AIM: To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS: HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2XYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12 that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating. pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein. RESULTS: C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray, of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function. CONCLUSION: The novel protein C12 is located in cell plasma, and its overexpression has no significant effect on the metabolism of liver cell. It interacts with many proteins in hepatocytes and may be involved in many processes of gene expression.
AIM: To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized. METHODS: HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2XYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12 that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating. pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein. RESULTS: C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray, of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function. CONCLUSION: The novel protein C12 is located in cell plasma, and its overexpression has no significant effect on the metabolism of liver cell. It interacts with many proteins in hepatocytes and may be involved in many processes of gene expression.
Authors: Robert C Duncan; Poonam Salotra; Neena Goyal; Natalia S Akopyants; Stephen M Beverley; Hira L Nakhasi Journal: Curr Mol Med Date: 2004-09 Impact factor: 2.222