CN115896043A - O-type foot-and-mouth disease vaccine candidate strain and construction method and application thereof - Google Patents
O-type foot-and-mouth disease vaccine candidate strain and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a candidate strain of O-type foot-and-mouth disease vaccine, a construction method and application thereof, belonging to the technical field of biological products. The invention utilizes the reverse genetic manipulation technology of foot-and-mouth disease virus, uses the gene infectious clone of the current epidemic strain O/XJ/CHA/2017L + P1 of the chimeric foot-and-mouth disease virus as a skeleton, and further uses the G-H ring gene of the foot-and-mouth disease virus O/SCGH/CHA/2016 strain to replace the equivalent gene thereof to construct a recombinant virus rHN/XJ/SCGH. Compared with the parental virus rHN/XJ, the time of 100 percent of infected cytopathic effect caused by the recombinant virus rHN/XJ/SCGH is shortened to 12h, the replication titer is obviously improved, the recombinant virus vaccine immunized pig can generate high-level protective cross neutralizing antibodies for the foot-and-mouth disease O type four-lineage epidemic viruses, and the antigen spectrum is expanded. Therefore, the recombinant virus rHN/XJ/SCGH constructed by the reverse genetic manipulation technology is suitable for serving as an FMD vaccine candidate strain and is used for effectively preventing and controlling the current O-type foot-and-mouth disease in China.
Description
Technical Field
The invention belongs to the technical field of biological products, and relates to a candidate strain of an O-type foot-and-mouth disease vaccine as well as a construction method and application thereof.
Background
Foot-and-mouth disease (FMD) is a virulent infectious disease that infects major livestock such as pigs, cattle and sheep. The disease has long prevalence in China and has great harm to the livestock breeding industry. In recent 10 years, the A-type FMD and the O-type FMD are mainly popular in China, wherein the A-type FMD tends to be controlled, and the O-type FMD still seriously harms the livestock breeding industry in China. Foot-and-mouth disease virus type O (FMDV), which has prevailed in recent years, was analyzed and found to be four lineage virus strains of mainly three topotypes (middle east-south asia (ME-SA), southeast asia (SEA), and classical chinese type (Cathay)), a Mya-98 lineage (SEA), panAsia lineage (ME-SA), ind-2001 lineage (ME-SA), and Cathay lineage, respectively. The complex situation that the O-type FMD multi-lineage virus strain is popular together currently in China aggravates virus variation, so that new variant virus strains continuously appear, epidemic situations are frequently caused, and unprecedented severe challenges are provided for prevention and control of FMD in China.
FMDV is a small RNA virus, multiple serotypes, highly variable genes and antigens, making FMD difficult to control. The different serotypes of FMDV lack cross-immune protection, and even though there are large differences in antigenicity between different viruses of the same serotype, the degree of serological cross-reactivity varies. For FMDV of the same serotype, when virus gene variation is accumulated to a certain extent, the antigenicity of the virus is changed, so that the antigen matching of the current vaccine and the epidemic strain is reduced or the antigen is not matched, and the immunoprotective efficacy of the vaccine is reduced or the immunity is failed. For example, since the O/Mya-98 lineage virus strain is epidemic in 2010 to the present, regular amino acid variation appears on the structural protein VP1, so that the antigen matching of the field epidemic strain and the vaccine strain is reduced. The popular swine-philic Cathay pedigree virus strain in China is inherited into four branches (old swine virus, new swine virus-1, new swine virus-2 and new swine virus-3), and the existing vaccine strain is not matched with the new swine virus-3 branch virus strain which is popular in recent years in antigen. Therefore, aiming at the complex situation that the O-type multi-lineage virus strains are popular together at present in China, the screening of vaccine candidate strains highly matched with all O-type virus strain antigens which are popular at present is urgently needed for the effective prevention and control of FMD in China.
The FMDV structural protein VP1 contains a highly variable G-H ring, which is the most important epitope for inducing a body to generate neutralizing antibodies and plays a very important role in vaccine immune protection. Researches show that G-H of different types of FMDV is embedded, and the antigen spectrum of FMD vaccines can be expanded. In view of the above, in order to expand the antigen spectrum of the O-type FMD vaccine, the invention further replaces G-H ring genes of the O-type foot-and-mouth disease classical vaccine strain and the Catheay lineage virus strain prevalent in China on the frameworks of the chimeric FMDV O/XJ/CHA/2017 (Ind-2001 lineage) virus leader protein L and structural protein P1 recombinant virus by the reverse genetic manipulation technology of FMDV, constructs the recombinant FMDV, and further researches the potential of the recombinant FMDV as a candidate strain of the O-type FMD vaccine.
Disclosure of Invention
The invention aims to provide an O-type FMD vaccine candidate strain and a construction method and application thereof, wherein the time of 100% infected cytopathic disease caused by the constructed recombinant FMDV is shortened to 12h, the replication titer is remarkably improved, and the recombinant virus vaccine immunized pigs can generate high-level protective cross neutralizing antibodies for epidemic viruses of four foot-and-mouth disease O-type pedigrees (Mya-98 pedigrees, panasia pedigrees, ind-2001 pedigrees and Cathay pedigrees), so that the antigen spectrum is expanded.
The O-type FMD vaccine candidate strain is obtained by taking the full-length infectious clone pQSA of the recombinant FMDV rHN/XJ as a framework and embedding the G-H loop gene of an FMDV O/SCGH/CHA/2016 strain; the full-length infectious clone pQSA of the recombinant FMDV rHN/XJ is obtained by taking the full-length infectious clone pOFS of an FMD vaccine strain O/HN/CHA/93 as a framework and embedding the L + P1 gene of an FMD epidemic strain O/XJ/CHA/2017. The nucleotide sequence of the G-H loop gene of the FMDV O/SCGH/CHA/2016 strain is shown as SEQ ID NO:5, respectively. The nucleotide sequence of the L + P1 gene of FMDV O/XJ/CHA/2017 strain is shown as SEQ ID NO:2.
the invention relates to a construction method of O-type FMD vaccine candidate strains, which comprises the following steps:
(1) Artificially synthesizing a recombinant plasmid pSK-Z123XJLP1 containing FMD epidemic strain O/XJ/CHA/2017L + P1 gene by taking FMD vaccine strain O/HN/CHA/93 semi-long plasmid pSK-Z123 as a template;
(2) Artificially synthesizing a recombinant plasmid pSK-Z123XJLP1/SCGH containing FMDV O/SCGH/CHA/2016 strain G-H loop gene chimeric by taking the recombinant plasmid pSK-Z123XJLP1 synthesized in the step (1) as a template; digesting the synthesized plasmid with SpeI and BglII restriction enzymes, recovering a target fragment of about 5400bp, and inserting the target fragment into a pOFS plasmid digested with the SpeI and BglII restriction enzymes to obtain a recombinant full-length plasmid pQSQ;
(3) Transfecting the NotI linearized recombinant full-length plasmid pQSQ to BSR/T7 cells, rescuing the recombinant FMDV and obtaining the O-type FMD vaccine candidate strain rHN/XJ/SCGH.
The invention also provides application of the O-type FMD vaccine candidate strain obtained by the construction method in preparation of a prevention and control FMD vaccine. Experiments show that G-H consisting of different amino acids is replaced on the framework of the same virus, so that the influence on CPE of 100% infected cells is different, and the influence on the replication level of the foot-and-mouth disease virus is different. Parental virus rHN/XJ and recombinant virus rHN/XJ/TURGH vaccine immunized pigs can only produce high-level and protective cross-neutralizing antibodies against three lineages of epidemic virus O/XJ/CHA/2017 (Ind 2001 lineage), O/Tibet/99 (Panasia lineage) and O/NXYCH/CHA/2018 (Mya-98 lineage), while recombinant virus vaccine rHN/XJ/SCGH immunized pigs can produce high-level and protective cross-neutralizing antibodies against four lineages of epidemic virus O/XJ/CHA/2017, O/Tibet/99, O/NXYCH/CHA/2018 and O/SCGH/CHA/2016 (Catay lineage), so that the recombinant virus vaccine rHN/XJ/SCGH expands the antigenic profile and is useful for preparing vaccines for preventing and controlling the potential of O type FMD.
In conclusion, the invention utilizes FMDV reverse genetic manipulation technology, uses infectious clone of L + P1 gene of chimeric FMDV O/XJ/CHA/2017 virus as a framework to construct recombinant virus rHN/XJ/SCGH replacing the gene of Catay pedigree epidemic virus O/SCGH/CHA/2016G-H in China, the time of 100 percent of infected cytopathic disease caused by the recombinant virus is shortened to 12H, the replication titer is obviously improved, animals immunized by the recombinant virus vaccine can generate high-level protective cross-neutralizing antibodies for four pedigree epidemic viruses of FMD, and the antigen spectrum is expanded. Therefore, the recombinant virus rHN/XJ/SCGH constructed by the reverse genetic manipulation technology is suitable for serving as an FMD vaccine candidate strain and is used for effectively preventing and controlling the current O-type FMD in China.
Drawings
FIG. 1 is a schematic genome diagram of an FMDV recombinant full-length plasmid (dark gray parts indicate L + P1 gene of FMDV O/XJKS/2017/CHA strain, light gray indicates the position of G-H);
FIG. 2 shows the cleavage identification of the recombinant plasmid PstI (M: DNA standard marker; 1;
FIG. 3 is an alignment chart of amino acids encoded by the G-H loop of the full-length recombinant plasmid;
FIG. 4 shows BSR/T7 cells after transfection of recombinant plasmids for 60h (A: normal BSR/T7 cells, B, C and D are BSR/T7 cells after transfection of plasmids pQSA, pQSD and PQSQ for 60h, respectively);
FIG. 5 shows indirect immunofluorescence results;
FIG. 6 is an electron micrograph of the recombinant FMDV (left: rHN/XJ; middle: rHN/XJ/TURGH; right: rHN/XJ/SCGH);
FIG. 7 is a one-step growth curve of recombinant viruses.
Detailed Description
The invention is further illustrated by the following specific examples.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Plasmids pOFS and semi-long plasmid pSK-Z123 containing the complete gene of FMD vaccine strain O/HN/CHA/93 (see the Evaluation of a genetic modified foot-and-mouse disease virus vaccine generated by reverse genetics, li et al, BMC vector Research 2012,8 published by Pinghua Li et al, 2012) were used for the construction of recombinant full-length plasmids. FMD epidemic strains O/SCGH/CHA/2016 (Catay lineage, genBank KX 161429.1), O/Tibet/99 (PanAsia lineage, genBank AJ 539138), O/XJ/CHA/2017 (Ind-2001 lineage, genBank MF 461724.1), O/NXYCH/CHA/2018 (Mya-98 lineage, genBank MH 791315.1) were stored in the national foot and mouth disease reference laboratory. The gene sequence of the virus was queried in genebank. The method for artificially synthesizing the recombinant vector is not particularly limited in the present invention, and an artificial synthesis method known in the art may be used. In the present example, the artificial recombinant vector was obtained from Jinzhi Biotechnology Ltd.
1. Construction of chimeric FMD epidemic strain O/XJ/CHA/2017L + P1 gene full-length clone
An FMD vaccine strain O/HN/CHA/93 semi-long plasmid pSK-Z123 is taken as a framework, and a recombinant semi-long plasmid pSK-Z123XJLP1 (synthesized by Jinwei Biotechnology Co., ltd.) containing the virus (O/XJ/CHA/2017) L + P1 gene is designed and synthesized according to the L and P1 nucleotide sequences (SEQ ID NO: 2) of an O/XJ/2017/CHA virus strain published in Genebank. The plasmid was digested with Spe I and Bgl II restriction enzymesAfter the digestion, the target fragment of about 5400bp was recovered and inserted into pOFS plasmid digested with the same enzyme to obtain the full-length plasmid pQSA of the L + P1 gene of the chimeric O/XJ/CHA/2017 virus. The system in which Spe I and Bgl II were digested was as follows: 10 Xbuffer H10. Mu.L, bglII 4. Mu.L, speI 4. Mu.L, recombinant plasmid 4. Mu.g, ddH 2 O was supplied to 100. Mu.L. The enzyme digestion system is incubated for 1 h-2 h at 37 ℃. The pQSA was subjected to enzyme cleavage with Pst I, and found that the bands of 838bp,4250bp and 6050bp were cut out and conformed to the expected sizes (see FIG. 2). The recombinant plasmid with correct enzyme digestion identification is sent to Jinzhi biotechnology Limited for sequence determination, and the result shows that the constructed recombinant plasmid contains expected replacement.
2. Construction of chimeric G-H Loop FMDV recombinant full-Length clone
The synthesized pSK-Z123XJLP1 plasmid is used as a template, and a plasmid pSK-Z123XJLP1/SCGH containing O/TUR/5/2009G-H loop chimeric gene and a plasmid pSK-Z123XJLP1/SCGH containing O/SCGH/2016G-H loop chimeric gene are designed and synthesized (synthesized by Jinwei Biotechnology Limited) according to the Panya lineage international vaccine strain O/TUR/5/2009 (GenBank KP 202878.1) published in Genebank and the Cathay lineage FMDV O/SCGH/CHA/2016G ring gene (amino acid 130-160 of VP 1) published in China. The synthesized plasmids were digested with Spe I and BglII restriction enzymes, respectively, and about 5400bp of the desired fragment was recovered and inserted into pOFS plasmid digested with the same enzymes, to obtain recombinant full-length plasmid and pQSD and pQSQ (see FIG. 1). The 2 recombinant plasmids were digested with Pst I, and as a result, bands of interest (838bp, 4250bp and 6050 bp) having the expected sizes were excised (see FIG. 2). The system in which Spe I and Bgl II were digested was as follows: 10 Xbuffer H10. Mu.L, bglII 4. Mu.L, speI 4. Mu.L, recombinant plasmid 4. Mu.g, ddH 2 O was supplemented to 100. Mu.L. The enzyme digestion system is incubated for 1-2 h at 37 ℃. The recombinant plasmid with correct enzyme digestion identification is sent to Jinzhi biotechnology Limited for sequence determination, and the result shows that the constructed recombinant plasmid contains expected replacement. Wherein the nucleotide sequences of the L + P1 genes of pOFS and pQSA are respectively shown in SEQ ID NO. 1 and SEQ ID NO. 2, the nucleotide sequences of the G-H loops of pQSA, pQSD and pQSQ plasmids are respectively shown in SEQ ID NO. 3, SEQ ID NO. 4 and SEQ ID NO. 5, and the corresponding amino acid sequences are respectively shown in SEQ ID NO. 6 and SEQ ID NO. 7And SEQ ID NO 8. The alignment of the amino acids encoded by the recombinant full-length plasmid G-H loop gene is shown in FIG. 3.
TABLE 1 partial sequence names and viruses corresponding to recombinant plasmids
3. Rescue of labeled viruses
Full-length recombinant plasmids pQSA, pQSD and pQSQ are prepared from QIAGEN Plasmid Midi Kits, and the Not I is linearized and then purified and recovered by a DNA fragment recovery kit to be used as a transfection template. When the conventionally cultured single-layer BSR/T7 cells grow to 70% -80%, liposome Lipofectamine is used TM 2000 mediated transfection (see the protocol for details). 2mL of DMEM medium containing 8% fetal bovine serum was added 5h after transfection, and the medium was incubated at 37 ℃ with 5% CO 2 The incubator was continued and the cells were observed for cytopathic effect (CPE).
The results show that: typical CPE appeared in all 3 full-length recombinant plasmids 60h after BSR/T7 cells were transfected, i.e., cells in a fibrous distribution became large and rounded (FIG. 4). After transfection for 68h, cells are harvested, after repeated freeze-thawing for 2 times, serial passage is carried out on BHK-21, and virus of each generation is preserved at-70 for later use. The rescued genetically engineered viruses were designated rHN/XJ, rHN/XJ/TURGH, and rHN/XJ/SCGH, respectively.
4. Identification of recombinant viruses
4.1 RT-PCR identification
The transfected supernatants were extracted with the RNAasy Mini Kit for total cytotoxic RNA, and the primers OZ1490 (+)/OZ 3980 (-) (OZ 1490 (+): GACAAGACCACGCCGTATT), OZ3980 (-): TGCATCTGTTGATGTGTGTC) RT-PCR respectively amplifies P1 gene segments of transfection supernatant, and the P1 gene segments are sent to Shanghai Sangni company for sequencing after purification and recovery, so as to verify the correctness of recombinant viruses. The sequencing result shows that: the rHN/XJ, rHN/XJ/TURGH and rHN/XJ/SCGH recombinant viruses all contain target replacement, which indicates that the recombinant FMDV containing the expected gene replacement is successfully constructed by the invention.
4.2, indirect immunofluorescence
BHK-21 monolayer cells were inoculated with rHN/XJ, rHN/XJ/TURGH and rHN/XJ/SCGH recombinant viruses, respectively, when they grew to 70% -80% confluency. The expression of 3B protein was detected by indirect immunofluorescence 6h after virus inoculation. The method comprises the following specific steps: (1) Discarding culture solution of virus-inoculated cells, rinsing with PBS (0.01 mol/L pH 7.2) for 3 times, adding 4% ice-cold paraformaldehyde, and fixing at room temperature for 30min; (2) PBS rinsing 3 times, adding 5% BSA room temperature blocking for 30min; (3) After rinsing with PBS for 3 times, respectively adding 1; (4) PBS was rinsed 5 times, FITC-labeled IgG secondary antibody diluted 1; (5) PBS rinsing 5 times, adding 0.5ug/ml DAPI staining for 10min, PBS washing 5 times, removing excessive DAPI, and taking pictures under confocal fluorescence microscope with normal cell control.
The results show that: BHK-21 cells inoculated with rHN/XJ, rHN/XJ/TURGH and rHN/XJ/SCGH can react with 3B monoclonal antibodies to generate specific green fluorescence, and no visible fluorescence can be seen when the control cells react with the 3B monoclonal antibodies (see figure 5), which indicates that the recombinant FMDV is successfully constructed, and the rescue of infectious recombinant FMDV is not influenced by the replacement of L + P1 or G-H loop genes.
4.3, electron microscope Observation
Respectively proliferating FMDV rHN/XJ, rHN/XJ/TURGH and rHN/XJ/SCGH 100mL in BHK-21 cells, freeze thawing for 2-3 times, inactivating with BEI, centrifuging at 12000rpm/min for 1h, collecting virus supernatant, and centrifuging at 35000rpm/min at 4 ℃ for 3h. The centrifuged pellet was resuspended in PBS (PH = 7.6) buffer, and observed under electron microscope after negative staining. The observation result of the electron microscope shows that: the morphology of 3 recombinant viruses was identical, with spherical virions of approximately 25nm diameter, identical to that of FMDV (FIG. 6).
5. Growth characteristics of recombinant viruses
5.1 time to CPE in 100% of infected cells due to recombinant virus
Confluent BHK-21 cells were inoculated with 10% of the transfected supernatants, harvested when 100% of the cells inoculated with the transfected supernatants exhibited typical CPE, repeated freeze-thawing for 3 times, and serial passage under the same conditions was continued, and the time until 100% of the infected cells exhibited typical CPE was observed after passage 5 (Table 2). The results of serial passages indicated: after the rescued 3 recombinant viruses were continuously transmitted to the 6 th generation, the time for the appearance of typical CPE tended to be stable, the time for 100% of infected cells to appear CPE by the parental virus was about 48h, the time for 100% of infected cells to appear typical CPE by the rHN/XJ/TURGH virus was about 15h, and the time for 100% of infected cells to appear typical CPE by the rHN/XJ/SCGH virus was about 12h. This result shows that: replacement of both FMDV vaccine strain O/TUR/5/2009 and the circulating strain O/SCGH/CHA/2016G-H significantly reduced the time to CPE in 100% of infected cells, and different G-H loops affected CPE differently in 100% of infected cells.
TABLE 2 time to CPE 100% of the cells after passage 5 of the recombinant virus (h)
5.2 one-step growth curves of recombinant viruses
Respectively diluting rHN/XJ, rHN/XJ/TURGH and rHN/XJ/SCGH of 6 th generation by 10 series, respectively inoculating viruses with different dilutions into full monolayer BHK-21 cells (200 ul/well, 6-well plate), placing in an incubator at 37 ℃, shaking once every 10min, adding 2mL of mixed solution of tragacanth (one part of 2 xMEM, one part of 1.2% of tragacanth and 1% of serum) after 1h, statically culturing, sucking and discarding the culture solution after 48h, washing with PBS for 1-2 times, adding a fixing solution (50% acetone and 50% methanol), fixing at room temperature for 30min, then staining with crystal violet for 1h, and calculating plaque forming unit (PFU/mL) of each virus after washing with clear water. The 6 th generation recombinant virus was inoculated into a confluent monolayer of BHK-21 cells at 1moi virus infection, the inoculated virus solution was discarded after 1 hour of adsorption, and after washing with MEM for 2 times, 5mL of MEM medium was added and the culture was continued in a 37 ℃ incubator. Samples were harvested at 4h, 8h, 12h, 16h and 20h after inoculation, and virus titers (PFU/mL) were determined on BHK-21 monolayers (6 well plates) after 3 repeated freeze-thaw cycles (experiments were performed in 2 replicates) as described above, and one-step growth curves of the virus were plotted (FIG. 7).
The results show that: compared with a parent virus rHN/XJ, the replication titer of the recombinant FMDV is remarkably improved within 4H-20H after the cells are infected by the recombinant viruses inserted with FMD vaccine strain O/TUR/5/2009 and epidemic strain O/SCGH/CHA/2016G-H loop, but the replication titers of the recombinant viruses rHN/XJ/SCGH inserted with the epidemic strain O/SCGH/2016G-H loop at different time are slightly higher than those of rHN/XJ/TURGH, which shows that the foot-and-mouth disease virus replication level is affected differently by replacing G-H containing different amino acid components on the same virus skeleton.
6. Preparation of FMDV inactivated vaccine
6.1 proliferation, inactivation and purification of FMDV
Recombinant viruses rHN/XJ/TURGH, rHN/XJ/SCGH and parental viruses rHN/XJ are respectively inoculated with 100% full monolayer adherent BHK-21 cells (175 mL cell bottles, 27mL inoculation solution +3mL virus solution), the culture is continued in a 37 ℃ incubator, when 100% infected cells have typical CPE, the viruses are respectively harvested, and each virus is harvested by about 500mL. The collected virus liquid is repeatedly frozen and thawed for 3 times, and then centrifuged for 1h at 8000rpm/min at 4 ℃ to remove cell debris. The collected viral supernatant was inactivated with 1-1.2% BEI at 30 ℃ for 28h. Inactivated virus antigens are subjected to inactivation safety inspection by using suckling mice and cells for 4 blind generations. After the virus antigen is qualified, purifying the virus particles by a sucrose density gradient centrifugation method, and detecting the 146S content of the virus antigen by a liquid chromatograph. The antigen concentration was diluted with PBS solution pH =7.6 to 12 μ g/mL.
6.2 preparation of vaccine
Placing an ISA201 adjuvant in a constant-temperature water bath kettle at 37 ℃ for preheating, slowly adding a proper amount of adjuvant into a virus antigen according to the ratio of the antigen to the adjuvant in a volume ratio =1, slowly shaking until the antigen and the adjuvant are not layered, and placing a prepared vaccine product (with the antigen concentration of 6 mug/mL) at 4-8 ℃ for storage.
7. Animal experiments
Selecting 15 healthy susceptible pigs (with O-type foot-and-mouth disease liquid blocking ELISA antibody titer less than 1, 6,3ABC antibody negative) at the age of 90 days, and dividing the pigs into groups A, B and C3, wherein each group comprises 5 pigs. Group A was immunized with the parental virus rHN/XJ vaccine, group B with the rHN/XJ/TURGH virus vaccine, and group C with rHN/XJ/SCGH. The ear root is injected intramuscularly, and the dosage is 2 mL/head. Blood is collected 28 days after all pigs are immunized, and serum is collected for later use.
The cross-neutralizing antibodies of different lineages of FMDV (O/SCGH/CHA/2016, O/Tibet/99, O/XJ/CHA/2017 and O/NXYCH/CHA/2018) in immune serum of three vaccines immunized with animals after 28 days are detected by a micro-neutralization experiment, which comprises the following specific steps:
a. all immune sera were inactivated for 30min at 56 ℃ before the assay;
b. taking inactivated serum, diluting the serum by serum-free cell culture solution on a 96-well micro-cell culture plate, and performing a series of dilution by multiple proportions from 1;
c. collecting virus liquid stored in refrigerator at-70 deg.C, and making 200TCID according to the measured toxicity value 50 Dilution (after mixing with equivalent amounts of serum, the titer was 100TCID 50 )；
d. Adding 50. Mu.L of diluted virus solution to each well of a 96-well plate containing diluted serum, and setting the content to 5% CO 2 A 37 ℃ incubator;
e. adding cell suspension serum virus to neutralize for 1h, taking out, adding 50 μ L cell suspension into each well (with monolayer growth degree within 24h, generally 100-150 ten thousand cells per ml), sealing with transparent adhesive tape, and culturing at 37 deg.C. After 48h, making proper judgment under a microscope, and after 72h, fixing and dyeing;
f. the following controls must be set up for each plate for each experiment: (1) positive and negative serum controls: the positive and negative serum controls are each provided with 2-4 wells, 50 μ L per well, the positive wells should be blue, and the negative wells should not be colored. (2) Virus regression test: firstly, making virus into 0.1, 1, 10, 100TCID 50 Dilutions, 2-4 wells per dilution, 50 μ l per well. Then 50. Mu.l of cell suspension per well was supplemented with 50. Mu.l of diluent. 0.1TCID 50 Should be blue, 100TCID 50 No coloration, otherwise the experiment did not hold. (3) Normal cell control: to avoid the culture plate itself causing experimental errors, 2-4 wells of normal cell control not inoculated with virus and serum should be set up on each plate, and the control should maintain good morphology and life characteristics throughout the experiment, and the staining is blue. (4) For the initial test, a serotoxicity control (equivalent to the lowest serum dilution in the test) is set in 2-4 holes, and no virus is added;
g. and judging the result when the virus regression test, the positive cell control, the negative cell control, the normal cell control and the serotoxicity control are all established. The test serum wells were judged to be negative for 100% of CPE and more than 50% of the cells were preservedThe guardian is positive; results of the fixed virus dilution serum neutralization assay were calculated, with the final dilution of serum being represented by the 50% end point of the serum/virus mixture, and the serum neutralization titer was calculated by Karber method, 1 10 ) Or more positive, 1.
The results of the serum neutralization experiments of the immunized animals show that: parental virus rHN/XJ and recombinant virus rHN/XJ/TURGH vaccine immunized pigs can only produce high-level and protective cross-neutralizing antibodies (more than or equal to 2.43 log) on three lineages of epidemic virus O/XJ/CHA/2017 (Ind 2001 lineage), O/Tibet/99 (Panasia lineage) and O/NXYCH/CHA/2018 (Mya-98 lineage) 10 ) However, none of them could generate protective cross-neutralizing antibodies (< 1.65 log) against the Catheay lineage circulating strain O/SCGH/CHA/2016 10 ) (ii) a The rHN/XJ/SCGH immune pig with the recombinant virus vaccine can generate high level and protection (> 2.47 log) on four lineages of epidemic virus O/XJ/CHA/2017, O/Tibet/99, O/NXYCH/CHA/2018 and O/SCGH/CHA/2016 (Cathay lineage) 10 ) Cross-neutralizing antibodies. The above results show that: the recombinant virus vaccine rHN/XJ/SCGH for immunizing chimeric Cathay topological strain G-H loop gene not only produces high-level and protective cross-neutralizing antibodies for the epidemic strains of the Panasia lineage, the Myanmar 98 lineage and the Ind2001 lineage, but also produces high-level and protective cross-neutralizing antibodies for the Cathay lineage epidemic strain O/SCGH/CHA/2016, and the vaccines prepared by the recombinant viruses of the parent virus and the chimeric classical vaccine strain O/TUR/5/2009G-H loop can not produce protective cross-neutralizing antibodies for the Cathay lineage epidemic strain O/SCGH/CHA/2016.
TABLE 3 neutralizing anti-shock potency (log) of porcine sera against homologous and heterologous viruses 10 )
Note: OIE stipulates that the neutralizing antibody titer is more than or equal to 1.65log 10 Typically for protection.
In conclusion, the invention uses FMDV reverse genetic manipulation technology to chimeric FMDV O/XJ/CHA/2017The virus L + P1 gene infectious clone is used as a framework to construct a recombinant virus rHN/XJ/SCGH replacing the O/SCGH/CHA/2016G-H gene of the Catay pedigree epidemic virus in China, the virus has short virus receiving time (12H) and high replication titer (1 × 10) 7.6 PFU/mL), the animal immunized by the virus vaccine can generate high-level protective cross-neutralizing antibodies for four-lineage epidemic viruses, and the antigen spectrum is expanded. Therefore, the recombinant virus rHN/XJ/SCGH constructed by the reverse genetic manipulation technology is suitable for serving as an FMD vaccine candidate strain and is used for effectively preventing and controlling the current O-type FMD in China.
The above description is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art to prepare FMD strains with expanded antigen spectrum by replacing the G-H loop of other circulating strains of the catay lineage with reverse genetic manipulation of FMDV, such improvements and modifications should be considered as the protection scope of the present invention without departing from the principle of the present invention.
Claims (6)
1. A candidate strain of a foot-and-mouth disease vaccine type O is characterized in that: the recombinant foot-and-mouth disease virus rHN/XJ full-length infectious clone pQSA is taken as a framework, and the G-H ring gene of an O/SCGH/CHA/2016 strain is embedded to obtain the recombinant foot-and-mouth disease virus strain; the full-length infectious clone PQSA of the recombinant foot-and-mouth disease virus rHN/XJ is obtained by taking the full-length infectious clone pOFS of a foot-and-mouth disease vaccine strain O/HN/CHA/93 as a framework and embedding the L + P1 gene of a foot-and-mouth disease epidemic strain O/XJ/CHA/2017.
2. The candidate strain for a foot-and-mouth disease type O vaccine of claim 1, wherein: the nucleotide sequence of the G-H loop gene of the O/SCGH/CHA/2016 strain is shown as SEQ ID NO:5, respectively.
3. The candidate strain for a aftosa vaccine as claimed in claim 1, wherein: the nucleotide sequence of the L + P1 gene of the O/XJ/CHA/2017 strain is shown as SEQ ID NO:2, respectively.
4. A method for constructing a candidate strain for the aftosa vaccine of type O according to any one of claims 1 to 3, comprising the steps of:
(1) Artificially synthesizing a recombinant plasmid pSK-Z123XJLP1 containing a foot-and-mouth disease epidemic strain O/XJ/CHA/2017L + P1 gene by taking a foot-and-mouth disease vaccine strain O/HN/CHA/93 semi-long plasmid pSK-Z123 as a template;
(2) Artificially synthesizing a recombinant plasmid pSK-Z123XJLP1/SCGH containing O-type foot-and-mouth disease virus O/SCGH/CHA/2016G-H loop gene chimeric gene by using the recombinant plasmid pSK-Z123XJLP1 synthesized in the step (1) as a template; digesting the synthesized plasmid with SpeI and BglII restriction enzymes, recovering a target fragment of about 5400bp, and inserting the target fragment into a pOFS plasmid digested with the SpeI and BglII restriction enzymes to obtain a recombinant full-length plasmid pQSQ;
(3) Transfecting the NotI linearized recombinant full-length plasmid pQSQ to BSR/T7 cells, rescuing the recombinant foot-and-mouth disease virus, and obtaining the O type foot-and-mouth disease vaccine candidate strain rHN/XJ/SCGH.
5. An application of the candidate strain of the O-type foot-and-mouth disease vaccine constructed based on the construction method of any one of claims 1 to 3 or 4 in the preparation of the O-type foot-and-mouth disease vaccine.
6. The application of the candidate strain of the type-O foot-and-mouth disease vaccine in the preparation of the type-O foot-and-mouth disease vaccine according to claim 5, is characterized in that: the prevention and control objects of the O-type foot-and-mouth disease vaccine are O/XJ/CHA/2017, O/Tibet/99, O/NXYCH/CHA/2018 and O/SCGH/CHA/2016 virus strains.
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