Expression of ORF2 protein of HEV genotype IV in Hansenula polymorpha.
- Author:
Cai-Xia SU
1
;
Mei-Rong GU
;
Ping ZHANG
;
Zhen-Ji JIN
;
Fan-Hong MENG
;
Er-Jia CHEN
;
Zhe YANG
;
Yong LIU
;
You-Chun WANG
Author Information
1. Department of R&D, Dalian Hissen Bio-Pharmaceutical INC., 116600, China. sucx@hotmail.com
- Publication Type:Journal Article
- MeSH:
Blotting, Western;
Electrophoresis, Polyacrylamide Gel;
Enzyme-Linked Immunosorbent Assay;
Gene Expression Regulation, Viral;
Genotype;
Hepatitis E;
immunology;
virology;
Hepatitis E virus;
genetics;
immunology;
metabolism;
Humans;
Pichia;
genetics;
Polymerase Chain Reaction;
Recombinant Proteins;
immunology;
metabolism;
Viral Hepatitis Vaccines;
immunology;
Viral Proteins;
genetics;
immunology;
metabolism
- From:
Chinese Journal of Biotechnology
2007;23(1):73-78
- CountryChina
- Language:Chinese
-
Abstract:
Hepatitis E, an acute infectious disease transmitted via the fecal-oral route, is caused by hepatitis E virus. However, no effective treatment currently exists for hepatitis E, and the only epidemic control approach is vaccination. But so for there are no commercial vaccine for hepatitis E available in the world. To find a new expression system to develop recombinant hepatitis E vaccine, in this study the expression system of methylotrophic yeast Hansenula polymorpha was used to express the gene encoding amino acid 112 - 607 of the open reading frame 2 (ORF2) of hepatitis E virus (HEV) genotype IV. In order to achieve high expression level, the coding sequence was optimized according to codon usage bias of Hansenula polymorpha and synthesized through overlapping PCR. Subsequently the gene was subcloned into the multi-copy expression vectors of Hansenula polymorpha, which include pDGXHP1.0 (MOX promotor), pDGXHP2.0 (MOX promotor) and pDGXHP2.1 ( FMD promotor). The series of one-copy and multi-copy recombinant plasmids were transformed into ATCC26012(Ura3-) by electroporation. The transformants were cultured in selection media MDL and screened for the existence of foreign gene by PCR. Then the strains were induced in MM media and the expression products were detected by SDS-PAGE, ELISA and Western blot assays to select the high-level expression strains. The result of SDS-PAGE showed that the HEV ORF2 expression product was accumulated up to 12% of total cellular protein and its molecular weight is 56kD. The expression product showed high immunoreactivity detected by ELISA and the highest titer is 1:2048. The result of Western blot demonstrated that the expression product could be specifically recognized by the polyclonal antibody against HEV. The successful expression of HEV ORF2 protein in Hansenula polymorpha provides foundation for the further development of recombinant subunit vaccine against hepatitis E.
