携反义二型基质金属蛋白酶基因的重组腺病毒的构建

Construction of Recombinant Adenovirus Vectors Carrying Antisense Matrix Metalloproteinase-2

目的 构建携带反义二型基质金属蛋白酶 (MMP2 )基因的重组腺病毒。方法 从新鲜肝癌组织中提取总RNA ,用RT PCR法合成MMP2cDNA序列中 5′端转录起始位点附近长约 5 0 0bp的基因片段 ,将此片段反义克隆到腺病毒载体 (AdEasy)系统的多克隆位点 ,经转染 2 93细胞生成携带反义MMP2基因的重组腺病毒Ad MMP2 AS。结果 成功构建并包装携带反义MMP2基因片段的重组腺病毒Ad MMP2 AS,病毒滴度达 1× 10 8/ml。结论 构建的重组腺病毒Ad MMP2 AS可望能有效地将反义MMP2基因片段导入人肝癌细胞株 ,为进一步研究肝癌浸润和转移机理以及探讨抑制肝癌浸润和转移的方法提供实验基础。

Objective To construct a recombinant adenoviral vector carrying antisense matrix metalloproteinase 2 (MMP2) for use in the gene therapy to inhibit the invasiveness and migratory capacity of hepatocellular carcinoma (HCC) cell line HepG2 in vitro and in vivo models. Methods Total RNA was extracted from HCC, and then a 500 bp fragment at the 5′ end of human MMP2 cDNA was synthesized by polymerase chain reaction (PCR) and was reversely inserted into the multiclone site (MCS) of the shuttle plasmid pAdTrack CMV,with the resultant plasmid and the backbone plasmid pAdEasy 1,the homologous recombination took place in the E.coli BJ5183 and the recombinant adenoviral plasmid carrying the antisense MMP2 gene was constructed and generated. The adenoviruses(Ad MMP2 AS ) were packaged and amplified in the HEK 293 cells.Then the viral titer was checked by GFP.ResultsThe recombinant adenovirus vector carrying antisense MMP2 was constructed successfully, the strong green fluorescence was observed in HEK 293 cells under a fluorescence microscopy. The viral titer was 1×10 8/ml. Conclusion The recombinant adenovirus Ad MMP2 AS constructed by us could introduce the antisense MMP2 into HepG2 effectively,which would provide experimental basis for reversing the overexpression of MMP2 in HCC and for inhibiting the invasiveness and migratory capacity of HepG2 in vitro and in vivo models.