人肝素酶基因正反义荧光真核表达载体的构建及肝癌细胞转染

Construction of sense and antisense human heparanase and green fluorescent protein eukaryotic co-expression vectors and expression in hepatic cancer cell lines

目的构建正义和反义人肝素酶绿色荧光蛋白真核共表达载体,并分别转染人低转移肝癌细胞株MHCC97-L及高转移细胞株MHCC97-H和HCCLM6。方法采用基因重组技术,用EcoRⅠ从pcDNA3-Hpa质粒上切下约1.7 kb的人肝素酶全长cDNA片段,然后连入pIRES2-EGFP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,并进一步测序确认。用脂质体法将肝素酶正义真核表达载体转入人低转移肝癌细胞株MHCC97-L,反义真核表达载体转入人高转移肝癌细胞株MHCC97-H和HCCLM6,24 h后荧光倒置显微镜下检测转染结果。结果经BamHⅠ酶切后,正义质粒形成5.3 kb和1.7 kb两条DNA片段,而反义重组质粒为6.5 kb和0.5 kb两条DNA片段,与理论计算值完全一致;测序结果进一步确认了方向的正确性。转染成功的肝癌细胞在倒置荧光显微镜下呈绿色荧光。结论成功构建了人肝素酶的正、反义真核表达载体,并成功转染人低、高转移肝癌细胞株,为进一步研究正、反义肝素酶基因转染对肝癌细胞的转移能力的影响奠定了基础。

Objective To construct sense and antisense human heparanase green nuorescent protein eukaryotic expression vectors and then transfect these vectors into hepatic cancer cell lines. Methods The human heparanase cDNA fragment contained in the pcDNA3 Hpa vector was cloned into the enhanced green fluorescent protein eukaryotic expression vector pIRES2-EGFP in cis-direction or trans-direction. The recombinant vectors were identified by digestion of BamH I and further by DNA sequencing. Then the recombinant were transfected to hepatic cancer cells with highly metastatic potential （ MHCC97-H and HCCLM6） or with lowly metastatic potential （MHCC97-L） by liposome method. After 48 h, the transfected cells were observed under fluorescent inverted microscope. Results After digested by BamH Ⅰ, two fragments of 5.3 kb and 1.7 kb were formed in sense fluorescent eukaryotic expression vector, while two other fragments of 6. 5 kb and 0. 5 kb were formed in antisense fluorescent vector, both of which were respectively named as pIRES2-EGFP-sHpa and pIRES2-EGFP-aHpa. The DNA sequencing also confirmed the linking direction of sense and antisense recombinant. Green fluorescence of the transfeeted cells could be observed under inverted fluorescence microscope at 48 h after transfection. Conclusion Human heparanase sense and antisense fluorescent eukaryotic expression vectors are successfully constructed and transfected to human hepatic cancer cell lines with different metastatic potential.