Screening the hepatitis B virus PreS1 associated proteins in yeast two-hybrid system

Objective To screen the interacted protein hepatitis B virus (HBV) PreS1 with human hepatocytes from normal human liver cDNA library by sos-recruitment system (SRS) and explore the mechanism of HBV endocytosis. Methods PCR was performed to amplify the gene of HBV PreS1 from the plasmid PCP10/HBV ayw subtype containing the whole fragment of HBV; the PCR product was cloned into yeast expression plasmid pSos, and reconstituted plasmid was tested by auto-sequencing assay and named pSos- PreS1. The pSos-PreS1 fusion protein expressed in the yeast cells was confirmed by Western blot after pSos- PreS1 was transfected into the yeast cell cdc25.Self-activation of the bait protein was determined by cotransformation of pSos- PreS1 and pMyr-Lamin C, and the cytoplasmic localization of the bait protein was verified by cotransformation of pSos- PreS1 and pMyr SB. Yeast cells co-transfected with pSos- PreS1 and the normal human liver cDNA library grew in selective nutrition and temperature. The true positive clones were submitted for sequencing. The results were submitted to the BLAST notebook of NCBI to seek homologous sequence. Results The yeast expression vector of HBV PreS1 gene was constructed successfully and its expression in yeast was verified. The recombinant bait plasmid did not have self-activation and toxicity to yeast cdc25H cells. Furthermore, the cytoplasmic localization of the bait protein was verified correctly. After yeast cells were co-transfected with pSos- PreS1 and the normal human liver cDNA library, 5 clones were positive and showed high homology with KCMF1, cyt C, vitamin D binding protein, Homo sapiens albumin (ALB) and Homo sapiens asialoglycoprotein receptor (ASGPR). Conclusion We obtained 5 proteins which may interact with HBV PreS1 protein by SRS. This is helpful for exploring the mechanism of HBV endocytosis.