ABSTRACT: To investigate the interaction between hepatitis C virus core protein and translin protein and its role in the pathogenensis of hepatocellular carcinoma and lymphoma.
With the components of the yeast two hybrid system 3, "bait" plasmids of HCV core the gene was constructed. After proving that hepatitis C virus core protein could be firmly expressed in AH109 yeast strains, yeast two- hybrid screening was performed by mating AH109 with Y187 that transformed with liver cDNA library plasmids-pACT2 and then plated on quadruple dropout (QDO) medium and then assayed for alpha-gal activity. Sequencing analysis of the genes of library plasmids in yeast colonies that could grow on QDO with alpha-gal activity was performed. The interaction between HCV core protein and the protein we obtained from positive colony was further confirmed by repeating yeast two - hybrid analysis and coimmunoprecipitation in vitro.
A gene from a positive colony was the gene of translin, a recombination hotspot binding protein. The interaction between HCV core protein and translin protein could be proved not only in yeast, but also in vitro.
The core protein of HCV can interact with translin protein. This can partly explain the molecular mechanism for hepatocellular carcinoma and lymphoma caused by HCV.
World Journal of Gastroenterology 03/2003; 9(2):300-3. · 2.47 Impact Factor
ABSTRACT: To investigate the biological function of augmenter of liver regeneration (ALR), we used yeast-two hybrid technique to detect proteins in hepatocytes interacting with ALR.
ALR bait plasmid was constructed by using yeast-two hybrid system 3, then transformed into yeast AH109. The transformed yeast was mated with yeast Y187 containing liver cDNA library plasmid in a 2XYPDA medium. Diploid yeast was plated on a synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-alpha-gal for selection and screening. After extracting and sequencing of the plasmid from blue colonies. Analysis was performed by bioinformatics.
Of 36 colonies sequenced, 14 are metallothionein, 12 albumin, and 3 selenoprotein P. One colony is a new gene with unknown function.
The successful cloning of gene of ALR interacting protein has paved the way for studying the physiological function of ALR and associated proteins.
Hepatobiliary & pancreatic diseases international: HBPD INT 03/2003; 2(1):81-4. · 1.08 Impact Factor
ABSTRACT: To study the function of augmenter of liver regeneration (ALR) as a regulatory factor that specifically stimulates hepatic cell regeneration, we constructed yeast expressive vector of ALR and expressed it in yeast cells.
Total RNA was extracted from HepG2 cells, and reverse transcription polymerase chain reaction (RT-PCR) was performed to amplify the coding region of ALR. The products were cloned into PGEM-T vector and sequenced, then cloned into PGBK T7 vector. The recombinant plasmid PGBK T7-ALR was transformed into yeast AH109. The yeast protein was extracted and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting hybridization technique.
DNA sequencing results confirmed that the coding region of ALR was correctly inserted into the yeast expression vector, and Western blotting assay showed that recombinant ALR was successfully expressed in yeast. Its molecular weight was identical to the theoretical value of 15 000 Da; the protein was found inside the yeast cells.
The successful expression of ALR in yeast cells makes it possible to study further on its biological function.
Hepatobiliary & pancreatic diseases international: HBPD INT 03/2002; 1(1):87-91. · 1.08 Impact Factor