CN117844768A - Recombinant foot-and-mouth disease virus carrying G-H loop epitope of Cathay lineage strain, construction method and application thereof - Google Patents
Recombinant foot-and-mouth disease virus carrying G-H loop epitope of Cathay lineage strain, construction method and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant foot-and-mouth disease virus carrying a Cathay lineage strain G-H loop antigen epitope, and a construction method and application thereof, and belongs to the technical field of biological products. Recombinant foot-and-mouth disease virus carrying G-H loop antigen epitope of Cathay lineage strain expresses recombinant VP1 protein on the basis of O-type foot-and-mouth disease virus strain rHN/TURVP 1; the recombinant VP1 protein is FMDV O/TUR/5/2009 strain VP1 structural protein inserted into G-H loop in Cathay lineage FMDV O/GZSD/CHA/2018 strain VP 1. By utilizing reverse genetic operation technology, a G-H loop gene with the length of 20 amino acids of a foot-and-mouth disease epidemic strain of a Cathay lineage is inserted into a recombinant FMDV full-length clone, so that the cross reactivity of foot-and-mouth disease viruses of the lineage is remarkably improved, and a new thought is provided for the research of foot-and-mouth disease vaccines.
Description
Technical Field
The invention belongs to the technical field of biological products, and particularly relates to a recombinant foot-and-mouth disease virus carrying a G-H loop epitope of a strain of a Cathay lineage, and a construction method and application thereof.
Background
Foot-and-Mouth Disease (FMD) is a major virulent infectious Disease that severely damages major livestock such as pigs, cattle and sheep. The effective prevention and control of foot-and-mouth disease not only plays an important role in developing agriculture and animal husbandry economy, guaranteeing food safety and maintaining national reputation, but also has important significance in controlling global foot-and-mouth disease.
In recent years, O-type FMD still seriously endangers livestock breeding industry in China, and genetic evolutionary relationship of Foot-and-mouth disease virus (FMDV) is analyzed, and four pedigree (Mya-98 pedigree (SEA), cathay pedigree, panasia pedigree (ME-SA) and Ind-2001 pedigree (ME-SA)) strains of the O-type FMDV which are mainly popular in China are found. Wherein the antigenic variation of the Cathay lineage strain of swine is greatest, has been inherited into four branches: old pig toxin, new pig toxin-1, new pig toxin-2 and new pig toxin-3, the existing vaccine strain is not matched with the antigen of the new pig toxin-3 branch strain popular in recent years, and a new vaccine strain needs to be screened.
Disclosure of Invention
Therefore, the invention aims to provide a recombinant foot-and-mouth disease virus carrying a G-H loop epitope of a strain of a Cathay lineage, and the invention inserts a G-H loop epitope gene with 20 amino acids (aa) length of the epidemic strain of the foot-and-mouth disease of the Cathay lineage into a constructed full-length clone of the recombinant FMDV by utilizing a reverse genetic operation technology of FMDV, so that the cross reactivity of the virus to the epidemic strain of the Cathay lineage can be remarkably improved, and a new idea is provided for researching novel FMD vaccines.
The invention provides a recombinant foot-and-mouth disease virus carrying a Cathay lineage strain G-H loop antigen epitope, which expresses recombinant VP1 protein on the basis of O-type foot-and-mouth disease virus;
the recombinant VP1 protein is FMDV O/TUR/5/2009 strain VP1 structural protein inserted with G-H loop in Cathay pedigree FMDV O/GZSD/CHA/2018 strain VP 1.
Preferably, the amino acid sequence of the G-H loop in the Cathay lineage FMDV O/GZSD/CHA/2018 strain VP1 is shown in SEQ ID NO: 1.
Preferably, the amino acid sequence of VP1 structural protein of FMDV O/TUR/5/2009 strain is shown as SEQ ID NO: 3.
Preferably, the G-H loop in the Cathay lineage FMDV O/GZSD/CHA/2018 strain VP1 is inserted upstream or downstream of the RGD of the VP1 structural protein G-H loop of FMDV O/TUR/5/2009 strain VP 1.
Preferably, the amino acid sequence of the recombinant VP1 protein is shown as SEQ ID NO: shown at 5.
The invention provides a construction method of the recombinant foot-and-mouth disease virus, which comprises the following steps:
synthesizing G-H loop genes in a Cathay pedigree FMDV O/GZSD/CHA/2018 virus strain VP1 into a half-length plasmid pSK-Z123/TURVP1 to obtain a recombinant half-length plasmid;
recovering a gene sequence containing VP1/G-H loop fusion genes from the recombinant semi-long plasmid;
cloning the gene sequence containing the VP1/G-H ring fusion gene into a pOFS/TURVP1 plasmid to obtain a recombinant plasmid pOFS/TURVP1/GZGH 20 ；
The recombinant plasmid pOFS/TURVP1/GZGH 20 Carrying out virus rescue to obtain the recombinant foot-and-mouth disease virus.
Preferably, the nucleotide sequence of the G-H loop gene in the Cathay pedigree FMDV O/GZSD/CHA/2018 strain VP1 is shown as SEQ ID NO: 2.
Preferably, the nucleotide sequence of the VP1/G-H loop fusion gene is shown as SEQ ID NO: shown at 6.
The invention provides a vaccine for preventing and controlling epidemic strains of O-type foot-and-mouth disease viruses, which comprises the recombinant foot-and-mouth disease viruses or the recombinant foot-and-mouth disease viruses and an adjuvant obtained by the construction method.
The invention provides application of the recombinant foot-and-mouth disease virus or the recombinant foot-and-mouth disease virus obtained by the construction method in constructing vaccines for preventing and controlling epidemic strains of O-type foot-and-mouth disease virus, wherein the epidemic strains of O-type foot-and-mouth disease virus comprise at least one of the following lineages: mya-98 lineage, cathay lineage, panAsia lineage and Ind-2001 lineage.
The invention provides a recombinant foot-and-mouth disease virus carrying a Cathay lineage strain G-H loop antigen epitope, which expresses recombinant VP1 protein on the basis of O-type foot-and-mouth disease virus; the recombinant VP1 protein is FMDV O/TUR/5/2009 strain VP1 structural protein inserted with G-H loop in Cathay pedigree FMDV O/GZSD/CHA/2018 strain VP 1. The invention inserts the VP120 amino acid long G-H loop of the FMDV O/GZSD/CHA/2018 strain of the Cathay pedigree into VP1 structural protein of the FMDV O/TUR/5/2009 strain to be expressed by utilizing reverse genetics means, and the replication capacity of foot-and-mouth disease viruses is not influenced, and the genetic stability of recombinant viruses is not influenced. Meanwhile, compared with the parent virus, the vaccine immune pigs prepared by the recombinant foot-and-mouth disease virus can obviously improve the cross reactivity of the recombinant virus to the Cathay lineage epidemic virus, and provide a new thought for the research of novel FMD vaccine.
Drawings
FIG. 1 is a schematic diagram of the genome of a recombinant plasmid, annotated: the dark grey part indicates the VP1 gene of FMDV O/TUR/5/2009 strain, and the arrow indicates the position of insertion;
FIG. 2 is a diagram of the identification of the PstI cleavage of the recombinant full-length plasmid; m, DNA standard marker;1, cleavage of pOFS/TURVP1 plasmid with pstI; 2 pOFS/TURVP1/GZGH 20 The plasmid was digested with pstI;
FIG. 3 shows BSR/T7 cells transfected with recombinant plasmid, A: normal BSR/T7 cells, B: BSR/T7 cells after 60h transfection with recombinant plasmid;
FIG. 4 is an indirect immunofluorescence result;
FIG. 5 is an electron microscope observation of recombinant FMDV;
FIG. 6 is a one-step growth curve for recombinant and parental viruses;
figure 7 is an average neutralizing antibody for 28 day porcine serum neutralization of different lineage strains, injected: the red dotted line indicates that antibody titer is equal to 1.65log 10 The method comprises the steps of carrying out a first treatment on the surface of the Neutralizing antibody titer of 1.65log or greater 10 Typically protection.
Detailed Description
The invention provides a recombinant foot-and-mouth disease virus carrying a Cathay lineage strain G-H loop antigen epitope, which expresses recombinant VP1 protein on the basis of O-type foot-and-mouth disease virus;
the recombinant VP1 protein is FMDV O/TUR/5/2009 strain VP1 structural protein inserted with G-H loop in Cathay pedigree FMDV O/GZSD/CHA/2018 strain VP 1.
In the present invention, the amino acid sequence of the G-H loop in the Cathay lineage FMDV O/GZSD/CHA/2018 strain VP1 is preferably as set forth in SEQ ID NO:1 (SSKYGDASANNVRGDLHVLA).
In the present invention, the amino acid sequence of VP1 structural protein of FMDV O/TUR/5/2009 strain is preferably shown as SEQ ID NO:3 (TTSAGESADPVTATVENYGGETQVQRRQHTDVSF ILDRFVKVTPKDQINVLDLMQTPAHTLVGALLRTATYYFADLEVAVKHEG NLTWVPNGAPETALDNTTNPTAYHKAPLTRLALPYTAPHRVLATAYNGNCKYGESHTTNVRGDLQVLDQKAARTLPTSFNYGAIKATRVTELLYRMKRAETYCPRPLLAIHPSEARHKQKIVAPVKQLL). In view of the many positions of FMDV structural protein VP1, insertion of some epitopes or tags can be tolerated without affecting recombinant expression of VP1 and rescue of live virus. Therefore, the position of insertion of the G-H loop in the Cathay lineage strain VP1 is not particularly limited, and any insertion position known in the art may be employed. The G-H loop in FMDV O/GZSD/CHA/2018 strain VP1 of the Cathay lineage is inserted upstream or downstream of RGD of the G-H loop in the VP1 structural protein of FMDV O/TUR/5/2009 strain. In the embodiment of the invention, the insertion position of the G-H loop in the VP1 strain VP1 of the Cathay lineage is upstream of RGD motif in the VP1 structural protein of FMDV O/TUR/5/2009 strain, and is particularly inserted between G and N of YNNNCK, and is particularly shown in FIG. 1. In the embodiment of the present invention, the amino acid sequence of the recombinant VP1 protein is preferably shown as SEQ ID NO:5 (TTSAGESADPVTATVENYGGETQVQRRQHT DVSFILDRFVKVTPKDQINVLDLMQTPAHTLVGALLRTATYYFADLEVAVKHEGNLTWVPNGAPETALDNTTNPTAYHKAPLTRLALPYTAPHRVLATAYNGSSKYGDASANNVRGDLHVLANCKYGESHTTNVRGDLQVLDQKAARTLPTSFNYGAIKATRVTELLYRMKRAETYCPRPLLAIHPSEARHKQKIVAPVKQLL).
The invention provides a construction method of the recombinant foot-and-mouth disease virus, which comprises the following steps:
synthesizing G-H loop genes in a Cathay pedigree FMDV O/GZSD/CHA/2018 virus strain VP1 into a half-length plasmid pSK-Z123/TURVP1 to obtain a recombinant half-length plasmid;
recovering a gene sequence containing VP1/G-H loop fusion genes from the recombinant semi-long plasmid;
cloning the gene sequence containing the VP1/G-H ring fusion gene into a pOFS/TURVP1 plasmid to obtain a recombinant plasmid pOFS/TURVP1/GZGH 20 ；
The recombinant plasmid pOFS/TURVP1/GZGH 20 Carrying out virus rescue to obtain the recombinant foot-and-mouth disease virus.
The invention synthesizes G-H loop genes in Cathay pedigree FMDV O/GZSD/CHA/2018 virus strain VP1 into half-length plasmid pSK-Z123/TURVP1 to obtain recombinant half-length plasmid.
In the present invention, the half-length plasmid pSK-Z123/TURVP1 is a recombinant plasmid comprising VP1 structural protein gene of FMDV O/TUR/5/2009 strain, and is disclosed in the prior art (Evaluation of immunogenicity and cross-reactive responses of vaccines prepared from two chimeric serotype O foot-and-mouth disease viruses in pigs and cattle, 2022).
In the present invention, the nucleotide sequence of the G-H loop gene in the Cathay lineage FMDV O/GZSD/CHA/2018 strain VP1 is preferably as set forth in SEQ ID NO:2 (agtagcaaatacggtgacgccagtgccaacaacgtcagaggcgaccttcacgtgttagct). The method of cloning is not particularly limited, and the cloning method described in the art may be employed. In the examples of the present invention, the recombinant semi-long plasmid was synthesized by the gene synthesis company.
After the recombinant half-length plasmid is obtained, the gene sequence containing the VP1/G-H ring fusion gene is recovered from the recombinant half-length plasmid.
In the present invention, the recovery method preferably includes an enzymatic cleavage method. The enzymatic cleavage method involves digestion with SpeI and BglII restriction enzymes, and collecting a 5400bp DNA fragment containing the VP1/G-H loop fusion gene. The nucleotide sequence of the VP1/G-H loop fusion gene is preferably shown as SEQ ID NO:6 (ACCACTTCTGCAGGTGAGTCAGCTGACCCCGTGACTGCCACTGTTGAGAACTACGGTGGCGAGACACAGGTCCAGAGACGCCAGCACACGGATGTCTCGTTTATATTGGACAGATTTGTGAAAGTGACACCAAAAGACCAAATTAATGTGTTGGACCTGATGCAAACCCCCGCCCACACTTTGGTAGGGGCGCTCCTCCGCACCGCCACCTACTACTTCGCTGATCTAGAGGTGGCAGTGAAACACGAGGGGAACCTTACCTGGGTCCCGAATGGGGCGCCCGAGACAGCGTTGGATAACACCACTAATCCAACGGCTTACCACAAAGCACCGCTCACCCGACTTGCACTGCCTTACACGGCACCGCACCGTGTCTTGGCTACCGCGTACAACGGGagtagcaaatacggtgacgccagtgccaacaacgtcagaggcgaccttcacgtgttagctAACTGCAAGTATGGCGAGAGCCACACAACCAATGTGAGAGGTGACCTGCAAGTGTTGGACCAGAAGGCGGCGAGAACGTTGCCTACTTCCTTTAACTACGGTGCCATCAAAGCTACCCGGGTGACCGAGTTGCTTTACCGCATGAAGAGGGCTGAAACATACTGCCCCCGGCCTCTTTTGGCTATTCACCCGAGCGAAGCAAGACACAAACAGAAGATAGTGGCACCTGTGAAGCAGCTTCTG).
After the VP1/G-H ring fusion gene is obtained, the gene sequence containing the VP1/G-H ring fusion gene is cloned into a pOFS/TURVP1 plasmid to obtain a recombinant plasmid pOFS/TURVP1/GZGH 20 。
The cloning method of the present invention is not particularly limited, and cloning methods well known in the art, such as cleavage and ligation methods, may be employed. Obtaining recombinant plasmid pOFS/TURVP1/GZGH 20 Thereafter, it preferably further comprises recombinant plasmids pOFS/TURVP1/GZGH 20 And (5) performing identification. The identification method is preferably that the Pst I endonuclease is used for enzyme digestion, and the result is that the target band which accords with the expected size is cut, which means that the recombinant plasmid pOFS/TURVP1/GZGH is successfully obtained 20 . Simultaneously adopting a sequencing means to detect the recombinant plasmid pOFS/TURVP1/GZGH 20 Insertion of the G-H loop epitope gene.
Obtaining recombinant plasmid pOFS/TURVP1/GZGH 20 The recombinant plasmid pOFS/TURVP1/GZGH is then introduced into the invention 20 Carrying out virus rescue to obtain the O-type foot-and-mouth disease vaccine strain.
The method of the present invention is not particularly limited, and can be carried out by methods known in the art, such as linearization of recombinant plasmid pOFS/TURVP1/GZGH 20 Post-transfection into BSR/T7 cells expressing T7RNA polymerase, and after cytopathic effect, virus solution was collected.
After the O-type foot-and-mouth disease vaccine strain is obtained, the difference condition of the exogenous gene is preferably identified by utilizing RT-PCR and indirect immunofluorescence detection. The results show that the O-type foot-and-mouth disease vaccine strain contains the insertion of the G-H loop gene.
In the invention, the O-type foot-and-mouth disease vaccine strain has good genetic stability, and is continuously transmitted for 10 generations in recombinant viruses, and a sequencing verification result shows that the structural protein and the inserted amino acid have no amino acid mutation. Meanwhile, the result of the one-step growth curve shows that the replication titer of the O-type foot-and-mouth disease vaccine strain is not remarkably different from that of the parent strain.
The invention provides a vaccine for preventing and controlling epidemic strains of O-type foot-and-mouth disease viruses, which comprises the recombinant foot-and-mouth disease viruses or the recombinant foot-and-mouth disease viruses and an adjuvant obtained by the construction method.
In the invention, the volume ratio of the recombinant foot-and-mouth disease virus to the adjuvant is 45-47:53-55, and more preferably 46:54. The adjuvant is preferably ISA201 adjuvant.
In the present invention, the method for preparing the vaccine preferably comprises the steps of: and (3) inactivating the recombinant foot-and-mouth disease virus, and then mixing the inactivated recombinant foot-and-mouth disease virus with an adjuvant according to a proportion until the recombinant foot-and-mouth disease virus and the adjuvant are not layered, thereby obtaining a vaccine finished product. The inactivation method is preferably to mix the collected virus liquid with BEI solution with the final concentration of 1-1.2% and inactivate the virus liquid at 30 ℃ for 28 hours. When mixing, the adjuvant is preferably subjected to a preheating treatment at 37 ℃. The vaccine is preferably stored at 4-8 ℃.
The invention provides application of the recombinant foot-and-mouth disease virus or the recombinant foot-and-mouth disease virus obtained by the construction method in constructing vaccines for preventing and controlling epidemic strains of O-type foot-and-mouth disease virus, wherein the epidemic strains of O-type foot-and-mouth disease virus comprise at least one of the following lineages: mya-98 lineage, cathay lineage, panAsia lineage and Ind-2001 lineage.
In the invention, neutralization experiments show that compared with the parental virus vaccine, the recombinant FMDV vaccine can generate protective average cross-neutralizing antibodies to epidemic strains of 4 lineages (Mya, panasia and Ind-2001 and Cathay lineages) after 28 days of immunization of pigs>2.0log 10 ) It was demonstrated that insertion of the 20aa long G-H loop of the Cathay lineage strain was significantly improved (p<0.05 Cross-reactivity of recombinant FMDV against viruses of this lineage, whereas the parental viral vaccine only produces protective cross-neutralizing antibodies to viruses of the Mya, panASia and Ind-2001 lineages>2.3log 10 ). The Cathay lineage epidemic strains include O/GZSD/CHA/2018 and O/GXCX/CHA/2018 strains.
The following examples are presented to illustrate in detail a recombinant foot-and-mouth disease virus carrying a G-H loop epitope of a strain of the Cathay lineage, and methods of constructing and using the same, but they are not to be construed as limiting the scope of the invention.
The half-length plasmid pSK-Z123/TURVP1 and the full-length plasmid pOFS/TURVP1 (see published article Evaluation of immunogenicity and cross-reactive responses of vaccines prepared from two chimeric serotype O foot-and-mouth disease viruses inpigs and cattle of 2022, which are commonly opened) inserted with VP1 structural protein of FMDV O/TUR/5/2009 strain were used for the construction of recombinant full-length plasmids. The O-type FMDV strain O/GZSD/CHA/2018 (Cathay pedigree), O/HB/HK/99 (Panasia pedigree), O/XJ/CHA/2017 (Ind-2001 pedigree), O/NXYCh/CHA/2018 (Mya-98 pedigree) O/GXCX/CHA/2018 (Cathay pedigree) is a public strain in China, wherein O/HB/HK/99 is disclosed on page P29 of the foot-and-mouth disease editions (Liu Xiangtao and other major editions); O/XJ/CHA/2017 (MF 461724.1), O/NXYCh/CHA/2018 (MH 791315.1) and O/GZSD/CHA/2018 (KX 161429.1), O/GXCX/CHA/2018 (MH 791316.1) are disclosed in Genebank. The full length plasmid pOFS/TURVP1 transfects the rescued virus rHN/TURVP1 as the parent virus (see co-opening of 2022 publication Evaluation of immunogenicity and cross-reactive responses of vaccines prepared from two chimeric serotype O foot-and-mouth disease viruses inpigs and cattle).
Example 1
Construction method of recombinant foot-and-mouth disease virus carrying 20 amino acid long G-H loop antigen epitope carrying foot-and-mouth disease epidemic strain of Cathay lineage
1. Construction of recombinant full Length clones
In this example, half-length clone pSK-Z123/TURVP1 inserted with VP1 structural protein of FMDV O/TUR/5/2009 strain is taken as a skeleton, and plasmid pSK-Z123/TURVP1/GZGH (Jin Wei, manufactured by Biotechnology Co., ltd.) containing 20aa long G-H loop gene insertion of the virus is designed and synthesized according to the published nucleotide sequence of VP 1G-H of FMDV O/GZSD/CHA/2018 strain of Cathay lineage in Genebank. The plasmid was digested with SpeI and BglII restriction enzymes, and the fragment of interest was recovered at about 5400bp and inserted into the pOFS/TURVP1 plasmid digested with the same enzymes to give a recombinant plasmid pOFS/TURVP1/GZGH 20 . The recombinant plasmid pOFS/TURVP1/GZGH 20 Cleavage with PstI resulted in a match with the expected size (see FIG. 2).
The recombinant plasmid with correct restriction enzyme identification was sent to Jin Weizhi Biotechnology Co., ltd for sequence determination, and the result shows that the constructed recombinant plasmid contains expected insertion. Wherein pOFS/TURVP1 and pOFS/TURVP1/GZGH 20 The nucleotide sequences of VP1 genes of (C) are shown in SEQ ID NO:4,SEQ ID NO:6, the corresponding amino acid sequence is shown in SEQ ID NO:3 and SEQ ID NO:5. the genome schematic of the recombinant full-length plasmid is shown in FIG. 1.
TABLE 1 partial sequence names and viruses corresponding to recombinant plasmids
2. Recombinant FMDV rescue
Full-length plasmid pOFS/TURVP1/GZGH prepared by conventional method 20 After linearization with Not I, the purified and recovered was used as a transfection template. After BSR/T7 cells cultured by six pore plates grow to 80 to 90 percent of full, liposome Lipofectamine is used TM 2000 medium linear plasmid transfection. 2mL of complete medium was added 5h after transfection and placed at 37℃in 5% CO 2 The incubator continues to cultivate. Cells were observed daily for cytopathic (CytopHathic Effect, CPE) appearance. Transfected cells were collected after 3 days of culture and serial passaging was performed after 3 times of repeated freeze thawing.
The results show that: typical cytopathic effects, i.e., enlargement and rounding of cells with fibrous distribution (see FIG. 3), were observed 60h after BSR/T7 cells were transfected with the recombinant full-length plasmid, and no enlarged, rounded cells were observed in control cells except individual dead cells. The recombinant virus obtained by rescue is named rHN/TURVP1/GZGH 20 。
3. Identification of recombinant viruses
3.1, RT-PCR identification
The transfected supernatant was used to extract total cytotoxic RNA using RNAasy Mini Kit, RT-PCR was performed using an OZ1490 (+)/OZ 3980 (-) primer pair (OZ 1490 (+): GACAAGACCACGCCGTATT, OZ3980 (-): TGCATCTGGTTGATGGTGTC) to amplify VP1 gene fragment, gel purified and recovered, and sent to Jin Weizhi biological Co., ltd for sequencing, to verify the correctness of the recombinant virus.
Sequencing results showed that: the recombinant virus contains the expected insertion, which means that the invention successfully constructs the recombinant FMDV containing the target gene insertion.
3.2 indirect immunofluorescence
BHK-21 monolayer cells (six-well plate) were grown to 70% -80% full and inoculated with parental virus and recombinant virus, respectively. The expression of FMDV nonstructural protein 3A was detected with indirect immunofluorescence 6h after virus inoculation. The detailed steps are as follows:
(1) The virus-inoculated cell culture solution was discarded, rinsed 3 times with PBS (ph=7.4), added with 4% ice-cold paraformaldehyde, and fixed at room temperature for 30min;
(2) Rinsing 3 times with PBS, adding 5% BSA, and sealing for 30min at room temperature;
(3) After PBS rinsing for 3 times, adding 1:500 diluted anti-FMDV nonstructural protein 3A and 3B monoclonal antibodies respectively for incubation for 1h at 37 ℃;
(4) Rinsing 5 times by PBS, adding FITC-labeled IgG secondary antibody diluted by 1:100, and incubating for 1h at 37 ℃;
(5) Rinsing with PBS for 5 times, adding 0.5ug/ml DAPI for 10min, washing with PBS for 5 times, removing excessive DAPI, photographing under confocal fluorescence microscope, and setting normal cell control.
The results show that: BHK-21 cells inoculated with the parent virus and the recombinant virus can act with the 3A monoclonal antibody to generate specific green fluorescence, and control cells can not see any visible fluorescence when acting with the 3A monoclonal antibody (see figure 4), so that the recombinant FMDV is successfully rescued.
3.3 observation by electron microscope
The BHK-21 monolayer adherent cells are respectively proliferated with 100mL of parent virus and recombinant virus, frozen and thawed for 2-3 times, centrifuged at 12000rpm/min for 1h to remove cell debris, and the supernatant is inactivated by BEI. Centrifugation was carried out at 35000rpm/min for 3 hours at 4℃after the completion of the inactivation. The centrifuged pellet was resuspended in PBS (ph=7.6) buffer and visualized by electron microscopy after negative staining.
And (3) the observation result of the electron microscope shows that: the recombinant virus was identical to the parental virus in morphology, with spherical virus particles of approximately 25nm in diameter, and completely identical to FMDV in morphology (see fig. 5).
Example 2
Growth characteristics of recombinant viruses constructed in example 1
1. Genetic stability analysis to rescue viruses
Inoculating the parent virus and the transfected supernatant into a grown BHK-21 monolayer cell (T25 cell bottle) according to an inoculum size of 10%, harvesting cells when 95-100% of cells inoculated with the transfected supernatant have typical cytopathic effect, repeatedly freezing and thawing for 3 times, continuing continuous passage, observing the time when the virus inoculated cells have more than 95% -100% of cells to have typical CPE after the 6 th generation, carrying out RT-PCR on the 5 th and 10 th generation viruses, and detecting the change of the structural protein amino acid of the rescued recombinant virus.
Results of serial passages are indicated: the time from the continuous transmission of the rescued recombinant viruses to the occurrence of typical CPE at the 6 th generation is stable (12-14 h). The recombinant virus is continuously transferred to the 10 th generation, and the sequencing verification result shows that the virus structural protein does not generate any amino acid mutation, and the virus structural protein has good genetic stability.
2. One-step growth curve of recombinant virus
Serial dilutions of 6 th generation parent virus and recombinant virus are respectively carried out for 10 times, then, different dilutions of virus are respectively inoculated with grown single-layer BHK-21 cells (200 ul/hole, 6 pore plate), the single-layer BHK-21 cells are placed in a 37 ℃ incubator for culture, shaking is carried out every 10min, 2mL of tragacanth mixed solution (2 XMEM, 1.2% tragacanth and 1% serum) is added after 1h for static culture, the culture solution is sucked and removed after 48h, MEM is used for washing 1-2 times, then, stationary solution is added for 30min at room temperature, crystal violet staining is carried out for 1h, clear water washing is carried out, and plaque forming units (PFU/mL) of each virus are calculated. Generation 6 of the parent virus and recombinant virus 1×10 6 PFU/mL virus infection was inoculated with a single layer of BHK-21 cells (25 mL flask), the inoculated virus solution was adsorbed for 1 hour, and after washing 2 times with MEM, 5mL of MEM medium was added to the flask, and the flask was incubated at 37℃for further incubation. Samples were collected 4h, 8h, 12h, 16h and 20h after inoculation, and after 3 times of repeated freeze thawing, virus titer (PFU/ml) was measured on BHK-21 monolayer cells (6 well plate) as described above (experiment was performed 2 times of repetition) and a one-step growth curve of the virus was drawn. The results show that: the 20 amino acid inserted recombinant virus was similar to the parental virus replication kinetics curves, indicating that insertion of 20aa long G-H upstream of RGD did not significantly affect the replication level of FMDV on BHK-21, as shown in fig. 6.
Example 3
Preparation of FMDV inactivated vaccine
The parent virus and the recombinant virus are respectively inoculated with 100% of full monolayer adherent BHK-21 cells (175 mL cell bottles, 27mL inoculation liquid and 3mL virus liquid), the parent virus and the recombinant virus are continuously cultured in a 37 ℃ incubator, when more than 100% of cells have typical CPE, the virus is harvested, repeatedly frozen and thawed for 3 times, and centrifuged at 6000rpm/min at 4 ℃ for 1 hour to remove cell debris. The collected viral supernatants were inactivated with 1-1.2% BEI at 30℃for 28h. And carrying out cell blind transfer for 4 generations on the inactivated virus antigen, and carrying out inactivation safety inspection. And (3) purifying the virus particles by a sucrose density gradient centrifugation method after the virus particles are qualified, and detecting the 146S content of the virus antigen by a liquid chromatograph. The ISA201 adjuvant is placed in a constant-temperature water bath kettle at 37 ℃ for preheating, a proper amount of adjuvant is slowly added into the virus antigen according to the ratio of antigen to adjuvant volume ratio=46:54, the virus antigen is slowly shaken until the antigen and the adjuvant are not layered, and the prepared vaccine product (the concentration of 146S antigen per part is 6 mug/mL) is placed in 4-8 ℃ for standby.
Example 4
Vaccine immunization
12 healthy and susceptible pigs (O-type foot-and-mouth disease liquid phase blocking ELISA antibody titer is less than 1:4,3ABC antibody is negative) of about 90 days old are selected and divided into 2 groups, and 6 groups are selected. One group of immunization parental virus rHN/TURVP1 vaccine and one group of immunization rHN// TURVP1/GZGH 20 The virus vaccine was inoculated at a dose of 2 mL/head (6. Mu.g). All pigs were bled weekly before and after immunization and serum was collected and placed at-20 ℃ for use.
Example 5
Virus neutralization assay
Serum neutralization after 28 days of immunization of animals with 2 vaccines was tested with a minivirus neutralization assay for cross-neutralizing antibody titers of four pedigree FMDV strains O/HB/HK/99, O/XJKS/2017, O/NXYCh/CHA/2018, O/GZSD/CHA/2018 and O/GXCX/CHA/2018 prevalent in our country, as follows: (1) immune serum is inactivated for 30min at 56 ℃;
(2) Taking inactivated serum, diluting the serum with serum-free cell culture solution on a 96-well micro-cell culture plate, and performing a series of multiple dilution from 1:4, wherein the content of each well is 50 mu L, and each dilution is 8 wells;
(3) Taking virus liquid stored in-70 ℃ refrigerator, and taking the virus liquid as 200TCID according to the determined toxicity price 50 Dilution (after mixing with equal amount of serum, its toxicity is 100TCID 50 )；
(4) 50 μl was added to each well of 96 empty plates containing diluted serumPlacing diluted virus liquid in 5% CO 2 Neutralizing for 1h at 37 ℃;
(5) After serum and virus act for 1h, 50 mu L of cell suspension (preferably, a monolayer grows in 24 h) is added into each hole, a transparent adhesive tape is used for sealing and covering, then the mixture is placed in a 37 ℃ incubator for culture, after 48h, appropriate judgment is carried out under a microscope, and after 72h, fixation and dyeing are carried out;
(6) The following controls were established per plate: (1) positive and negative serum controls: the positive and negative serum controls were each provided with 8 wells, 50 μl per well, the positive wells were blue and the negative wells were not colored. (2) Virus regression test: the virus was first treated as 0.1, 1, 10, 100TCID 50 Dilutions were performed, 8 wells per dilution, 50 μl per well. Then 50. Mu.l of the cell suspension was added to each well. 0.1TCID 50 Should be blue, 100TCID 50 No staining, otherwise the experiment was not true. (3) Normal cell control: to avoid experimental errors caused by the culture plates, 8-hole normal cell controls without virus and serum are set on each plate, and the controls keep good morphology and living characteristics all the time in the whole experiment and are dyed blue;
(7) The determination is made when all of the viral regression test, positive, negative, and normal cell controls are established. Judging negative when 100% CPE appears in the serum hole to be detected, and judging positive when more than 50% of cells appear in the protector; the final dilution of serum was calculated as the 50% endpoint of the serum/virus mixture as calculated from the results of the fixed virus dilution serum neutralization assay, the serum neutralization titer was calculated by the Karber method, neutralization titers of 1:45 or higher were positive, 1:16-1:32 were suspected to be reworked, and titers of 1:11 or lower were negative.
The result of the neutralization experiment shows that: the parent virus vaccine can generate protective average neutralizing antibodies to Mya, panasia and Ind-2001 lineage epidemic strains 28 days after immunization of pigs>2.3log 10 ) But cannot produce protective average neutralizing antibodies to Cathay lineage strains<1.0log 10 ) Whereas recombinant virus rHN/TURVP1/GZGH 20 Protective average cross-neutralizing antibodies can be generated on epidemic strains of 4 lineages after 28 days of immunization of pigs>2.0log 10) (see FIG. 7), demonstrating that insertion of the 20aa long G-H loop of the Cathay lineage strain significantly increases (p)<0.05 Recombinant FM)Cross-reactivity of DV against viruses of this lineage.
The results of the present invention show that: inserting a Cathay pedigree foot-and-mouth disease epidemic virus O/GZSD/CHA/201820aa long G-H epitope gene on the skeleton of a parent FMDV rHN/TURVP1, wherein the replication performance of the recombinant virus is not obviously affected, and the recombinant virus can be stably inherited; compared with the parent virus, the vaccine immune pigs prepared by the recombinant virus obviously improve the cross reactivity of the recombinant virus to the Cathay lineage epidemic virus, and provide a new thought for the research of novel FMD vaccine.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A recombinant foot-and-mouth disease virus carrying a G-H loop epitope of a strain of the Cathay lineage, characterized in that recombinant VP1 protein is expressed on the basis of a strain of the foot-and-mouth disease O rHN/TURVP 1;
the recombinant VP1 protein is FMDV O/TUR/5/2009 strain VP1 structural protein inserted with G-H loop gene in Cathay pedigree FMDV O/GZSD/CHA/2018 strain VP 1.
2. The recombinant foot-and-mouth disease virus according to claim 1, wherein the amino acid sequence of the G-H loop in strain VP1 of the Cathay lineage FMDV O/GZSD/CHA/2018 virus is as set forth in SEQ ID NO: 1.
3. The recombinant foot-and-mouth disease virus according to claim 1, wherein the amino acid sequence of VP1 structural protein of strain FMDV O/TUR/5/2009 is as set forth in SEQ ID NO: 3.
4. The recombinant foot and mouth disease virus according to claim 1, wherein the G-H loop gene in strain VP1 of FMDV O/GZSD/CHA/2018 of the Cathay lineage is inserted upstream or downstream of the RGD of the G-H loop of the VP1 structural protein of strain FMDV O/TUR/5/2009.
5. The recombinant foot-and-mouth disease virus according to any one of claims 1 to 4, wherein the amino acid sequence of the recombinant VP1 protein is as set forth in SEQ ID NO: shown at 5.
6. The method for constructing recombinant foot-and-mouth disease virus according to any one of claims 1 to 5, comprising the steps of:
synthesizing G-H loop genes in a Cathay pedigree FMDV O/GZSD/CHA/2018 virus strain VP1 into a half-length plasmid pSK-Z123/TURVP1 to obtain a recombinant half-length plasmid;
recovering a gene sequence containing VP1/G-H loop fusion genes from the recombinant semi-long plasmid;
cloning the gene sequence containing the VP1/G-H ring fusion gene into a pOFS/TURVP1 plasmid to obtain a recombinant plasmid pOFS/TURVP1/GZGH 20 ；
The recombinant plasmid pOFS/TURVP1/GZGH 20 Carrying out virus rescue to obtain the recombinant foot-and-mouth disease virus.
7. The construction method according to claim 6, wherein the nucleotide sequence of the G-H loop gene in the Cathay lineage FMDV O/GZSD/CHA/2018 strain VP1 is set forth in SEQ ID NO: 2.
8. The construction method according to claim 6, wherein the nucleotide sequence of the VP1/G-H loop fusion gene is shown in SEQ ID NO: shown at 6.
9. A vaccine for preventing and controlling epidemic strains of O-type foot-and-mouth disease virus, which is characterized by comprising the recombinant foot-and-mouth disease virus according to any one of claims 1 to 5 or the recombinant foot-and-mouth disease virus and the adjuvant obtained by the construction method according to claims 6 to 8.
10. Use of the recombinant foot-and-mouth disease virus according to any one of claims 1 to 5 or the recombinant foot-and-mouth disease virus obtained by the construction method according to claims 6 to 8 for constructing a vaccine for controlling epidemic strains of foot-and-mouth disease virus of type O comprising at least one lineage of: mya-98, cathay, panAsia and Ind-2001.
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