The Study About The Composition And Structure Of HEV And The Expression Of Its ORF2in Sf9Cells

Hepatitis E（HE）was caused by hepatitis E virus, which is transmitted withcontaminative water and food by fecally-orally. The clinical symptom of HE wassimilar to HA. China is the HE endemic area. The percentage of acute HE in acute viralhepatitis was10%and the fatality rate was1.4-5.3%. The data was shown that theprevention of HEV was important.The study has two parts, part one was the analysis and comparison of HEV fromdifferent source, then the compsition of virus was analyzed; part two was the expression,purification and characterization of HEV VLP produced in insect cells.Part One:The absence of enough HEV hampered detailed analysis of the structure andprotein composition of the hepatitis E virion. The study of HEV was mainly based ontwo approaches: the transfection of infectious HEV cDNA clones and the expression ofindividual viral proteins. The study of HEV structure protein (ORF2and ORF3protein)shown that ORF2can bind RNA and assemble into HEV; ORF3protein release HEVfrom infected cells. Emerson also speculated ORF3protein improve the virus infect ofcell to cell. The study about HEV structure shown that the virion from different sourcehave different structure. The virus from cell culture supernatant and serum have similarstructure, ORF2protein bind RNA and ORF3protein is responsible for virus egresscells on the surface, the virus steal lipid when they released into supernatent. The virusfrom feces have no ORF3or lipid on the surface because of the digestion of bile andenzyme of digestive tract. However, there were some questions about HEV still remainunknown,such as the form of ORF2protein in the virus was not confirmed, whetherORF3protein is on the surface of virus from cell culture supernatant, what is the effectof ORF3protein and lipid on binding of virus to cell.In this study, hepatitis E virus from a robust HEV cell culture system was produced.Then the virus from culture supernatant and from the feces of infected monkeys at thepeak of virus excretion were purified by ultra-centrifugation. The common feature ofthe two samples after ultracentrifugation was that the ORF2protein was separated fromthe HEV RNA. And the HEV infectivity was varied by HEV RNA content of fractionsafter ultracentrifugatin. This was mean that most ORF2protein was not formed the virusparticle and was in the non-VLP form. The density of RNA-rich particles from fecesand from the culture supernatant were different. After treatment of the samples with NP40to remove lipid, the density of the particles from the cell culture supernatant wassame to the density of those from feces. This suggests that the virus in the cell culturesupernatant was enveloped with lipid and banded at a lower density.3%of virions from the cell culture were captured by the beads bound withanti-ORF3protein MAb and this increased to20%after treatment with NP40. This thendecreased to1.2%after treatment with NP40,2-ME and pronase E. This suggested thatthe ORF3protein on the virus surface was covered in lipid and there was little ORF3protein on the lipid surface. However, the percentage of RNA captured from the virionsfrom the feces was0.3%, with and without treatment with NP40. This was indicatesthere was very little ORF3protein on the surface of the virions from feces.The virions, with or without these treatments were incubated with cells to analyzethe influence of lipid and ORF3protein on binding to the cells. The virions from fecesand the virions without ORF3protein show the strongest binding, the binding capacityof the virions before and after delipidation was similar but was lower than the virionsfrom feces and those without ORF3protein. This implies that the lipid on the surfacehas no influence on the binding of the virions to cells but the ORF3protein interfereswith binding.We found that the ORF2protein from cell culture system was glycosylated, with anapparent molecular weight of88kDa, and was not infectious in PLC/PRF/5cells. TheORF2protein in this part can bind to and protect HEV RNA from digestion by RNase A.The RNA-ORF2product has a similar sedimentation coefficient to the virus from feces.The result suggested that the ORF2protein captured from cellculture supernatant maybe the capsid protein of native HEV.Part two:The native HEV was spherical particles without envelope observed using EM. Theviral size was27-34nm. The size of HEV VLP was27nm and the particle was alsospherical. This was mean that the VLP is similar to native HEV in morphology. Thegranular structure makes VLP have more immunogenic in immune to body. The VLP isbetter than non-VLP in immunogenic effective as HEV vaccine cadidate.The study constructed the126-621aa of4genotype HEV ORF2in ORF2-rAcNPV.Then the HEV VLP was expressed in infected sf9cells. The condition of expresstionand purification of VLP was optimized, the VLP produced using the scheme wasevaluated as vaccine candidate. The infection of express parameters, MOI, VLP harvest time and cell concentration,was investigated after the insect cells was infected with ORF2-rAcNPV. We found HEVVLPs production in the cells reached the maximum at120hpi with MOI=5and2.0×106cells/ml. The maximum yield of VLP was up to0.072mg/ml.Two forms, VLP and non-VLP, were found in the sf9cells when the ORF2126-621aa was expressed in the cells. The non-VLP will reduce the productionimmunity on account of its non granularity. So the production of non-VLP should bedecreased durig the expression optimization and be removed during the VLPpurifacation because it will reduce the production immunity.In order to seperate and purify HEV VLP from the non-VLP and cell impurities,the study use the ultrasonic method to lysate sf9cells and then the centrifugation wasused to remove the cell debris. The ultracentrifugation was used to remove the non-VLPfrom the production of cell lysate. The sample was dialyzed to loading buffer of ANXSepherose FF to remove the high concentration sucrose after the ultracentrifugation andthe VLP was also fixation during the dialyze process. The dialyzed sample was purifiedusing ANX Sepherose FF and the VLP was separated from other impurities. The resultwas shown that the VLP purity was increased from40.4%to97.7%using SDS-PAGEand the purity was100%using SEC-HPLC after the ANX Sepherose FF.Then we analyzed and evaluated the VLP physicochemical property, purificationand immuno response in mice. The result showed that the HEV VLP acquired fromscheme has high purity and strong immuno response. The result suggested that weestablished a simple and efficient production of HEV VLP expression and purificationscheme. This part study produced the preliminary research for further industrializedproduction of HEV vaccine.In summary, the structure and composition of HEV from different source wasanalyzed.The effect on virus binding to cells of the composition was detected using cellbinding assay. On the other hand, the capsid protein was expressed using insect cell.Then the VLP was pruducted and purifired as the HEV vaccine candidate.The study first analysis the ORF3content on the surface of HEV from cell culturesupernatant, ORF3protein obstruct the binding effect of virus to cell but lipid have noinflunce on binding effect, the result of study also first suggested that the glycosylateORF2protein may be the capsid protein in HEV particle. The study also optimized the scheme of HEV VLP expression and purification, and then evaluated the VLP producedusing the scheme.