Cloning, Expression Of Multi-genes Of Foot-and-Mouth Disease Virus (FMDV) And Immunogenicity Of Genetic Engineering Vaccines Against FMDV

Foot-and-mouth disease (FMD) caused by FMD virus (FMDV) is oneof the most contagious animal virus diseases. In this study, by means ofmolecular biology, immunology, molecular virology, biochemistry andbioinformatics et al, multi-genes of FMDV Chinese isolate O/NY00 werecloned, sequence analyzed and expressed, and immunogenicity of 2recombinant Fowl-pox viruses (rFPV) and another 2 DNA vaccines againstFMDV was evaluated.FMDV is the prototype member of the Aphthovirus genus of the familyPicornaviridae, whose genome is about 8500 bases in length. From 5'terminal to 3'terminal, there are 5'UTR (including S-fragment, poly(C)tract, pseudoknot, cre and IRES), L^pro, structural proteins precursor P1region (encoding VP4, VP2, VP3, VP1), nonstructural proteins precursorP2 region (encoding 2A, 2B and 2C) and P3 region (encoding 3A, 3B,3Cand 3D), 3'UTR in line. And it was generally believed that completegenome information of virus would contribute to understanding themolecular characteristic of FMDV and R&D of new vaccines and diagnosistechniques.In experiment first, 3 genome fragments of FMDV Chinese isolateO/NY00 were cloned and sequenced by hemi-nested RT-PCR. Andsequence information of part 5'UTR (including pseudoknot, cre and IRESstructure regions), full length of L^pro gene (651nt), P2 (1416nt) and P3(2721nt) regions were acquired. With the sequence of P1-2A (2208nt)obtained in previous study, 7731nt sequences comprising approximately95% of the whole virus genome was obtained and banked into Genbank(Access number: AY333431).Using Clarke's method, 4 conservative pseudoknot structures werefound in pseudoknot region of O/NY00 genome, whereas only 3pseudoknots were identified in same region of O/TAW/97, which is theprototype member of Cathay topotype of FMDV serotype O. This differentmight be a new molecular mark to distinguish PanAsia strains from Cathaytopotype strains. Comparison of nucleotide and amino acid sequenceshomology revealed that IRES of O/NY00 had higher homology withO/SKR/2000 (prototype member of PanAsia strain) than O/TAW/97,O/AKESU/58 and O1/Kauf/66, which suggested that PanAsia strain hasunique molecular characteristic. Sequence analysis and homologycomparison also showed that in nonstructural protein encoding region,although Lpro and 3A gene had low homology (<90%), other genes werevery conservative (homology of amino acid sequence>95%), and variationof Lpro concentrated in 5'terminal of gene, whose influence on activity ofLpro was limited. Further more, no deletion was found in 3A gene. Many research demonstrated that detection of antibodies tononstructural proteins (NSP) 2C, 3A, 3B, and the polyprotein 3ABC ofFMDV, could differentiate infection from vaccination in FMD. Morerecently, 2 fragments in 3A (amino acid residues 70¹40) and 3B (aa 1⁶0)were identified, which could induce strong antibody response to poly-protein 3ABC. Therefore, In this experiment, a truncated 3ABC gene (afragment of 170aa encoded polyprotein from 70aa of N terminal of 3A to20aa of N terminal of 3C) –3ABCt was cloned by PCR and expressed inPichia pastoris. In experiment second, the adaptability of foreign proteins to Pichiapastori expression system was analyzed firstly. Results showed that 3ABCtsuited to Pichia pastori expression system. Therefore, recombinantexpression plasmid pPIC9K-3ABCt was constructed by inserting of FMDV3ABCt (525bp) into yeast expression vector pPIC-9k. Secondly, plasmidpPIC9K-3ABCt was lineared by BglII, and transformed into GS115 cellsby electroporation. Positive clones were selected with MD/MM plates andconfirmed by PCR and RT-PCR. Finally, expression product of 3ABCt wasanalyzed by SDS-PAGE and Western blot. The results showed thatexpression product could be secreted into media and existed in dimer form,which accounted for about 14% of the total supernatant protein. And theWestern blot results showed that expression product could be detected by3ABCt antiserum, which proved the good antigen specificity of expressedprotein. In previous studies, 4 genetic engineering vaccines, including arecombinant Fowl-pox virus (rFPV) vUTAL3CP1 co-expressing P1-2A and3Cpro gene of FMDV, another rFPV vUTA2-OAT and a DNA vaccineplasmid pVRI-OAT co-expressing chimeric Foot-and-Mouth Disease Virusmultiple epitopes gene and pig IL-18 gene, another DNA vaccine plasmidpVIRIL18P1 co-expressing P1-2A gene and pig IL-18 gene, wereconstructed successfully. The results of experimental immunity researchesin mice showed these recombinant vaccines could effectively inducespecific humor and cellular immunity against FMDV. On these bases, theimmunogenicity of these recombinant vaccines in guinea pigs, pigs andcattle were evaluated in experiment third. Firstly, vUTAL3CP1 and vUTA2-OAT were multiplied in CEF cellrespectively. Then, the inheritance stabilities and expression of foreigngenes of passage 3 and passage 5 viruses were examined and confirmed byPCR and RT-PCR. Secondly, FMDV VP1 gene was expressed in E coli. And theexpressed protein was purified by inclusion bodies washing and preparativeelectrophoresis. Results showed that purification ratio was about 91.4%,and production of target protein could reach 25 mg per litter bacteriaculture. Thirdly, C terminal (130²13aa) of FMDV VP1 was amplified, andcloned into the mammalian expression vector pDisplay. An expressionplasmid pDisplay-mVP1 was constructed and transfected into PK-15 cell...