Expression of human gene 5 transactivated by pre-S1 protein of hepatitis B virus in yeast cell / 中华传染病杂志

Objective To study the exact function of human gene 5 transactivated by pre-S1 protein of hepatitis B virus(PS1TP5)by investigating the gene expression of PS1TP5 in yeast cells. Methods Reverse transcription-polymerase chain reaction(RT-PCR)was performed to amplify the gene of PS1TP5 using the mRNA of HepG2 cells as template and the gene was cloned into pGEM-T vector.The gene of PS1TP5 was cut from pGEM-T-PS1TP5 vector and cloned into yeast expressive plasmid pGBKT7,then pGBKT7-PS1TP5 was transformed into yeast cell AH109.The yeast protein was isolated and analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western hybridization.Results PS1TP5 gene was successfully amplified and identified by DNA sequencing.The digested fragment was cloned into pGBKT7 vector and transformed into yeast cell AH109.The results of SDS-PAGE and Western assay showed that the relative molecular weight of the expressed product was about 36 950,and PS1TP5 protein existed in yeast cells.Conclusion The findings suggest that PS1TP5 can be successfully expressed in yeast cell.

Screening and identification of the genes trans-regulated by hepatitis B virus pre-S1 protein with microarray assay / 解放军医学杂志

Objective To evaluate the influence of expression of pre S1 protein on the genomic expression of hepatitis B virus (HBV) infected hepatocyte with microarray. Methods The differentially expressed genes between the hepatoblastoma cell line HepG2 transfected by pcDNA3.1(-) and pcDNA3.1(-)-preS1, were respectively compared by cDNA microarray technique. The HBV pre-S1 coding DNA fragment was amplified with polymerase chain reaction (PCR) technique by using G376-7 plasmid DNA containing the full length of HBV genome as the template. The expressive vector of pcDNA3.1-preS1 was constructed by routine molecular biological methods. HepG2 cells were transfected by pcDNA3.1(-) and pcDNA3.1-preS1, respectively, using FuGENE6 Transfection Reagent. The total RNA was isolated and reversely transcribed. Results The cDNAs were subjected for microarray screening with 1152 cDNA probes. From the scanning results, it was found that 30 genes were up-regulated and 38 genes were down-regulated by pre-S1 protein of HBV. Conclusion The expression of pre-S1 protein affected the genomic expression spectrum of HBV infected hepatocyte(HepG2 cell line).

Cloning of human gene 5 trans-activated by pre-S1 protein of hepatitis B virus / 解放军医学杂志

Objective To clone a new human gene 5 trans-activated by pre-S1 protein of hepatitis B virus (HBV), PS1TP5, and explore its structure and function by bioinformatics analysis. Methods PS1TP5 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) technique by using HepG2 cDNA as template and inserted into pGEM-T vector by TA cloning. Recombinant eukaryotic expression vector pcDNA TM 3.1/myc-His A-PS1TP5 had been constructed by subcloning, followed by restriction enzyme digestion analysis and sequencing. Bioinformatic methods were used to analyze its possible physical and chemical characters, structure, and function. Results PS1TP5 was successfully amplified and cloned into pGEM-T and pcDNA TM 3.1/myc-His A vector by RT-PCR from HepG2 cDNA. The new gene had been confirmed by sequencing after PCR identification and restriction enzyme digestion and named as PS1TP5 because of its trans-active function. The sequence for the PS1TP5 gene had been deposited into GenBank, the accession number was AY427953. Bioinformatics analysis showed that its ORF was 438bp and translated a protein of 145 aa. Conclusion A new gene-PS1TP5 has been recognized, and its recombinant eukaryotic expression vector (pcDNA TM 3.1/myc-His A-PS1TP5) has been constructed. These results will certainly bring some new clues for the study of the biological function of new gene and pathogenesis of chronic hepatitis B.