δ gE/gI + P3054 is very safe for suckling piglets. Meanwhile, the detection result of the antibody shows that: the recombinant virus rPRV JS-2012- Δ gE/gI + P3054 can generate specific antibodies against P30 and P54 proteins of African swine fever virus 14 days after piglet inoculation (FIG. 12 and FIG. 13), and is expected to provide immune protection for the African swine fever virus.
TABLE 2 clinical recordings at various times after inoculation of piglets with rPRV JS-2012- Δ gE/gI + P3054
Figure BDA0003628400750000111
a is the number of days at body temperature greater than or equal to 40.5 ℃.
The above-mentioned embodiments only express the implementation manner of the present invention, and the description thereof is specific and detailed, but not to be understood as the limitation of the patent scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Shanghai veterinary research institute of Chinese academy of agricultural sciences & lt 110 >
Gene engineering vaccine for serially expressing African swine fever virus P30 and P54 genes and recombinant pseudorabies virus and application thereof
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CGAGCTCGCGACCGACGCCCAGCCCGTGAACC
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CATTAATAACTAGGACCGCTGGGCGTGGATCGCACCC
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CAGCTTAAGGGTCGGCGCCCCAGGTTCCCATAC
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CCGCTCGAGCAGCTCCACGCGCCCGCTGTAGTT
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ATGGATTTTATTTTAAATATATC
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TTACAAGGAGTTTTCTAGGTCTTT
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AGGAGGTGACCGAGGAGGAGGC
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CGCTGGCAGGTGAGTGTATGGG

Claims (6)

1. A recombinant pseudorabies virus genetic engineering vaccine for serially expressing genes P30 and P54 of African swine fever virus is characterized in that exogenous genes P30 and P54 of the African swine fever virus are serially expressed in the recombinant pseudorabies virus genetic engineering vaccine, and gI and gE genes in the pseudorabies virus are deleted.
2. The recombinant pseudorabies virus genetic engineering vaccine as claimed in claim 1, which is strain JS-2012- Δ gI/gE + P3054.
3. The recombinant pseudorabies virus strain serially expressing the genes P30 and P54 of African swine fever virus of claim 1, wherein the virus strain (JS-2012- Δ gI/gE + P3054 strain) is preserved in China Center for Type Culture Collection (CCTCC) at 16 months 4 in 2022, and the preservation number is CCTCC No. V202230 at the eight-channel preservation center of Wuhan university No. 299 in Wuchang district, wuhan City, hubei province.
4. A preparation method of a recombinant pseudorabies virus genetic engineering vaccine comprises the following steps:
(1) Constructing a recombinant vector, designing primers to amplify homologous recombination left and right recombinant arms, carrying out enzyme digestion on a CMV promoter-EGFP gene fragment and an SV40 polyA signal sequence fragment, and sequentially assembling the left recombinant arm-CMV promoter-EGFP gene-SV 40 polyA signal sequence-right recombinant arm to obtain a recombinant transfer vector;
(2) Extracting total DNA of a pseudorabies virus JS-2012-delta gI/gE strain, and cotransfecting the total DNA and the recombinant vector in the step (1) to obtain a recombinant pseudorabies virus containing an EGFP fluorescent label;
(3) Constructing a recombinant vector carrying P30 and P54, wherein the recombinant vector replaces the EGFP gene in the recombinant vector in the step (1) by fused P3054 protein;
(4) Extracting the total DNA of the recombinant pseudorabies virus obtained in the step (2), and cotransfecting the total DNA and the recombinant vector carrying P30 and P54 in the step (3) to obtain the recombinant pseudorabies virus not containing EGFP fluorescent markers, wherein the recombinant pseudorabies virus is the recombinant pseudorabies virus genetic engineering vaccine JS-2012-delta gI/gE + P3054 strain.
5. A vaccine composition comprising the recombinant pseudorabies virus genetically engineered vaccine JS-2012- Δ gI/gE + P3054 strain admixed with an adjuvant or a pharmaceutically acceptable carrier.
6. The use of the recombinant pseudorabies virus genetic engineering vaccine JS-2012- Δ gI/gE + P3054 strain as defined in claim 1 in the preparation of a vaccine for preventing or treating pseudorabies and swine fever.
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