Expression And Characterization Of Capsid Protein Of Genetype ? Hepatitis E Virus

Hepatitis E virus(HEV)is responsible for large scale epidemics and rampant sporadic cases of acute viral hepatitis in various regions of the world.Hepatitis E(HE)is widely prevalant in China,genetype ? HEV is a zoonotic and can infect both human and domestic animal.Development on the difference of infection mechanism between genetype ? and genetype ? HEV is becoming the focus of the research of scholars from various countries.Two runcated genetype ? HEV genes of Open Reading Frames 2(ORF2)were amplified by PCR,and subcloned to E.coli expression vector.The recombinant plasmids were identified by restriction enzyme analysis,and then transformed into E.coli ER2566.The recombinant strains were induced with IPTG.SDS-PAGE result showed that the two proteins were expressed in the form of inclusion body,and the relat?e molecular masses were about 24 kDa and 27 kDa,respect?ely,the two proteins were named p24 and p27.SDS-PAGE result indicated that the inclusion body,which purified by washing,could mainly dissolved in 4 mol/L urea solution.Result of western Blot displayed that both proteins could strongly react with the HEV monoclonal antibody 8C11 and HE posit?e serum,and the two protein exited as dimer and monomer.The purity of recombinant protein was over 99%after ion exchange chromatography and gel chromatography.The recovery ratio of p24 protein was about 26%,while that of p27 was about 31%.Result of indirect ELISA showed that the detection rate of them were the same as anti-HEV-IgG diagnostic kit(Wan Tai corporation)when the p24 and p27 were used as coating antigen to detect the HE positive serum and negative serum,respectively.Native-PAGE and Western Blot showed that most p27 were existed between Stacking gel and Separating gel,which implyed that p27 can form a high molecular weight polymore.Dynamic light scatter detected the average hydration radius of p24 and p27 were 7 nm and 22 nm,respectively.p27 existed as 20nm diameter virus like particles(VLP)when observed with transmission electronic microscope and atomic force microscope,while p24 canot form the particle.Result of indirect ELISA demonstrated that the median effective dose(ED50)of p27 was 0.59?g when p27 absorbed to Freund's complete adjuvant was used to immunize Babl/c mice with different dose.These result implyed that p27 maybe a favourable immunogenicity.p27 protein,expressed from Ecoli,could mimic the structure of genetype? HEV capsid protein,which provides a basis for the further development of the difference of infection mechanism between genetype ? and genetype I HEV.