Molecular Characterization, Rescuing Of FMDV And Study Of Multivalence Genetically Engineered Vaccine

Foot-and-mouth disease (FMD) caused by FMD virus (FMDV) is one of the most contagious animal virus diseases. At present, FMD is spreaded in all of the world. The virus exists in the form of seven different serotype: O, A, C, Asia1, and South African Territors1 (SAT1), SAT2 and SAT3. Serotype O, A and Asia1 FMD once happened in history of China, and are often epidemic in the country around China and Southeast Asia. Our country has been threatened by Serotype O, A and Asia1 FMD at present.FMDV is the prototype member of the Aphthovirus genus of the family Picornaviridae. The genome is a 8.5kb long ssRNA in the positive orientation. From 5'terminal to 3'terminal, there are 5'UTR (including S-fragment, poly(C) tract, pseudoknot, cre and IRES), Lpro, structural proteins precursor P1 region (encoding VP4, VP2, VP3, VP1), nonstructural proteins precursor P2 region (encoding 2A, 2B and 2C) and P3 region (encoding 3A, 3B,3C and 3D), 3'UTR in line, and VP1 possesses the characteristic of distinguishing different serotype and subtype.The stamping out strategy is adopted by developed country, and vaccination strategy is adopted by developing country. The vaccination strategy often is adopted by part of developed country because of stamping out strategy causing heavy economics loss. To day, the vaccination with inactivated FMDV is a major means to prevent and control this disease in most developing countries. However, the inactivation processes are technically challenging and sometimes can lead to incomplete inactivation, which is considered as a risk to massively employ the conventional vaccine So newly developed genetic recombinant vaccine have become alternative characterized by the advantages of being safer and more economic.In this study, by means of molecular biology, immunology, molecular virology, biochemistry and bioinformatics et al, the whole genes of FMDV Cathy isolate O/LZ were cloned, sequenced and analyzed, and the O/LZ isolate FMDV was rescued. Base on the above study, and FMD epidemic in China, a multiple-epitope foot and mouth disease virus (FMDV) antigen, named OAAT has been designed and constructed. The gene encoding OAAT was expressed in e coil, and the immunogenicity of OAAT has been also studied by DNA vaccine and rFPV vaccine.It was generally believed that complete genome information of virus would contribute to understanding the molecular characteristic of FMDV and R&D of new vaccines and diagnosis techniques. The whole genome fragments of serotype O FMDV isolate O/LZ were cloned, sequenced by RT-PCR except for poly(C)and poly(A), and banked into Genbank (Access number: DQ248888).The genome of O/LZ isolate is 8014nt, including partical 5'UTR(including pseudoknot, cre and IRES structure regions, 1041bp) , full length of Lpro gene(603bp), P1(2011 bp), P2 (1464 bp ) and P3(2804 bp).Comparison between O/LZ and other reference isolates indicates that O/LZ strain have a close evolutionary relationship with O/HNK/2002, O/ES/2001, O/CHP/TW/97, etc, O/LZ should be classified to Cathay topotype. Futhermore, comparison of deduced amino acid residues at the antigenic sites with PanAsia strain of FMDV serotype O ME-SA topotype, shows there is a obviouslly topotypical amino acid difference at Site 1 and Site 3, and the 3A gene was deleted 15aa, besides, 5'UTR and Lpro gene have variation, but the key point amino acids motif of Petiole of loop-step structure and binding site of protein factor in IRES. Using Clarke's method, 3 conservative pseudoknot structures were found in pseudoknot region of O/LZ genome, whereas 4 pseudoknots were identified in same region of O/NY00, which is the prototype member of PanAsia topotype of FMDV serotype O. This different might be a new molecular mark to distinguish Cathay topotype strains from PanAsia strains. The above results suggested that O/LZ strain has different molecular biology characteristic, comparing with other FMDV isolate strains.The conventional molecular biology measure hasn't been able to ensure the study of the pathogenicity and immunogenicity of FMDV because the pathogenicity and immunogenicity of FMDV is very easily variation under selective pressure Reverse genetics was used to study molecular biology characteristic of FMDV rather than the conventional molecular biology measure. The measure of reverse genetics is important to study virus in molecular level analyzing and changing virus in order to control the replication, pathogenicity and vaccine study of FMDV.In this paper, four cDNA fragments covering the foot-and-mouth disease virus (FMDV) O/LZ genome were amplified and cloned into pMD18T-vector, constructing four recombinant plasmid pMD18–S,pMD18–I,pMD18-P1 and pMD18-P2. The sequence of cDNA was analyzed by TaKaRa Biotechnology(Dalian)Co.,Ltd.The above sequences are right ,then the IS was amplificated by fusing PCR with the pMD18–S and pMD18–I as template, and the IS specific primers was designed, which T7 promoter was introduced upstream of S, 6 C were introduced upstream of I , As same time, d the antisense primer of I include the only NruI of the cDNA of FMDV genome.The P3 was amplificated with the pMD18–P2 as template, and the 15 A was added to at the 3′- utmost sequece of P3 by renew designing primers of P3. The amplified fragments were cloned into the pMD18T-vector at first and then subcloned into pBluescript II SK(- ) to constructe the recombinant plasmid PCKS-F containing a full-length genomic cDNA of FMDV. The Cloned cDNA derived from the genome of FMDV O/LZ strain used as template was transcripted to produce RNA with T7 RiboMAXTM Express Large Scale RNA Production System. The RNA were transfected to BHK cells. The type cytopathic effect which were caused by rescusing FMDV was observed. Furthermore, the rescusing FMDV were verified by RT-PCR, IFA and observation of immunoelectron microscope. The results suggested that the FMDV were successfully rescued. This study have paved the way for vaccines against FMDV, pathogenic mechanism and so on.In this paper, we report the design and construction of a multiple-epitope foot and mouth disease virus (FMDV) antigen, designated OAAT. This recombinant antigen consists of the structural protein VP1 genes from serotypes A and O FMDV, 5 major VP1 immunodominant epitopes from two genotypes of Asia1 serotype, and 3 Th2 epitopes originating from the non-structural protein, 3ABC gene and structural protein VP4 gene.The OAAT gene cloned into pET-28a(+) to generate pET28a-OAAT. After the pET28a-OAAT was transferred BL21, the interest protein were expressed in the form of inclusion body whose protein level is 18% of total of bacterium protein. The protein was purified by cutting the part of polyacrylamide gel that contained interest protein after washing inclusion body. This study provides a significant step in the further development of new FMDV vaccines and detection text.Furthermore, to study immunogenic of OAAT and the effect of Staphylococcal enterotoxin A (SEA) or IL18 as a genetic adjuvant, three recombinant plasmids, p-OAA , pVAX1-OAAT, pVR-IL18-OAAT (briefly named as pOAAT-IL18) and p AXI-SEA-OAAT (briefly named as p -SEA), were constructed using/or not SEA or IL18 as a genetic adjuvant, and the animals experiments were performed. Expressions of target gene from these plasmids in HeLa cell were verified by western-blot and indirect immunoflurescent assay. BALB/c mice were immunized intramuscularly with these DNA vaccines three times every 2 weeks. We found that pVAX1-OAAT, pOAAT-IL18, and p–SEA could induce simultaneously specific CTL activity and the antibodies against serotypes A, Asia1, and O FMDV. Furthermore, the pSEA-OAAT could induce considerably higher antibodies titer than the pOAAT and pOAAT-IL18, but the pOAAT-IL18 enhancing the cell immunity was higher than the other. The results demonstrated OAAT may be a potential antigen that can be used to develop various multivalence recombinant vaccines against FMDV. Furthermore, SEA and IL18 may be an effective genetic adjuvant for DNA vaccine.The gene producing OAAT and encoding pig Interleukin-18 (IL-18) are inserted into the fowlpox virus (FPV) expression vector pUTA-16-LacZ to produce recombinant expression plasmid pUTAL-OAAT-IL18, and respectively regulated by ATI-P7.5×20 promoter and P7.5×16 promoter. The recombinant expression plasmids are then co-transected into CEF cells. The recombinant fowlpox viruses-OAAT-IL18 (rFPV-OAAT-IL18) is produced by three cycles with the BrdU and verified by RT-PCR and IFA. BALB/c mice were immunized with the recombinant virus by muscular inoculation, and the level of antibodies, the number of spleen T lymphocyte subgroups and cytotoxicity activity of specific CTL were examined after three times every week.The results showed that both of the recombinant fowlpox virus could increase the percentage of T cell subgroups and elicited cytotoxicity activity of specific CTL which were significantly higher than that of the control.The immunized mice could produce the the antibodies against serotypes A, Asia1, and O FMDV.The only genetically engineering vaccine was used, the results weren't good. So we study the prime-boost vaccine programme in FMDV as same time we study the gene adjuvant, which the immunity methods of prime-boost was performed in control HIV and verified to be latent good methods. In this paper, we evaluate the feasibility of prime-boost immunization with rFPV-OAAT-IL18 and pSEA-OAAT. The results showed that prime-boost immunization could enhance the level of humoral immunity and cell immunity, but the titer of antibody is similar inactivated vaccine.