Seek protein which can interact with hepatitis B virus X protein from human liver cDNA library by yeast two-hybrid system.
OBJECTIVE
To seek the X associated protein (XAP) with the constructed bait vector pAS2-1X from normal human liver cDNA library.


METHODS
The X region of the HBV gene was amplied by PCR and cloned into the eukaryotic expression vector pAS2-1. The reconstituted plasmid pAS2-1X was transformed into the yeast cells and the expression of X protein (pX) was confirmed by Western blot analysis. Yeast cells were cotransformed with pAS2-1X and the normal human liver cDNA library and were grown in selective SC/-trp-leu-his-ade medium, the second screen was performed with the LacZ report gene. Furthermore, segregation analysis and mating experiment were performed to eliminate the false positive and the true positive clones were selected for PCR and sequencing.


RESULTS
Reconstituted plasmid pAS2-1X including the anticipated fragment of X gene was proved by auto-sequencing assay. Western blot analysis showed that reconstituted plasmid pAS2-1X expressed BD:X fusion protein in yeast cells. Of 5 x 10(6) transformed colonies screened,65 grew in the selective SC/-trp-leu-his-ade medium, 5 scored positive for beta-gal activity, and only 2 remaining clones passed through the segregation analysis and mating experiment. Sequence analysis identified that two clones contained similar cDNA fragment:GAACTTGCG.


CONCLUSIONS
The short peptide(glutacid-leucine-alanine)is a possible required site for XAP binding to pX. Normal human liver cDNA library has difficulties in expressing the integrated XAP on yeast cells.
Wang
Xiao-Zhong
XZ
Department of Gastroenterology, Union Hospital of Fujian Medical University, Fuzhou 350001, Fujian Province, China. drwangxz@pub6.fz.fj.cn
Jiang
Xiang-Rong
XR
Chen
Xiao-Chun
XC
Chen
Zhi-Xing
ZX
Li
Dan
D
Lin
Jian-Yin
JY
Tao
Qi-Min
QM
D001483:Base Sequence; D015153:Blotting, Western; D015723:Gene Library; D005820:Genetic Testing; D006801:Humans; D008099:Liver / Q000737:chemistry / Q000502:physiology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D012441:Saccharomyces cerevisiae; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D054334:Viral Regulatory and Accessory Proteins
D010446:Peptide Fragments; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein