OBJECTIVE: To explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre Slprotein of HBV by two-hybrid system.METHODS: Yeast expression plasmids encoding fusion proteins of full length or ...OBJECTIVE: To explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre Slprotein of HBV by two-hybrid system.METHODS: Yeast expression plasmids encoding fusion proteins of full length or portions of Pre Sl ofHBV and DNA binding domain of yeast protein GAL4 were constructed and used to transform yeastreporter strain SFY526. Reporter gene product β-galactosidase activity was assayed as a measure oftranscriptional activation in yeast, Mammalian expression plasmid encoding fusion proteins of full lengthPre Sl and DNA binding domain of GAL4 was constructed and used to cotransfect hepatoma cell lineHuh-7 together with CAT reporter plasmid. Cell extracts were assayed for CAT activity by thin-layerchromatography.RESULTS: The fusion proteins of full length Pre Sl protein and GAL4 DNA binding domain presentedtranscriptional activation function in yeast. The transcription activating sequence was localized to the 21 to47 amino acids of Pre Sl protein. Fusion proteins of full length Pre Sl and GAL4 DNA binding domaindid not show transcriptional activation function in mammalian cells.CONCLUSIONS: The transcription activating sequence of HBV Pre Sl protein in yeast overlaps thehepatocyte receptor binding site. The transcriptional activation function of HBV Pre Sl protein in yeastmay prevent researchers from using yeast two-hybrid system to clone HBV receptor interacting with Pre Slprotein. However, the Pre Sl protein does not show transcriptional activation function in mammaliancells. Mammalian two-hybrid system may be a practical method to clone the HBV hepatocyte receptorinteracting with Pre Sl protein.展开更多
The large protein of the hepatitis B virus (HBV) envelope was subdivided intothe preSl region with 108-115 amino acids, the preS2 region with 55 amino acidsand the S region with 226 amino acids. It was believed that t...The large protein of the hepatitis B virus (HBV) envelope was subdivided intothe preSl region with 108-115 amino acids, the preS2 region with 55 amino acidsand the S region with 226 amino acids. It was believed that the preSl region cotainedthe hepatocyte attachment sites of the virus as well as multiple highly immunogenicT-cell and B-cell epitopes. The antibody response to the preSl peptide was also oneof the most important serological markers of HBV infection.展开更多
基金This work was supported by a grant from the National Natural Science Foundation of China (No. 39600006).
文摘OBJECTIVE: To explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre Slprotein of HBV by two-hybrid system.METHODS: Yeast expression plasmids encoding fusion proteins of full length or portions of Pre Sl ofHBV and DNA binding domain of yeast protein GAL4 were constructed and used to transform yeastreporter strain SFY526. Reporter gene product β-galactosidase activity was assayed as a measure oftranscriptional activation in yeast, Mammalian expression plasmid encoding fusion proteins of full lengthPre Sl and DNA binding domain of GAL4 was constructed and used to cotransfect hepatoma cell lineHuh-7 together with CAT reporter plasmid. Cell extracts were assayed for CAT activity by thin-layerchromatography.RESULTS: The fusion proteins of full length Pre Sl protein and GAL4 DNA binding domain presentedtranscriptional activation function in yeast. The transcription activating sequence was localized to the 21 to47 amino acids of Pre Sl protein. Fusion proteins of full length Pre Sl and GAL4 DNA binding domaindid not show transcriptional activation function in mammalian cells.CONCLUSIONS: The transcription activating sequence of HBV Pre Sl protein in yeast overlaps thehepatocyte receptor binding site. The transcriptional activation function of HBV Pre Sl protein in yeastmay prevent researchers from using yeast two-hybrid system to clone HBV receptor interacting with Pre Slprotein. However, the Pre Sl protein does not show transcriptional activation function in mammaliancells. Mammalian two-hybrid system may be a practical method to clone the HBV hepatocyte receptorinteracting with Pre Sl protein.
文摘The large protein of the hepatitis B virus (HBV) envelope was subdivided intothe preSl region with 108-115 amino acids, the preS2 region with 55 amino acidsand the S region with 226 amino acids. It was believed that the preSl region cotainedthe hepatocyte attachment sites of the virus as well as multiple highly immunogenicT-cell and B-cell epitopes. The antibody response to the preSl peptide was also oneof the most important serological markers of HBV infection.
