MMP-9反义RNA真核表达载体的构建及鉴定

Construction and identification of the eukaryotic antisense RNA expression vector of MMP-9

目的构建MMP-9反义RNA真核表达载体。方法提取胃癌组织总RNA,按照GenBank中检索的MMP-9基因cDNA序列,设计并合成反义MMP-9基因片段引物,进行逆转录聚合酶链反应(RT-PCR),先将扩增产物克隆至pGEM-T载体,再将其反向亚克隆到真核表达质粒pcDNA3.1中,然后对阳性克隆进行酶切鉴定和测序分析。结果PCR扩增出的反义片段与预期长度相符。构建的MMP-9反义RNA表达质粒pcDNA3.1-MMP-9分别作PCR及双酶切(BamH和Hind)鉴定,证实其中有目的片段完整插入,插入片段测序结果与反义MMP-9序列设计完全一致。结论成功构建了MMP-9反义RNA真核表达载体pcDNA3.1-MMP-9,为进一步研究MMP-9蛋白分子的生物学功能和以MMP-9为靶点的恶性肿瘤的基因治疗研究奠定了基础。

Objective To construct eukaryotic antisense RNA expression vector of MMP-9 and to identify the clones.Methods A pair of antisense MMP-9 primers were designed and synthesized according to the MMP-9 cDNA sequence in Genbank.Using the primers of antisense MMP-9 gene and the total RNA extracted from the tissues of gastric carcinoma,reverse transcriptase-polymerase chain reaction (RT-PCR) was performed,the product of which was cloned into pGEM-Teasy vector.The cloned gene was subcloned reversely into eukaryotic expression vector pcDNA3.1.Finally,the recombinant was confirmed by PCR,restriction endonuclease digestion (BamHⅠand HindⅢ) and nucleotide sequencing.Results A 225bp DNA fragment was amplified by PCR as expected.It was verified by PCR and partial nucleotide sequencing that the constructed eukaryotic antisense RNA expression vector pcDNA3.1-MMP-9 was correct.Conclusion The results of this study lay the foundation for further studying the biological functions of MMP-9 protein and target gene therapy of anticancer.