乙型肝炎病毒X基因DNA竞争子的构建及鉴定

Construction and Identification of Competitor of HBV xDNA

目的构建乙型肝炎病毒X基因DNA(xDNA)竞争子L180,为竞争PCR定量分析xDNA打下基础。方法以扩增的xDNA片段为模板,用引物1434和Lx PCR扩增竞争子DNA,并将扩增产物克隆到pUCm-T载体,转化感受态大肠杆菌DH5α,酶切鉴定和测序,筛选阳性竞争子质粒,并建立竞争PCR定量分析xDNA的方法。结果所构建xDNA竞争子L180,经测序及PCR定量分析证实,PCR扩增的产物即为目的xDNA。初步建立了竞争PCR定量分析xDNA的方法。结论已成功构建乙肝病毒xDNA竞争子。

Objective To construct the competitor L180 of HBV xDNA and provide a basis for the quantitative analysis of xD- NA by competitive PCR. Methods The competitor DNA was amplified from xDNA fragment by PCR using primers 1434 and Lx, and cloned into vector pUCm-T, then transformed to E. coli DH5α. The positive competitor plasmid was screened by digestion with restriction endonuclease and nucleotide sequencing,then quantitatively analyzed by competitive PCR. Results The digestion with restriction endonuclease and nucleotide sequencing proved the amplified product as competitor L180 of HBV xDNA ,and the competitive PCR for quantitative analysis of xDNA was primarily developed. Conclusion The competitor of HBV xDNA was successfully constructed.