Screening and identification of interacting proteins with hepatitis B virus core protein in leukocytes and cloning of new gene C1.
OBJECTIVE
To investigate the biological function of HBcAg in pathogenesis of HBV replication in peripheral blood mononuclear cells (PBMCs).


METHODS
HBcAg region was amplified by polymerase chain reaction (PCR) and HBV HBcAg bait plasmid pGBKT7-HBcAg was constructed by routine molecular biological methods. Then the recombinant plasmid DNA was transformed into yeast AH109. After the HBV core protein was expressed in AH109 yeast strains (Western blot analysis), yeast-two hybrid screening was performed by mating AH109 with Y187 containing leukocyte cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) (QDO) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) (TDO). The second screening was performed with the LacZ report gene ( yeast cells were grown in QDO medium containing X-alpha-gal). The interaction between HBV core protein and the protein obtained from positive colonies was further confirmed by repeating yeast-two hybrid. After plasmid DNA was extracted from blue colonies and sequenced, the results were analyzed by bioinformatic methods.


RESULTS
Eighteen colonies were obtained and sequenced, including hypermethylated in cancer 2 (3 colones), eukaryotic translation elongation factor 2 (2 colones), acetyl-coenzyme A synthetase 3 (1 colone), DNA polymerase gamma (1 colone), putative translation initiation factor (1 colone), chemokine (C-C motif) receptor 5 (1 colone), mitochondrial ribosomal protein L41 (1 colone), kyot binding protein genes (1 colone), RanBPM (1 colone), HBeAg-binding protein 3 (1 colone), programmed cell death 2 (1 colone). Four new genes with unknown function were identified.


CONCLUSIONS
Successful cloning of genes of HBV core protein interacting proteins in leukocytes may provide some new clues for studying the biological functions of HBV core protein.
Department of Infectious Diseases, The First Affiliated Hospital of Medical College of Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, China. linshumei123@126.com
Shu-Mei
SM
Lin
Jun
J
Cheng
Yin-Ying
YY
Lu
Shu-Lin
SL
Zhang
Qian
Q
Yang
Tian-Yan
TY
Chen
Min
M
Liu
Lin
L
Wang
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics; D003001:Cloning, Molecular; D019295:Computational Biology; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D006512:Hepatitis B Core Antigens / Q000235:genetics / Q000378:metabolism*; D006515:Hepatitis B virus / Q000235:genetics; D006801:Humans; D007763:Lac Operon; D007962:Leukocytes / Q000378:metabolism* / Q000821:virology; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020798:Two-Hybrid System Techniques; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D015003:Yeasts / Q000235:genetics
D002352:Carrier Proteins; D006512:Hepatitis B Core Antigens; D011506:Proteins; D014758:Viral Core Proteins