Construction of cDNA library of hepatitis B virus with X protein C-terminally truncated 40 amino acids by suppression subtractive hybridization method / 第二军医大学学报

ABSTRACT

Objective:To construct subtractive cDNA library from human hepatocellular carcinoma cells transactivated by C-terminally truncated 40 amino acids using suppression subtractive hybridization(SSH) technique and to clone the associated genes.Methods: Huh-7 cells were separately transfected with pcDNA3(-) harboring the sequence of HBx protein C-terminally truncated 40 amino acids and pcDNA3(-) harboring the full length sequence of HBx protein vectors.The total RNAs were isolated from the transfected Huh-7 cells and were reversely transcripted into double strand cDNAs.After the cDNAs were digested with restriction enzyme RsaⅠ,they were divided into 2 groups and were ligated to the special adaptor 1 and adaptor 2R,respectively.The tester cDNAs were then hybridized with driver cDNAs twice and the products were amplified twice by nested PCR technique.The PCR products were connected with pUCm-T plasmid vectors to establish the subtractive library.Amplification of the library was carried out with E.coli strain JM109.The inserts of cDNAs were sequenced and analyzed in GenBank with Blast search.Results: The subtractive cDNA library was successfully constructed.The amplified library contained 154 positive clones,and colony PCR showed that these clones contained 200-800 bp inserts;some fragments coded proteins involved proto-oncogenes,cell signaling genes,cell growth factor genes,cell apoptosis genes,metabolism and protein synthesis genes.Conclusion: Subtractive cDNA library has been successfully constructed by SSH technique,which may help to clone novel genes transactivated by HBx C-terminally truncated 40 amino acids and to explore the molecular mechanism of hepatoma pathogenesis.