[Screening the hepatitis B virus PreS1 associated protein by the yeast two-hybrid system].
OBJECTIVE
To screen the hepatitis B virus PreS1 associated protein from normal human liver cDNA library by the yeast two-hybrid system and explore the role of PreS1 protein in the infection of hepatitis B virus (HBV).


METHODS
PCR was preformed to amplify the PreS1 gene containing EcoRI and PstI from HBV positive serum, and the production was inserted into plasmid pAS2-1 after digesting with the former two restricted endonuclease, then the bait vector pAS2-1-PreS1 was verified by auto-sequencing assay. The PreS1-BD fusion protein expressed in the yeast cells was confirmed by western blot, after pAS2-1-PreS1 was transfected into the yeast cell AH109. Yeast cells co-transfected with pAS2-1-PreS1 and the normal human liver cDNA library grew in selective SC/-trp-leu-his-ade2 medium, and the second screening was performed with LacZ report gene. Furthermore, segregation analysis and mating experiment were done to eliminate the false positive, then the true positive clones were submitted for PCR and sequencing. The results were submitted to the BLAST notebook of World Wide Wed Site NCBI to seek homologous sequence.


RESULTS
Bait vector pAS2-1-PreS1 included the anticipated fragment of PreS1 gene. Western blot showed that pAS2-1-PreS1 could correctly express PreS1-BD fusion protein in the yeast cells. After yeast cells co-transfected with pAS2-1-PreS1 and the normal human liver cDNA library, 97 colonies grew in the selective SC/-trp-leu-his-ade2 medium, only one clone was positive and showed high homology with Homo sapiens nascent-polypeptide-associated complex alpha polypeptide.


CONCLUSIONS
Bait vector pAS2-1-PreS1 is successfully constructed, and nascent-polypeptide-associated complex alpha polypeptide protein expressed in hepatocyte can interact with PreS1 by the yeast two-hybrid system.
Li
Dan
D
Department of Gastroenterology, Union Hospital Affiliated to Fujian Medical University, Fuzhou 350001, China.
Wang
Xiao-zhong
XZ
Chen
Zhi-xin
ZX
Huang
Yue-hong
YH
D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D000074401:Peroxins; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism; D015003:Yeasts / Q000235:genetics*
D018076:DNA, Complementary; D006514:Hepatitis B Surface Antigens; C076587:PEX4 protein, S cerevisiae; D000074401:Peroxins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014759:Viral Envelope Proteins; C050806:presurface protein 1, hepatitis B surface antigen