Construction and expression of “bait plasmid” with hepatitis C virus E2 protein in yeast two hybrid system

Objective: Using hepatitis C virus (HCV) E2 gene without transmembrane domains to construct the bait vector, which can express E2 protein in yeast cell, and can be used in yeast two hybrid as “bait plasmid” to look for the gene from the cDNA library, which expresses the protein that can interact with the HCV E2 protein. Methods: PCR was performed to amplify the HCV E2 gene fragment E2 661 from the vector pKK223 E2 constructed by our institute. The production of the amplification was inserted into the “bait” plasmid pAS2 1 after the digestion with the restricted endonuclease of EcoR I and Pst I. After verified by restriction endonucleases, the plasmid was transformated into the yeast cell. PCR was used to verify whether the plasmid was transformed into yeast. The E2 protein expressed in the cell was confirmed by Western blot. Using colony lift filter assay to verify the constructed plasmid alone could not activate the reporter gene in the yeast cell. Results: Digested by two endonucleases, the recombined vectors pAS2 1 E2 661 produced anticipated fragment. PCR verified that there was E2 fragment in the yeast. Having assayed by Western blotting, it was showed that the yeast cell transformated with pAS2 1 E2 661 vector had positive signal which could not be seen in the control. Tested by the colony lift filter assay, the plasmid and negative plasmid pLAM5' 1 could not activate LacZ reporter gene in the yeast, while the cell transformed with positive control plasmid pCL1 the carrying whole GAL4 gene turned to blue, which indicated that the reporter gene was activated. Conclusion: Bait plasmid was constructed correctly and could express HCV E2 661 proteins in the yeast cell but could not activate transcription of LacZ reporter gene alone. The plasmid can be used in yeast two hybrid. (J Beijing Med Univ, 2000,32:113 116)