The Characterization Of The VP1 Gene And The 2A Gene, Protein Of Enterovirus Type 71

Enteorviurs71(EV71) is the most described serotype of the genus Enteroviurs(family Picornaviridae).It is a common cause of hand,foot,and mouth disease, herpangina, aseptic meningitis, encephalitis, poliomyelitis-like paralysis, neurogenic pulmonary edema (NPE) and a lot of epidemics correlated with nerve system. In recent years it appeared serious infections of central nervous system in the Asia-pacific region, even death. So the pathogenicity, pathogenesis, diagnosis and vaccine research of EV71 were valued.The National Institute Institutes of Health decided to put HFMD into Class C infectious diseases on the Law of the People's Republic of China on the Prevention and Treatment of Communicable Diseases to manage on May 2,2008.why can EV71 lead to a few severe or death cases, what are toxicity determining factors.The gene for VP1 of EV71 is the major antigenic determinant,VP1 gene sequences can not only be basis of classification of different serotype in Enterovirus,but also can be used as Classification reference in RNA Circoviridae.Only 2A produces toxic to Saccharomyces in the 11 kinds of virus protein of EV71 from the previous studies.We know that 2A is a protein hydrolysis enzyme,in normal,it can inhibit the growth of host after intestinal virus infection, one of whose mechanism is resolving eIF4G of the host to inhibit Protein Synthesis.Some thesis pointed out that 2A protein of poliomyelitis virus could restrain the growth of Saccharomyces cerevisiae.perhaps 2A cut some protein transcription factors which were used for transcription.Transient expression of 2A protease could cause apoptosis, eukaryon initiation factor 4GI could be cracked after infection of EV71 2A,and 4GI was the key factor of host protein synthesis.ObjectiveThe research intended to study if there were differences in the nucleotide sequences of VP1 genes,2A gene and the predictable structure of 2A protein between mild cases and severe cases of EV71.Methods RD cells were cultivated for viruses isolation,used specificity primers by retrovirus polymerase chain reaction (RT-PCR) for gene amplification with the purpose of determination of EV71. Specific primers were used for amplification of VP1 and 2A gene separately.After VP1,2A gene sequencing of EV71,the homology comparsion and the phylogenetic tree construction were completed through the software of DNAMAN.We made simulated protein structure using amino acid sequence by homology modeling methods and chimera software and discussed the differences of secondary,tertiary structure of the protein.At last we cloned 2A gene and constructed recombinant plasmid pIRES2-EGFP-2A.Results1. Culture of RD cells and Viruse isolation:RD cells were successfully cultivated free of contamination.Cell pathological changes (CPE) caused by enterovirus shows cells shrinkage, endochylema granulation, the cell nucleus pyknosis and finally cell rupture.2. Differential diagnosis of EV71 by RT-PCR:The RT-PCR results indicated that 30 isolates were EV71,13 of 30 isolates were from clinical specimens of patients with light symptoms of hand-foot and mouth,the other were from clinical specimens of patients with heavy symptoms of complications.3. VP1,2A gene amplification:We successfully obtained the amplified products of VP1 and 2A gene from 5 light strains and 5 heavy strains with specific primer pairs,which were 1082 and 539 base pairs respectively.4. VP1,2A gene sequencing of EV71,the homology comparsion and the phylogenetic tree construction:10 correct PCR products were sequenced and compared with that of previously isolated 5 EV71 Chinese isolates available from GenBank (fuyangEU703814.1, xi anHM003207.1,shandongEU753418.1,shenzhenFJ607337.1, henanGU366191.1) by homogeneity and phylogenetic tree analyses.The homogeneity of VP1 and 2A genes of the 10 EV71 isolated strains and 5 previously isolated strains were between 94.7%～99.4% and 93.6%-99.3% respectively, with the representative isolates of A and B genotypes was between 81.0%～84.6% and 78.4%～82.2% respectively.The data suggested that all of the 10 Chinese isolates belong to EV71 genotype C.There were only 87.8%～90.2% homology among these 10 strains and the representative strains of C1,C2,C3 sub-genotypes of EV71 but 96.8%～99.6% homology among these 10 strains and the representative strains of C4 sub-genotypes of EV71,this suggested that these 10 Chinese isolates composed the C4 sub-genotype,of the C genotype, that formed a single branch in the phylogenetic tree.5. Simulated protein structure graph of 2A:We made simulated protein structure of 2A using amino acid sequence by chimera software and found that there were three different domains of 2A protein between number 20 and 57,the other four pairs of light and heavy strains also had differences but no consistency structure features.6. The clone of 2A:We successfully acquired 2A clons and confirmed them by PCR and sequencing.The sequence results certified we had gotten these clones successfully.7. Construction of recombinant plasmid pIRES2-EGFP-2A:We chose one of light and heavy cases respectively,inserted the 2A gene to pIRES2-EGFP.We sucessfully constructed recombinant plasmid pIRES2-EGFP-2A and identified by PCR, double-enzyme cleavage,agarose gel electrophoresis and sequencing methods.Conclusions1. All of the isolates belonged to EV71 genotype C and had close genetic relationship with isolated strains of other provinces in Mainland China.2. VP1,2A gene had close genetic relationship in 10 strains respectively, there was no difference between clinical specimens of patients with light or heavy symptoms.3. There were differences in secondary, tertiary structure of the 2A protein from light and heavy isolates but I did't find any consistency protein structure features.