[Expression and characterization of envelope protein 2 gene of hepatitis G virus in Pichia pastoris].
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.
Wang
Zhuo-Hua
ZH
College of Life Sciences, Wuhan University, Wuhan 430072, China.
Ye
Kai
K
Xu
Hong
H
Ma
Hui-Wen
HW
Tong
Li-Heng
LH
Peng
Xi-Liang
XL
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D029407:GB virus C / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression; D005818:Genetic Engineering; D005982:Glutathione Transferase / Q000235:genetics; D006508:Hepatitis Antibodies / Q000097:blood / Q000276:immunology; D006801:Humans; D010843:Pichia; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012549:Schistosoma japonicum / Q000201:enzymology; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D000956:Antigens, Viral; D006508:Hepatitis Antibodies; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C107619:glycoprotein E2, GB virus C; D005982:Glutathione Transferase