Prokaryotic Expression And Generation Of Monoclonal Antibodies Of ORF2 Gene Of Swine Hepatitis E Virus

Hepatitis E is an acute,self-limiting viral disease which caused by Hepatitis E virus(HEV).Hepatitis E presents a major outbreak in parts of Asia and northern Africa, It is also responsible for the sporadic acute hepatitis in developed countries.The virus is transmitted primarily via the fecal-oral way,epidemics of hepatitis E are large associated with fecal contamination of drinking water.HEV may be particularly severe among in young adults in these regions, it is accounts for the mortality rates reaching as high as 20% in pregnant women.Antibody(anti-HEV) against HEV is widely found in the body of non-human primates,rodents,domestic animals such as swine and chicken.they all appear to be reservoirs of the virus.lt further proved this HEV disease might be zoonotic.Hepatitis E virus (HEV) is a single-stranded positive-sense RNA virus that is not enveloped.it is now the sole member of the Hepaviridae family.The viral RNA is approximately 7.2 kb in size and contains three partially overlapping open reading frames (ORFs). ORF1 encodesa non-structural protein while ORF2 encodes the putative capsid polypeptide and ORF3 encodes a small immunogenic phosphoprotein that associates with the cytoskeleton.The comprehensive studies have been improved the ORF2-encoded protein contains strong antigenic region located at the C terminus.In approximately two thirth C terminus region of the ORF2 capsid protein,HEV-CP was riched in B-cell antigenic epitopes.Each ORF2 protein contains 3 linear domains, S(118～313aa), P1(314～453aa), and P2 (454-end). This research segmentation express the four linear domains of pig source HEV ORF2 gene to get four fusion protein, The comprehensive studies have been conducted to further elaborate the self-assembly function of S structure domain protein, confirming the function of PI structure domain in the maintaining capsid proteins stability, and confirming the existence of the mechanism of strong antigenic region located at P12 protein.It is help to screen potential receptor binding sites of P2 structure domain,which is not only useful for investigating the functional and structure relationship of different structure domains but also useful for diagnosis of HEV infection and developing safe and effective ELISA diagnosis kit against the disease.ORF2 gene of Hepatitis E virus was obtained by RT-PCR, The ORF2 capsid protein, HEV-CP was encoded by HEV ORF2 containing a total of 674 amino acids.Three pairs of specific primers were designed according to the distribution of linear domains of HEV-CP and molecular biological characteristics results predicted biological software to amplify the four fragments:S,P1,P2 and P12.The four DNAs was subcloned to pET-32a(+) to construct pET-S,pET-P1,pET-P2and pET-P12 fusion expression vectors.The four positive recombinant fusion expression vectors were transformed into appropriate express bacterium Rosetta(DE3) E.coli or BL21(DE3) and expressed by different induction of IPTG. SDS-PAGE was performed to analyze the four recombinant fusion proteins, Results showed that the protein was highly expressed in E.coli, and the molecular weights of His-S, His-P1, His-P2 and His-P12 fusion proteins were 41 kDa,35 kDa,45 kDa and 58 kDa.Fusion protein mainly exists in the form of inclusion body Immunoreactivity between each four expressed proteins was detected by Western blot. Results proved that each protein was successfully purified and better immunoreactivity was detected between each protein and their immune sera. The expressed fusion proteins His-P12 were purified and utilized to immunize experimental animals to produce monoclonal antibodies (mAbs).Five hybridoma cell lines designated steadily secreting monoclonal antibody (McAb) against His-P12 protein of swine Hepatitis E virus were screened out by indirect ELISA and Western Blotting.This experiment provide fundamental research for screen the potential receptor binding and Indirect ELISA.