Construction and identification of a yeast two-hybrid bait vector for screening of Homo sapiens CD4-binding proteins
To construct and identify a yeast two-hybrid system bait vector for screening of Homo sapiens CD4-binding proteins. Methods CD4 cDNA has been amplified by reverse transcription-PCR(RT-PCR), cloned into pAS2-1 vector and then transformed into Saccharamyces oerevisiae strain Y190. To find β-galactosidase, the self-activating effect was tested by filter assay. The known ligand of CD4 encoded by HIV, gp120 was cloned into the prey vector pACT2 and then transformed into Y190/pAS2-l-CD4. Its β-galactosidase activity of the recombinant cells were assayed. Results CD4 cDNA has been cloned and used to construct recombinant bait vector hpAS2-1-CD4. The pAS2-1-CD4 transformed into Y190 didn't show its self-activation effect. The Y190 transfonned with pAS2-1-CD4 and pACT2-gp120 showed β-galactosidase activity. Expressed proteins of these two vectors can interact each other. Con-clusiom The yeast two-hybrid bait vector offers an applicable platform for screening the proteins, especially the viral ligands that combined with CD4.