Human Enterovirus71Capsid Protein Gene Synthesis And The Prokaryotic Expression Analysis

Human enterovirus71is a member of the enterovirus a species within the genusEnterovirus of the family Picornavirus. Infection of EV71can cause hand, foot andmouth disease, herpes herpangina, aseptic meningitis, encephalitis and polio-likeparalytic disease and other nervous system diseases.Historically, the outbreaks ofHFMD (hand, foot and mouth disease) in European countries and the United States havebeen spontaneous and small in scale. Noticeably, there were also two large outbreakswith high mortality rates in Bulgaria (1975) and Hungary (1978). In recent yearsnumerous large outbreaks of HFMD have occurred in eastern and southeastern Asiancountries and regions, including Malaysia, Singapore, Taiwan, Japan, South Korea,Vietnam and mainland China.HFMD has become an emerging disease in China since March2008. Furthermore,rapid mutation rates result in the emergence of new subgenotypes every few years. Todate,11EV71subgenogroups have been identified based on comparison of their VP1sequence: A, B1-B5, C1-C5. The Asian pandemics have been associated withco-circulation of different genetic lineages and the emergence of novel strains.TheEV71virus particle consists of a naked icosahedral capsid composed of the fourstructural proteins VP1-4surrounding a single-stranded positive-strand RNA of about7.4kb. The viral RNA contains a single open reading frame coding for a polyproteinwhich is autocatalytically cleaved after translation. P1, P2, and P3are three distinctregions on the polyprotein that encode the structural proteins (P1) and the sevenaccessory proteins2A-C and3A-D (P2&P3).In recent years, several EV71vaccine candidates, including live-attenuated virus,inactivated whole virus, recombinant viral protein, virus-like particle and DNA vaccine,have been evaluated in animal studies. The vaccine studies in animal models havedemonstrated that neutralizing antibodies may play a critical role in protecting micefrom the viral challenge. EV71vaccine clinical trials have been approved recently and will be soon carried out in China. Other than the target populations, it is difficult topredict what antibody titer will be considered as a protective level in the clinical trials.the lack of approved antigens, antibodies (ELISA or chemiluminescence) reagents,resulting in the evaluation of vaccine activity and antibody titer inaccuracy andincomparability.we have constructed three prokaryotic expression plasmids which wereinduced to express in E.coli to produce recombinant mutants. and the immunogenicityof vP1protein was preliminary evaluated, which is helpful for the research of VP1subunit vaccine and diagnostic kits. The whole study consists of three parts as follows:Part I EV71-VP1，EV71-VP0，EV71-VP3gene fragments in vitro synthesized ofHuman enterovirus71Amino acid sequence for the following protein was obtained from Gene Bank(EV71/Fuyang.Anhui.CHN/19.08/6strain). All primers designed for SequentialOE-PCR about the seven genes were based on codon optimized gene sequence. Thesynthesis of full-length fragment TA cloning to pMD18T vector, after sequencingdetermination and final revision of the synthesis, the synthesis of the gen wassuccessfully.Part II The Expression of the three prokaryotic expression plasmidsDNA fragments of EV71-VP1，EV71-VP0，EV71-VP3were obtained through PCRand were cloned onto vector pMD18-T. After being sequenced, the correct ones werecloned onto expression vector pET-32a to construct prokaryotic expression plasmids:pET-32a-VP1，pET-32a-VP0，pET-32a-VP3。Then all the plasmids were transformedinto E.coli BL21(DE3) and induced to express by IPTG. pET-32a-VP1，pET-32a-VP0athigh expression levels except pET-32a-VP3. And use pET-32a-VP1Immune NewZealand rabbits and BABL/C mice and get the antiserum, the immunogenicity of VP1protein was preliminary evaluated by ELISA. m1．ELISA testify the anti-sera ofpET-32a-VP1protein from rabbtis and the serum of patient with HFMD could react specifically with pET-32a-VP1，pET-32a-VP0，and the titres were1：128000，1：64000respectively and its reaction with PET32a protein was weak.Part III Gene synthesis，cloning，prokaryotic expression and analysis of chikungunyavirus E2glycoprotein and capsid COn-line software ExPasy was used to predict transmembrane domain of E2proteinand optimized gene sequence encoding E2protein was obtained according to aminoacids of the protein from GenBank; Primers were designed to synthesize E2geneaccording to the principle of OE-PCR and prokaryotic expression plasmids offull-length E2protein and its mutant were constructed. After being transfromed intoE.coli BL21(DE3), two plasmids in E.coli were induced to express by IPTG and proteinexpression was identified by SDS-PAGE. In the study, the gene encodingCHIKV-E2(1-404) protein was synthesized by OE-PCR and two prokaryotic expressionplasmids, pET21b-E2(1-404) and pET21b-E2(1-350), were constructed successfully;The plasmids were induced by IPTG and SDS-PAGE result showed that the productionof the deletion mutant E2(1-350) was much higher than that of pET21b-E2(1-404). Thehydrophobic transmembrane domain(351-378aa) of E2protein plays an important rolein E2production in E.coli and production of the mutant with the domain deleted issignificantly higher than that of full-length E2protein.