正、反义Heparanase基因荧光真核表达载体的构建及胰腺癌SW1990细胞转染

Construction of Sense and Antisense of Human Heparanase and Green Fluorescent Protein Eukaryotic Co-expressing Vectors and Their Expression in Pancreatic Cancer Cell Lines SW1990

目的构建正、反义人肝素酶绿色荧光蛋白真核共表达载体,并转染人胰腺癌SW 1990细胞.方法用EcoR I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EcoR I酶切位点上,经BamH I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、反义真核表达载体转染人胰腺癌SW 1990细胞,24 h后荧光倒置显微镜下检测转染结果.结果经BamH I酶切后,正义质粒形成5.3 kb和1.7 kb两条DNA片段,反义重组质粒为6.5 kb和0.5 kb两条DNA片段,与理论计算值一致;测序结果进一步确认了方向的正确性.转染成功的胰腺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正、反义真核表达载体,并成功转染人胰腺癌细胞株SW 1990,为进一步研究正、反义肝素酶基因转染对胰腺癌细胞的转移能力的影响奠定基础.

Objective To construct sense and antisense of human heparanase and green fluorescent protein eukaryotic expressing vectors and then transfect these vectors into Pancreatic Cancer Cell Lines SW1990. Methods The human Heparanase cDNA fragment contained in the PeDNA3 Hpa vector was cloned into the enhanced green fluorescent protein eukaryotic expressing vector pIRES2 -EGFP in cis -direction or trans -direction. The recombinant vectors were identified by digestion of BamH I and further identified by DNA sequencing. The recombinant were transfected to pancreatic cancer cells SW1990 by liposome method. After 24 h, the transfected cells were observed under fluorescent inverted microscope. Results After digested by BamH I, two fragments with the length of 5.3 kb and 1.7 kb were formed in sense fluorescent eukaryotic expressing vector, while two other fragments with the length of 6. 5 kb and 0. 5 kb were formed in antisense fluorescent vector. The DNA sequencing was also confirmed the linking direction of sense and antisense recombinant. Green fluorescence of the transfected cells could be observed under fluorescent inverted microscope after 48 h of transfection. Conclusion Human heparanase sense and antisense fluorescent eukaryotic expressing vectors are successfully constructed and transfected to human pancreatic cancer cell lines.