Construction of cDNA library of hepatitis B virus with X protein C-terminally truncated 40 amino acids by suppression subtractive hybridization method
- Author:
Li WANG
1
Author Information
1. Department of Pathology
- Publication Type:Journal Article
- From:
Academic Journal of Second Military Medical University
2006;27(10):1081-1084
- CountryChina
- Language:Chinese
-
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 Rsa I, 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.
