Expression and characterization of envelope protein 2 gene of hepatitis G virus in Pichia pastoris.
- Author:
Zhuo-Hua WANG
1
;
Kai YE
;
Hong XU
;
Hui-Wen MA
;
Li-Heng TONG
;
Xi-Liang PENG
Author Information
1. College of Life Sciences, Wuhan University, Wuhan 430072, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Antigens, Viral;
genetics;
immunology;
isolation & purification;
GB virus C;
genetics;
immunology;
Gene Expression;
Genetic Engineering;
Glutathione Transferase;
genetics;
Hepatitis Antibodies;
blood;
immunology;
Humans;
Pichia;
Recombinant Fusion Proteins;
genetics;
immunology;
isolation & purification;
Schistosoma japonicum;
enzymology;
Viral Envelope Proteins;
genetics;
immunology;
isolation & purification
- From:
Chinese Journal of Biotechnology
2002;18(2):187-192
- CountryChina
- Language:Chinese
-
Abstract:
A cDNA fragment locating at the putative envelop protein 2(E2) region of GBV-C/HGV fused with Schistosoma japonicum, glutathione S-transferase(GST) was amplified with PCR from plasmid pGEX-E2. The amplified DNA fragment was inserted into plasmid pGEX-5X-1, at the downstream of the coding sequences of GST, in the same reading frame with the gene of GST. The fusion gene fragment of GST-E2 was amplified with PCR, using the recombinant plasmid pGEX-5X-1-E2 as the template. The amplified 1324 bp DNA fragment of GST-E2 was inserted into Pichia pastoris expression vector pPIC9K in reading frame with alpha-factor secreting signal peptide. The plasmid pPIC9K-GST-E2 was transformed into Pichia pastoris GS115 with electroporation. The transformants (His+ Muts) were selected and induced to express the 54kD GST-E2 fusion protein, which could be specially recognized by both the antisera directed against E2 and against GST. The GST-E2 fusion protein was purified with Sepharose 4B glutathione affinity chromatography to a purity of 95%. The expression was optimized to achieve the highest expression level of GST-E2 fusion protein which was accumulated up to 50% of total proteins in the culture supernatant. The GST-E2 protein derived from the recombinant Pichia pastoris was proved possessing antigenicity and high specificity by ELISA, probed with sera from the patients infected by GBV-C/HGV.
