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  • Title: [Prokaryotic expression of hepatitis C virus envelope 1 gene and application of the expressed product].
    Author: Gao J, Tao Q, Ma D.
    Journal: Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi; 2001 Mar; 15(1):20-3. PubMed ID: 12526296.
    Abstract:
    OBJECTIVE: To express the HCV E1 gene in E. coli cells and to demonstrate its clinical significance in detection of anti-HCV E1 antibodies. METHODS: The expression vector was constructed by ligation of HCV E1 sequence, which was amplified by RT-PCR methods from 50 microliters of HCV RNA positive serum using primers specific to the HCV E1 sequence, to the prokaryotic expression vector PMS-31b transfected POP2136 at 16 degrees C for 16 hours. The recombinant plasmid was screened out and characterized by restriction enzyme analysis. The bacteria containing the recombinant plasmid was induced at 42 degrees C for 4 hours, and the recombinant protein was visualized by SDS-PAGE. The specificity of the recombinant protein was determined by Western blot assay. After purification of the expressed protein, this protein was coated on the plate with the concentration of 2 micrograms/ml in pH 9.6 buffer at 4 degrees C for overnight, and the serum specimen was tested at the dilution of 1:20 by ELISA. RESULTS: There were 2 fragments could be seen on the SDS-PAGE after digestion of the RT-PCR product with Sma I. And there emerged one fragment of 356 bp after digesting the recombinant plasmid with Sma I and Xba I. A band of 30,000 could be seen on the SDS-PAGE after the induction of bacteria containing the recombinant plasmid pMS-E1 at 42 degrees C for 4 hours. The Western blot assay showed that the expressed band could react with the anti-HCV positive serum. The ELISA result indicated that there were 28.9% (26/90) anti-HCV positive serum were anti-HCV E1 positive, but 3.9% (3/76) were positive in the anti-HCV negative serum. CONCLUSION: The HCV E1 sequence from HCV RNA positive serum has been expressed in E. coli. The expression rate is about 17% of the total protein of the bacteria. This protein possessed good specificity and may be used in the diagnosis of HCV infection.
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