Cloning Of Xeroderma Pigmentosum Complementation Group B (XPB) Gene And Construction Of Mammalian Expression Recombinant PcDNA3.1(+)-XPB

Object XPB protein, also named excision repair cross complementing (ERCC) 3 protein, is a ubiquitous important acidic protein in the cells of a broad range of organisms. XPB protein is the essential component of cellular nucleotide excision repair pathway which is essential for the maintenance of organism genome integrity, and is the largest p89 subunit of transcription factor TFIIH which is involved in both basal and activated eukaryotic gene transcription. XPB protein presents 3′→5′ATP-dependent single-stranded DNA (ssDNA) helicases and plays a dual central role in the cellular nucleotide excision repair pathway as well as in RNA polymerase II transcription by unwinding and opening the DNA around both a promoter in RNAP II transcription and /or a lesion in NER functions. Recent studies have demonstrated that the expression of hepatitis B virus X proein (HBx) in the hepatic cell is derectely and inderectely associated with a down-regulation of endogenous XPB and p53 at both mRNA and protein levels. To investigate the expression, biological function and molecular mechanism of XPB in chronic hepatitis and the development of hepatocellular carcinoma, we cloned XPB cDNA full-length code sequence and construct a mammalian expression recombinant plasmid pcDNA3.1 (+)-XPB, which will provide the basis for next establishing a transgenic HepG2 cell line stably expressing wild-type XPB gene and for further working on carcinogenesis gene therapy especially for hepatocellular carcinoma gene therapy. Methods Human XPB cDNA was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from total RNA isolated from cultured HeLa cells maintained in DMEM+ 10% FBS, and was inserted into the HindIII and BamHI sites of a mammalian expression plasmid pcDNA3.1(+), which contains cytomegalovirus (CMV) promoter and T7 promoter and contains ampcillin and neomycin resistance genes, to construct recombinant plasmid pcDNA3.1(+)-XPB. The recombinant pcDNA3.1(+)-XPB was transformed into competent JM-109 cell for positive screening and propagation followed by plasmid preparation and purification, and then performed by the analysis of different restriction endonuclease digestion and automate sequencing. Results The XPB cDNA was particularlly amplified with primers we designed as expected. The recombinant of pcDNA3.1(+)-XPB was structurally confirmed by analysis of restriction endonuclease digestion and sequencing. The inserted XPB cDNA segment in the vector was sense orientation as a wild type XPB gene and its sequence was structurally confirmed to be consistent with that of the published data (GenBank access NM000122). Conclusion The cDNA of human XPB can be successfully cloned and inserted into mammalian expression vector pcDNA3.1(+). The newly constructed recombinant is useful for investing the biological function of XPB in gene therapy of hepatocellular carcinoma.