Explore The Protein That Can Interact With Hepatitis B Virus PreS1 Protein By Yeast Two-Hybrid System

Objective: This study was designed to seek the hepatitis B virus preSl associated protein from normal human liver cDNA library by yeast two-Hybrid system and explore the role of preSl gene in the infection of HBV.Methods: PreSl gene including EcoRI and PstI endoenzyme sites was obtained by PCR. Double enzyme digestion was carried out for vector pAS2-l and PCR product of preSl gene. Both fragments were connected by ligase and transferred to Eco-JM105. Reconstitute plasmid sequence was tested by auto-sequencing assay and was named as pAS2-l-preSl. Reconstituted plasmid was transformated into the yeast cell AH 109 by Liac-mediated transformation. The preS 1-BD fusion protein expressed in the yeast cell was confirmed by Western blot. Yeast cells cotransformed with pAS2-l-preSl and the normal human liver cDNA library grew in selective SC/-trp-leu-his-ade media, the second screen was performed with the LacZ report gene. Furthermore, segregation analysis and mating experiment were done to eliminate the false positive and the true positive clones were submitted for PCR and sequencing. The results were submitted to the BLAST notebook of World Wide Wed site NCBI in order to seek homologous sequence.Results: Reconstituted plasmid pAS2-l-preSl included the anticipatedfragment of preSl gene was proved by auto-sequencing assay. Having assayed by Western blot, it was showed that reconstitute plasmid pAS2-l-preSl can correctly express preSl-BD fusion protein in the yeast cell. After yeast cells cotransformed with pAS2-l-preSl and the normal human liver cDNA library, 97 grew in the selective SC/-trp-leu-his-ade medium Of 5X106 colonies screened, 17 scored positive for 3 -gal activity, only one remaining clones past through the segregation analysis and mating experiment and showed high homology to Homo sapiens nascent-polypeptide-associated complex alpha polypeptide (NACA)0Conclusions: Eukaryotic vector pAS2-l-preSl was successfully reconstituted , the protein which can interact with preS 1 in vivo founded by yeast two-hybrid system is nascent-polypeptide-associated complex alpha polypeptide.