Identification And Genotyping Of Enterovirus 71 And Prokaryotic Expression And Purification Of Human Cytomegalovirus Envelope Protein UL115

1.Identification and genotyping of enterovirus 71 infection and associated outbreak of Hand,Foot,and Mouth Disease in Shawo of China in 2012Infection of enterovirus 71(EV71)and associated hand,foot,and mouth disease(HFMD)are recognized as emerging public health issues worldwide.Hundreds of thousands of children are annually infected with EV71 and develop HFMD in China alone.Studies the virological characteristics of EV71 are critical to prevent and control the outbreaks of HFMD.In this report,we studied an outbreak of 105 HFMD cases in Shawo Township of China between September to October 2012.By using reverse transcription PCR technology,real-time quantitative reverse transcription PCR technology,virus isolation and culture technology to detect the existing of enterovirus,then sequenced and analyzed the geng of enterovirus 71’s VP1 gene.At last,we constructed the phylogenetic tree for enterovirus EV71 to confirm the genotyping of enterovirus 71.More than 90% of cases were children younger than 9 years old,with over 50%of cases aged 3–6 years old.Laboratory studies detected a high prevalence of EV71 and suggested EV71 as the most common enterovirus causing HFMD in Shawo.Of66 enterovirus-positive samples,48 were Enterovirus 71 positive,while 16 were Coxsachie A 16 viruspositive.Sequencing analysis showed that the EV71 strains from Shawo belong to the C4 subgenotype,and are phylogenetically more related to those from the distant city of Nanchang than those from the nearby city of Wuhan with distinct variations.More girls were found to be associated with EV71 in Shawo whereas more boys were associated with EV71 in Wuhan and Nanchang.Our studies further the understanding of the molecular epidemiological features of HFMD and infection by enteroviruses in China,and it has a great significance to prevent and control the outbreak of enterovirus.2.Prokaryotic expression and purification of human cytomegalovirus envelope protein UL115Human cytomegalovirus(HCMV)is a member of the herpesvirus family which infects most populations worldwide.For persons with immunocompromised or immune suppression may cause morbidity and even mortality after infected HCMV.HCMV’s envelope protein is very complicated and comprises more than 20 envelope proteins,they are located in the outermost layer of the virus,which related with virus tropism,adsorption,fusion and progeny virus packaging,maturation,and transmission.Understanding the envelope proteins’ structure,biological functions of HCMV,which can provide a new insights into the biological mechanism of the virus,and deeply understand the relationship between virus and host,provide a new ideas and guidance to design and develop an antiviral drugs.Besides,it has an important clinical significance and researching foundation to develop a virus vaccine.UL115(g L)protein is a member of the envelope protein,UL115 envelope protein as an antigen witch has a good immunogenicity.As a result,it can be as a materials to developing a HCMV antigen monoclonal antibody and designing a rapid diagnostic kit.Therefore,studying the biological function of HCMV-UL115 has a great value to elucidate the pathogenic mechanism of HCMV and develop virus vaccine.In this study,the UL115 gene was cloned by conventional PCR technology,the recombinant cloning strain and recombinant expression strain of UL115 gene was constructed.The recombinant protein of UL115 was expressed successfully.After the experimental identification,the recombinant protein has expressed in a form of inclusion bodies in prokaryotic cells.Upon the study,the expression conditions of the expressed strain has been tentatively optimized.The better conditions for expressing the recombinant protein in this experiment are as follows: 31℃,0.03 m M/L IPTG,inducting 9 hours.The recombinant protein was obtained after purification by nickel column.Then used the BCA method to quantify the preliminarily purified recombinant protein,the recombinant protein’s concentration can reach 0.105 μg/μl.In this study,it has successfully constructed the prokaryotic expression system of UL115 recombinant protein.Besides,the expression conditions were optimized.This study lays a foundation of researching monoclonal antibodies biological function of UL115,and has an import meaning to studying HCMV diagnosis kit and vaccine,what’s more,it can provides relevant experimental experience for prokaryotic expression of HCMV’s other envelope protein.