CN113106071A - HQ06 epitope mutated swine fever candidate marker vaccine strain and application thereof - Google Patents
HQ06 epitope mutated swine fever candidate marker vaccine strain and application thereof Download PDFInfo
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Abstract
The invention discloses a candidate marked vaccine strain of hog cholera with HQ06 epitope mutation and application thereof. The invention takes a C strain as a framework, and the single point mutation of 3 key amino acids of the HQ06 epitope of the E2 protein117F、119G and122p, 3 mutant viruses were constructed. The 3 strain mutant viruses constructed by the invention lose the reactivity with a monoclonal antibody (mAb) HQ06, retain the reactivity with other antigenic sites of CSFV E2 protein, and keep the biological characteristics consistent with that of the C strain; wherein, rHCLV-E2P122A can reach the growth titer of parental virus in vitro and can reach the growth titer of parental virus in rabbits and pigsThe rHCLV-E2P122A strain can be developed into a swine fever marker vaccine by inducing an organism to produce an antibody aiming at CSFV E2 and a neutralizing antibody aiming at CSFV but losing inducing the organism to produce a specific antibody aiming at HQ06 epitope.
Description
Technical Field
The invention relates to a swine fever marked vaccine strain, in particular to a swine fever candidate marked vaccine strain with HQ06 epitope mutation and application thereof, belonging to the field of swine fever marked vaccine strains.
Background
Classical Swine Fever (CSF) is a highly contagious disease of pigs characterized primarily by high fever retention and extensive bleeding, causing significant economic losses to the swine industry in many countries around the world. The world animal health Organization (OIE) lists them in the Notifiable (Notifiable) List of animal diseases in OIE. In China, "China middle and long term animal epidemic prevention and control program (2012 and 2020)," one type of animal epidemic disease "ranks swine fever as one of five types of animal epidemic diseases that are preferentially prevented and controlled.
Classical Swine Fever Virus (CSFV), which is a member of the Flaviviridae (Flaviviridae) Pestivirus genus (Pestivirus), is the etiological agent of CSF. CSFV is a single-stranded positive-stranded RNA virus whose genome has a total length of about 12.3kb and comprises a5 'non-coding region (UTR), a long open reading frame and a 3' UTR. The reading frame encodes a polyprotein of 3898 amino acids, which is cleaved into 12 mature viral proteins including 4 structural proteins by cellular and viral proteases (C, E)rnsE1 and E2) and 8 non-structural proteins (N)proP7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), wherein E isrnsE1 and E2 are envelope proteins of the virus (Heimann M, Roman-Sosa G, Martoglio B, et al. core protein of pestivirus processed at the C terminal by signal peptide enzyme [ J].J Virol,2006,80(4):1915-1921.Gottipati K,Ruggli N,Gerber M,et al.The structure of classical swine fever virus N(pro):a novel cysteine autoprotease and zinc-binding protein involved in subversion of type I interferon induction[J].PLoS Pathog,2013,9(10):e1003704.)。
At present, Chinese mainly takes immunization measures to prevent the swine fever, and the commonly used vaccine is a lapinized hog cholera attenuated vaccine strain (C strain or HCLV strain). The vaccine is formed by cultivating CSFV virulent strain in rabbits for 480 generations continuously in the 50 th 20 th century by Chinese scholars, integrates high safety and good immunogenicity, can simultaneously induce humoral immunity and cellular immunity, is safe to domestic pigs and wild pigs of various ages, and can protect pigs of different ages against attack of classical swine fever virulent virus (Jiang Huaji, childhood, Shenrong display, classical swine fever lapinized attenuated vaccine-half century review [ J ] Chinese agricultural science 2005,38(8): 1675-. Because the C strain has stable performance, safety and good immune effect, the C strain is internationally recognized as a safe and effective attenuated vaccine. Early in the 60's of the 20 th century, C strain was introduced from the middle to eastern europe and asian friendship, called "K strain" or "LC strain", and subsequently passed to western europe and the lamic, which made a significant contribution to the control of swine fever in the world. To date, strain C is still used in many countries of the world, and the status and value of strain C are not changeable.
With the continuous development of the purification work of the swine fever in the global scope, part of European and American countries have completely eliminated the swine fever, and China has also brought the purification work of the swine fever into the schedule. According to requirements of the national middle and long-term animal epidemic disease prevention and treatment plan (2012-2020), the department of agriculture organizes and formulates national swine fever prevention and treatment guidance opinions (2017-2020) in 2017, and aims to achieve the swine fever purification standard of all pig farms and partial areas in China and further expand the purification area range of the swine fever by the end of 2020 on the basis of continuously improving the epidemic prevention capability of farms (households). At present, two strategies for purifying the swine fever can be used for reference, one is a strict killing policy implemented in European and American countries, and the other is a marker vaccine matched with a corresponding differential diagnosis detection method. As is known, China is a big world pig-raising country, strict killing policies are not suitable for purifying Chinese swine fever, and the purification work of Chinese swine fever is carried out by relying on a marker vaccine to be matched with a screening mode. However, in the present stage, the swine fever attenuated vaccine C strain does not have the characteristic of distinguishing wild virus infection and vaccination (DIVA), and the technical deficiency limits the purification of Chinese swine fever to a great extent. At the present stage, how to develop the C strain independently researched and developed in China into the marked vaccine strain is to continuously exert the advantages in the work of preventing and controlling the swine fever is the direction of the research and development efforts.
The development of marker vaccines has mainly been to label or differentiate viruses by inserting, deleting or mutating a certain point of the original strain at the level of the viral gene using a reverse genetics platform (Dong Xiao-nan, Chen Ying-hua]Vaccine,2006,25(2): 205-. CSFV structural protein ErnsAnd E2 epitopes that stimulate the body to produce neutralizing antibodies (Sun Yongfang, xuluo, Zhangqiang, etc.. Swine fever Virus epitopes research progress [ J]Chinese veterinary journal, 2017,53(7):72-75.), leading to a modification of ErnsOr the E2 protein, is one of the strategies for constructing a candidate strain of a marker vaccine. A monoclonal antibody HQ06 aiming at E2 protein is prepared in the early stage of our laboratory, the epitope is a conserved linear B cell epitope in CSFV, and the recognized minimal motif is772LFDGTNP778Wherein117F、119G and122p is 3 key amino acids of HQ06 epitope (Peng W, Hou Q, Xia Z, et al.identification of a conserved linear B-cell epitope at the N-terminal of the E2 glycoprotein of a classified sweet virus by phase-displayed peptide library [ J]Virus Res,2008,135, 267-. The discovery of HQ06 epitope provides a sufficient research basis and theoretical basis for the development of the marker vaccine.
The marker vaccine as one of effective measures for purifying the swine fever plays a key role in the aspects of prevention, control and purification of the swine fever, and the development of the marker vaccine based on the C strain is very important for the purification and complete eradication of the swine fever.
Disclosure of Invention
The invention mainly aims to provide a candidate marked vaccine strain of the hog cholera with HQ06 epitope mutation.
The above object of the present invention is achieved by the following technical solutions:
the invention takes C strain full-length infectious clone pHCLV as a framework, and 3 key amino acids of HQ06 epitope of E2 protein117F、119G and122p is subjected to single point mutation, infectious clones pHCLV-E2F117A, pHCLV-E2G119A and pHCLV-E2P122A are respectively constructed, and then three swine fever candidate marker vaccine strains rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A with HQ06 epitope mutation are obtained through rescue by a reverse genetic operation method.
The method for constructing the mutant virus rHCLV-E2F117A comprises the following steps: (1) mutating F at the 117 th site of the epitope of HQ06 of the classical swine fever virus C strain to A; cloning the mutated target fragment into pHCLV to obtain infectious clone; (2) the constructed infectious clone is transfected by a reverse genetic operation method and rescued to obtain mutant virus rHCLV-E2F 117A.
The construction method of the mutant virus rHCLV-E2G119A comprises the following steps: (1) mutating 119 th G of the epitope of HQ06 of CSFV C strain to A; cloning the mutated target fragment into pHCLV to obtain infectious clone; (2) the constructed infectious clone is transfected by a reverse genetic operation method and rescued to obtain mutant virus rHCLV-E2G 119A.
The construction method of the mutant virus rHCLV-E2P122A comprises the following steps: (1) mutating P at the 122 th position of the epitope of the CSFV C strain HQ06 into A; cloning the mutated target fragment into pHCLV to obtain infectious clone; (2) the constructed infectious clone is transfected by a reverse genetic operation method and rescued to obtain mutant virus rHCLV-E2P 122A.
The characteristics of the 3 mutant viruses in rabbits are evaluated in vivo by inoculating the rabbits through the ear veins. Test results show that the 3 mutant viruses can induce the rabbits to generate a typing thermal reaction after being inoculated with the rabbits, can replicate in the spleen of the rabbits, can induce the rabbits to generate an anti-CSFV E2 antibody and an anti-CSFV neutralizing antibody, and stably exist in rabbit bodies without a back mutation phenomenon; thus, the 3 mutant viruses in this study retained the biological properties and good immunogenicity of strain C in rabbits.
In order to verify whether the obtained 3 strains of mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A lost reactivity with mAb HQ06, SK6 cells were inoculated with the 3 strains of mutant viruses for immunofluorescence assay. The results showed that the 3 mutant viruses lost reactivity with mAb HQ06 while being able to react with rabbit anti-CSFV E2 polyclonal antibody and mAb WH 303.
The in vitro growth characteristics of 3 strains of mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A are evaluated on SK6 cells; the test results show that mutation of a single amino acid site affects the replication of the virus, and the replication levels of rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A are relatively delayed compared with rHCLV, but at 60h, the virus titer of the mutant virus rHCLV-E2P122A is consistent with that of rHCLV.
To evaluate the genetic stability of the mutant viruses, 3 strains of mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A, which were successfully rescued, were serially passaged on SK6 cells for 20 passages. The gene of the 20 th generation mutant virus is amplified by RT-PCR and sequenced, and the sequencing result shows that the mutation site of the 3 mutant viruses is kept stable.
The invention further tests the biological characteristics and immunogenicity of the 3 mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A in rabbits, and the test result shows that the 3 mutant viruses constructed by the invention have similar biological characteristics and immunogenicity to the C strain in rabbits.
The indirect immunofluorescence results confirmed that no antibodies against the HQ06 epitope were detected in rabbit sera vaccinated with rHCLV-E2P122A, whereas antibodies against the HQ06 epitope were detected in rabbit sera vaccinated with rHCLV-E2F117A, rHCLV-E2G119A and strain C. In addition, the invention carries out PCR amplification on the virus RNA extracted from the spleen of the rabbit to obtain a corresponding target fragment of the CSFV E2 region, and a sequencing result proves that the mutant virus is stable in the rabbit body and does not have a back mutation phenomenon.
According to the results of rabbit experiments, the invention further selects rHCLV-E2P122A from 3 strains of mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A to be used for inoculating weaned pigs, and after inoculation, the body temperature of the pigs is measured every day to observe clinical symptoms; the results show that rHCLV-E2P122A maintains the attenuated property of strain C; blood is collected from pigs weekly after inoculation, anti-CSFV E2 antibody in pig serum is detected by a CSFV antibody ELISA detection kit (IDEXX), and the detection result shows that the immunogenicity of the mutant virus rHCLV-E2P122A in weaned pigs is the same as that of the C strain, but the mutant virus loses the capability of inducing the generation of HQ06 epitope antibody.
The invention submits the rHCLV-E2P122A marker vaccine candidate strain to a patent approved organization for preservation, the microorganism preservation number is CGMCC NO.18219, and the classification name is as follows: hog cholera lapinized virus vaccine marker strain; the preservation time is as follows: year 2019, month 10, day 28; the preservation unit is: china general microbiological culture Collection center; and (4) storage address: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
In conclusion, the invention constructs 3 mutant viruses by using a C strain as a framework and mutating 3 key amino acids of the HQ06 epitope of the E2 protein at a single point. The 3 mutant viruses all lose the reactivity with the mAb HQ06, but retain the reactivity with other antigenic sites of the CSFV E2 protein, the biological characteristics of the 3 mutant viruses are consistent with those of the C strain, and the 3 mutant viruses can induce the production of antibodies against CSFV E2 and neutralizing antibodies against CSFV, but do not induce the production of antibodies against HQ06 epitope of rabbits and pigs. In conclusion, rabbit and pig experiments show that the marker vaccine candidate strain rHCLV-E2P122A endows the DIVA characteristic of the C strain on the basis of keeping the good immunogenicity of the C strain.
The 3-strain swine fever marker vaccine candidate strain constructed by the invention provides good attempt and beneficial exploration for developing a swine fever marker vaccine, and particularly, the rHCLV-E2P122A candidate strain has nearly consistent biological characteristics with the C strain and can be developed into the swine fever marker vaccine.
Drawings
FIG. 1 shows the construction strategy of the full-length infectious clones pHCLV-E2F117A, pHCLV-E2G119A and pHCLV-E2P 122A.
FIG. 2 shows the result of detecting the CSFV antigen of 3 mutant viruses.
FIG. 3 shows the results of indirect immunofluorescence assay to determine the reactivity of 3 mutant viruses with mAb HQ 06.
FIG. 4 shows growth curves of 3 mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P 122A.
FIG. 5 shows the sequencing results of the 20 th generation of mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P 122A.
FIG. 6 shows indirect immunofluorescence detection of antibodies against HQ06 epitope in rabbit serum.
FIG. 7 shows that the 3 mutant viruses did not back-mutate in rabbit spleen.
FIG. 8 shows the detection of anti-CSFV E2 antibody in swine blood serum by classical swine fever virus E2 antibody detection kit (IDEXX).
FIG. 9 shows indirect immunofluorescence detection of HQ06 epitope-resistant antibodies in pig serum.
FIG. 10 shows the sequencing result of CSFV E2 gene amplified by RT-PCR of viral RNA extracted from pig serum.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Experimental example 1 construction, identification and in vivo and in vitro biological characterization of the hog cholera virus C-strain mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A
1 materials and methods
1.1 plasmids, cells and viruses
Full Length of C Strain used in the present inventionThe infectious clone pHCLV is experimentally constructed and preserved by the inventor. Porcine kidney cells (SK6 and PK15) and human embryonic kidney cell 293T cell line (293T) used in the experiment were purchased and stored by the inventors' laboratory. SK6 cells were plated on 5% CO with DMEM medium containing 4% fetal bovine serum (antibody against BVDV and BVDV not included)2Was cultured at 37 ℃ in an incubator, and PK15 and 293T cells were cultured in a DMEM medium containing 10% fetal bovine serum (without BVDV and BVDV antibodies) in 5% CO2And (3) at 37 ℃. CSFV Shimen strain (Shimen, SM) is preserved in the laboratory of the inventor, and CSFV attenuated vaccine strain (rHCLV) is obtained by early-stage rescue in the laboratory of the inventor (Zhang Ling-Zheng, Liyongfeng, Xielibao, Sun Yuan, Wang Xiao, vengji. construction and identification of recombinant classical swine fever lapinized attenuated vaccine strain expressing porcine circovirus type 2 Cap protein [ J]The Bioengineering journal, 2018,34(02): 216-. Hog cholera vaccine (strain C) was purchased from Haerbinidae Biotechnology Ltd.
1.2 construction of infectious clones pHCLV-E2F117A, pHCLV-E2G119A and pHCLV-E2P122A
The full-length infectious clone pHCLV of the C strain is taken as a framework, single point mutation is carried out on 3 key amino acids of HQ06 epitope of E2 protein, and infectious clones pHCLV-E2F117A, pHCLV-E2G119A and pHCLV-E2P122A are constructed (figure 1). The primers in the table 1 are used to mutate single amino acid sites of HQ06 epitope respectively to obtain target fragments, and the target fragments are introduced into pHCLV by standard molecular cloning technology to obtain 3 infectious clones. Thereafter, the 3 infectious clones obtained were identified using restriction endonucleases and secondary sequencing.
TABLE 1 primers used for the construction of pHCLV-E2F117A, pHCLV-E2G119A and pHCLV-E2P122A
1.3 rescue of mutant viruses
3 mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A were rescued by using a reverse genetic operation platform established earlier in the laboratory of the inventors (Li C, Huang J, Li Y, et al. effective and stable recovery of cl)Organic swing boiler virus from bound cDNA using an RNA polymerase II system, Arch Virol,2013,158(4): 901-907). The specific operation steps are as follows: mu.g of pHCLV-E2F117A, pHCLV-E2G119A and pHCLV-E2P122A were transfected into SK6 cells, and the cells were serially passaged for 7 times and then freeze-thawed for 3 times to harvest the virus. Detection of E of 5 th and 7 th mutant viruses with CSFV antigen detection kit (IDEXX)rnsAnd (4) expressing the protein. Meanwhile, the viral genome of the rescued mutant virus is extracted, and the CSFV E is amplified by a RT-PCR methodrnsE1, E2 and NS5B genes, and carrying out sequencing identification.
1.4 Indirect immunofluorescence assay
Rescued 3 mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A were inoculated into SK6 cells grown to 60% -80% monolayer and the cells were changed after 2 h. Washing with DMEM for 2 times, adding fresh DMEM containing 2% fetal calf serum, and placing in 5% CO2And continuing culturing in a cell culture box at 37 ℃. After 48h, the supernatant was discarded, and the cells were fixed with-20 ℃ pre-cooled absolute ethanol at 4 ℃ for 20 min. Thereafter, mAb HQ06 (prepared by the inventor's laboratory), mAb WH303 (given by the Royal violator) and rabbit polyclonal antibody against CSFV E2 (prepared by the inventor's laboratory) were added, and after 2 hours at 37 ℃, they were washed 5 times with PBS, and FITC-labeled goat anti-mouse IgG (or goat anti-rabbit IgG) (Invitrogen) diluted 1:300 was added, and they were put in a wet box and allowed to act at 37 ℃ for 45 minutes, washed 5 times with PBS, and then observed under an inverted fluorescence microscope.
1.5 one-step growth Curve
3 mutant viruses (rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A) and C were inoculated in a 24-well plate plated with SK6 cells at a dose of 0.01 multiplicity of infection (MOI), respectively, and after infection at 37 ℃ for 2 hours, the culture was replaced with DMEM containing 2% fetal bovine serum, and the cells were placed in a medium containing 5% CO2The culture was continued in an incubator at 37 ℃. The virus was harvested at 12h, 24h, 36h, 48h, 60h and 72h, the titer of the harvested virus was determined by the Reed-Mulch method using an indirect immunofluorescence assay (Reed LJ, Mulch H.A single method of simulating fine procedure ends. am J Hyg,1938,27:493 497.) and using TC per mlID50And (4) showing. The test was repeated 3 times, and the mean and standard deviation were calculated.
1.6 Virus genetic stability analysis
The successfully rescued mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A are continuously passaged in SK6 cells, genes of 20 th generation mutant viruses are extracted after the passage to 20 th generation, and CSFV E2 is amplified by RT-PCR and then sequenced.
1.7 Rabbit inoculation test
26 new 12-week-old zealand white rabbits were randomly divided into 5 groups for inoculation, 6 rabbits each in three test groups (group a, group B and group C), and 4 rabbits each in two control groups (group D and group E). All rabbits were subjected to adaptive feeding for a period of 3d while the body temperature of the test rabbits was measured for 3d continuously, and the average value thereof was taken as the basal body temperature, followed by the immunization test.
Group a (a1, a2, A3, a4, a5 and a6), group B (B1, B2, B3, B4, B5 and B6) and group C (C1, C2, C3, C4, C5 and C6) were given equal doses (10) by intravenous injection into the ear-margin, respectively4TCID50Ml) of rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A, and control groups D (D1, D2, D3 and D4) and E (E1, E2, E3 and E4) were inoculated with strain C and DMEM, respectively (Table 2).
After 24h of strain inoculation, the rectal temperature of the rabbits is recorded every 6h to judge the rabbit typing thermal reaction. And 3d after inoculation, randomly selecting half of rabbits in each group for euthanasia, collecting spleens of the rabbits, and detecting the RNA copy number of the virus in the spleens of the rabbits by using quantitative RT-PCR. Within 2w of inoculation, sera from all rabbits were collected at 3d intervals and tested against CSFV E2 antibody in rabbit sera using CSFV antibody ELISA test kit (IDEXX). Detecting HQ06 antibody and neutralizing antibody levels in all rabbit sera collected in the assay using indirect immunofluorescence assay and serum neutralization assay
1.8 quantitative RT-PCR and sequencing
After the spleen of the rabbit is taken, the spleen tissue is ground, the virus RNA is extracted by a TRIzol method, and then the level of the virus RNA in the spleen of the rabbit is detected by an RT-qPCR method (Zhang XJ, Han QY, Sun Y, et al. development of a triple TaqMan real-time RT-PCR assay for differential detection of LV-type and HCvaccne strains of a classical swine wind turbine virus and a viral vector virus 1.Res Vet Sci,2012,92(3): 512-. The specific operation is as follows: 20 μ L of RT-PCR reaction was prepared, containing 2.5 μ L of cDNA, 1 μ L each of 10 μ L of Premix, 86S and 166R, and 0.5 μ L of HCLV-FAM (10 μ M) probe. The conditions were pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s and annealing/extension at 60 ℃ for 45s (40 cycles). Experiments for each sample were performed in 3 replicates. The RNA copy number of the viral genome was calculated by a standard curve. Meanwhile, RT-PCR amplification is carried out on virus RNA extracted from the spleen of the rabbit to obtain CSFV E2 gene, and whether the mutant virus carries out back mutation in the rabbit is analyzed.
1.9 domestic pig inoculation test
13 weaned piglets of 8 weeks of age and similar size were randomly divided into 3 groups, 5 pigs each in two test groups (group a and group B) and 3 pigs in a control group (group C). All pigs were first acclimatized for stage 3d during which time CSFV antigens and antibodies were detected using IDEXX CSFV antigen and antibody detection kits, respectively, to ensure that all test pigs were CSFV negative and were tested for no BVDV infection using PCR. Meanwhile, the body temperature of the test pig was measured continuously for 3 days, and the average value thereof was taken as the basal body temperature, followed by the immunization test.
Groups A (#86, 87, 88, 89, and 90) and B (#91, 92, 93, 94, and 95) were neck injected with equal doses (10) respectively4TCID50Perhead) of rHCLV-E2P122A and strain C, and control group C (#96, 97 and 98) were injected with 2mL of DMEM and a second immunization was performed 2w after inoculation (Table 3), and clinical symptoms of each animal were carefully observed daily. Meanwhile, blood was collected from pigs weekly after inoculation, anti-CSFV E2 antibody was detected in the sera of domestic pigs using CSFV antibody ELISA test kit (IDEXX), the level of neutralizing antibody in the sera was detected using neutralization assay, HQ06 antibody in the sera of domestic pigs was detected using indirect immunofluorescence assay. Meanwhile, virus RNA in pig serum is extracted by a TRIzol method, a virus E2 gene is amplified by RT-PCR, and whether the mutant virus carries out back mutation in a domestic pig body is analyzed.
1.10 detection of CSFV antibody
The serum samples of all rabbits and pigs collected in the test process are used for detecting the CSFV E2 antibody level by using a swine fever antibody detection kit (IDEXX), and the specific operation method is shown in the specification. The level of neutralizing antibodies in serum was determined using the eu recommended neutralization assay. The constructed p3xFLAG-CMV-6HQ06e is used for transfecting 293T cells, and after 48 hours, the cells are fixed by 4% paraformaldehyde for 20min and subjected to an indirect immunofluorescence test. The specific operation is as follows: rabbit serum, pig serum, mAb HQ06 and rabbit anti-Flag IgG (Sigma) were added, respectively, and after 2h at 37 ℃ the mixture was washed 1 time with PBS, FITC-labeled goat anti-rabbit IgG (or goat anti-mouse IgG or rabbit anti-pig IgG) (Invitrogen) diluted 1:300 was added, and the mixture was allowed to stand in a wet box at 37 ℃ for 45min, washed 2 times with PBS, and observed under an inverted fluorescence microscope.
1.11 statistical analysis
Statistical analysis was performed on all data using SPSS statistical software to compare differences between groups. Wherein, the difference is not significant when p is more than or equal to 0.05, the difference is significant when p is less than 0.05, and the difference is very significant when p is less than 0.01.
2 results of the test
2.1 obtaining of mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A
The obtained 3 infectious clones pHCLV-E2F117A, pHCLV-E2G119A and pHCLV-E2P122A were transfected into SK6 cells respectively, and the continuous passage was used to rescue 3 mutant viruses, and the IDEXX CSFV antigen detection kit was used to detect the supernatants of the SK6 cells of the 5 th and 7 th generations, and the results showed that the rescued virus E6 of the 7 th generationrnsProtein antigens were positive (fig. 2).
Rescued viral genomic RNA was extracted according to viral RNA extraction instructions, followed by amplification of the gene of interest by RT-PCR and sequencing. The result shows that the target gene of the 3 mutant virus genomes is consistent with the expected size, and the sequencing result shows that the target gene of the mutant virus has no other mutation. The results show that the mutant virus with three key amino acid mutations of HQ06 epitope of CSFV E2 protein is obtained.
2.2 mutant viruses lost reactivity with MAb HQ06
In order to verify that the obtained 3 strains of mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A lose the reactivity of mAb HQ06, SK6 cells were inoculated with the 3 strains of mutant viruses to carry out an indirect immunofluorescence assay. The results showed that the 3 mutant virus was able to react with the rabbit polyclonal antibody against CSFV E2 and mAb WH303, but lost reactivity with mAb HQ06 (figure 3).
2.3 growth kinetics and genetic stability of mutant viruses
The assay evaluated the in vitro growth characteristics of 3 mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A on SK6 cells. The results show that mutations at a single amino acid site affect viral replication, and that the replication levels of rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A are relatively late compared to rHCLV, but at 60h the viral titer of mutant virus rHCLV-E2P122A was consistent with that of rHCLV (FIG. 4).
To evaluate the genetic stability of the mutant viruses, 3 strains of mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A, which were successfully rescued, were serially passaged on SK6 cells for 20 passages. The genes of the 20 th generation mutant virus were amplified by RT-PCR and sequenced, and the sequencing results showed that the mutation sites of 3 mutant viruses remained stable (FIG. 5).
2.4 biological Properties and immunogenicity of mutant viruses in rabbits
To examine the biological properties and immunogenicity of the 3 mutant viruses rHCLV-E2F117A, rHCLV-E2G119A and rHCLV-E2P122A in rabbits, rabbits were vaccinated according to the grouping in Table 2 and the indices in the table were determined. The results show that 3 mutant viruses can cause the rabbit stereotyped heat reaction as the C strain; the replication of the virus can be detected in the spleen of the rabbit, and the replication levels of different strains have no obvious difference; at 7d post immunization, anti-CSFV E2 antibody in rabbit sera of all vaccinated viruses turned positive (Table 2). Neutralization assay results showed that neutralizing antibodies were detectable in sera from rabbits vaccinated with strain C and mutant virus at both 7d and 14d post immunization (Table 3). The data show that the 3 mutant viruses constructed by the invention have similar biological properties and immunogenicity to the C strain in rabbits.
Indirect immunofluorescence results confirmed that HQ06 antibody was not detected in rabbit sera vaccinated with rHCLV-E2P122A, whereas HQ06 antibody was detected in rabbit sera vaccinated with rHCLV-E2F117A, rHCLV-E2G119A and strain C (FIG. 6). Meanwhile, the experiment carries out RT-PCR amplification on the virus RNA extracted from the spleen of the rabbit to obtain the CSFV E2 gene, and the sequencing result proves that the mutant virus is stable in the rabbit body and does not have the phenomenon of back mutation (figure 7).
TABLE 2 established thermal response and replication in spleen of rabbits following mutant virus inoculation
TABLE 3 neutralizing antibody data in rabbit sera
2.5 immunogenicity of mutant Virus rHCLV-E2P122A in weaned piglets
According to the results of the rabbit experiment, the mutant rHCLV-E2P122A was inoculated into weaned pigs as described in the materials method. After inoculation, the temperature of the pigs was measured daily and observed for the presence of febrile symptoms. The pigs were bled weekly after vaccination and serum from the pigs was tested for CSFV E2 antibody using a CSFV antibody ELISA test kit (IDEXX). The results showed that anti-CSFV E2 antibody in the sera of pigs started to turn positive 14d after inoculation with the virus rHCLV-E2P 122A; at 28d post inoculation, all pigs in the test group had fully raised anti-CSFV E2 antibody (fig. 8). The results of the neutralization test show that neutralizing antibodies can be detected in the serum of test pigs 3w after immunization, and the neutralizing antibody titer in the serum of test pigs 4w after inoculation can reach 1:25 (Table 4). The above data indicate that the mutant virus rHCLV-E2P122A is as immunogenic as strain C in weaned piglets.
Indirect immunofluorescence results confirmed that no HQ06 antibody was detected in the sera of pigs vaccinated with rHCLV-E2P122A, whereas HQ06 antibody was detected in the sera of pigs vaccinated with strain C (fig. 9). Meanwhile, the experiment carries out RT-PCR amplification on the CSFV E2 gene of the virus RNA extracted from the serum of the domestic pig, and the sequencing result proves that the mutant virus rHCLV-E2P122A is stable in the domestic pig body and does not have the phenomenon of back mutation (figure 10).
TABLE 4 neutralizing antibody data in weaned piglet serum
SEQUENCE LISTING
<110> Harbin veterinary institute of Chinese academy of agricultural sciences (Harbin center of Chinese center of animal health and epidemiology)
HQ06 epitope mutated hog cholera candidate marker vaccine strain and application thereof
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Claims (7)
1. A mutant virus rHCLV-E2P122A of a swine fever C strain is characterized in that the construction method comprises the following steps: (1) mutating proline (P) at position 122 of epitope of HQ06 of classical swine fever virus C strain to alanine (A); cloning the mutated target fragment into pHCLV to obtain infectious clone; (2) the constructed infectious clone is transfected by a reverse genetic operation method and rescued to obtain mutant virus rHCLV-E2P 122A.
2. The mutant virus rHCLV-E2P122A according to claim 1, wherein the accession number for the microorganism is CGMCC NO. 18219.
3. A mutant virus rHCLV-E2F117A of a swine fever C strain is characterized in that the construction method comprises the following steps: (1) mutating phenylalanine (F) at the 117 th position of the epitope of the swine fever virus C strain HQ06 into alanine (A); cloning the mutated target fragment into pHCLV to obtain infectious clone; (2) the constructed infectious clone is transfected by a reverse genetic operation method and rescued to obtain mutant virus rHCLV-E2F 117A.
4. The mutant virus rHCLV-E2G119A of the swine fever C strain is characterized in that the construction method comprises the following steps: (1) mutating 119 th glycine (G) of epitope of HQ06 of CSFV C strain to alanine (A); cloning the mutated target fragment into pHCLV to obtain infectious clone; (2) the constructed infectious clone is transfected by a reverse genetic operation method and rescued to obtain mutant virus rHCLV-E2G 119A.
5. A mutant virus according to claim 1, 3 or 4, wherein the cell is an SK6 cell.
6. Use of the mutant virus of claim 1, 3 or 4 for the preparation of a swine fever marker vaccine.
7. Use of the mutant virus rHCLV-E2P122A as claimed in claim 2 in the preparation of swine fever marker vaccine.
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