人肝素酶基因正反义腺病毒表达载体的构建及鉴定

Construction and identification of sense and antisense human heparanase adenovirus expression vector

目的:构建人肝素酶正义和反义腺病毒表达载体.方法:用EcoRI从pcDNA3-hpa质粒上切下约1.7kb的人肝素酶全长cDNA片段,然后连入pDC315质粒的EcoRI酶切位点上,经BamHI酶切鉴定出正义和反义表达载体,并对正义重组质粒进一步采用测序鉴定其方向性.结果:经BamHI酶切后,正义质粒形成4.3+1.4kb两条带,而反义重组质粒为5.1+0.4kb两条带,与理论计算值完全一致;测序结果与GeneBank报告的肝素酶序列完全一致.结论:成功构建了人肝素酶的正、反义腺病毒表达载体,为进一步研究正、反义肝素酶基因转染对肿瘤细胞的影响奠定了基础.

AIM: To construct an adenovirus expressing vector of sense and antisense human heparanase gene. METHODS: The human heparanase cDNA fragment contained in the pcDNA3-hpa vector was cloned into the adenovirus expressing vector pDC315 in cis-direction or trans-direction using DNA recombinant technology. The recom-binant vectors were identified by digestion of BamH I. The sense recombinant vector was further identified by DNA sequencing. RESULTS: After digested by BamH I, two fragments which lengthened 4.3 and 1.4 kb were formed in sense recombinant vector (pDC315-sHpa), while two fragments which lengthened 5.1 kb and 0.4 kb were formed in antisense vector (pDC315-aHpa). Electrophoresis results were completely coincident with theoretical calculation. pDC315-sHpa DNA sequence was identical to the heparanase sequence published in the Gene Bank. CONCLUSION: The sense and antisense human heparanase adenovirus expressing vectors are successfully constructed.