肝素酶反义RNA真核表达载体的构建及鉴定

Construction and Identification of Eukaryotic Expression Vector for Antisense Heparanase

目的构建肝素酶(heparanase,Hpa)反义RNA真核表达载体。方法提取胃癌组织总RNA,按照GenBank中检索出的Hpa基因cDNA序列,设计并合成反义Hpa基因片段的引物,进行RT-PCR,并将扩增产物克隆至pGEM-T载体,PCR鉴定及测序证实后,双酶切pGEM-T-antiHpa重组体回收目的片段,再将其反向亚克隆到真核表达质粒pcDNA3.1中,并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Hpa反义RNA表达质粒pcDNA3.1-antiHpa,分别作PCR及双酶切(BamHⅠ和HindⅢ)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Hpa序列设计完全一致。结论成功构建了Hpa反义RNA真核表达载体pcD-NA3.1-antiHpa,此结果为进一步研究Hpa蛋白分子的生物学功能和以Hpa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 Hpa and to identify the clones. Methods A pair of antisense Hpa primers were designed and synthesized according to the Hpa cDNA sequence in GenBank. Using the primers of antisense Hpa 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 - T vector. After identification by PCR and nucleotide sequencing, the cloned gene was digested by restrictive endonuclease and subcloned reversely into eukaryotic expression vector pcD- NA3.1. Finally, the recombinant was confirmed by PCR, restriction endonuclease digestion （BamH Ⅰ and HindⅢ ） and nucleotide sequencing. Results A 108 bp 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-antiHpa was correct. Conclusion The results of this study lay the foundation for further studying the biological functions of Hpa protein and target gene therapy of anticancer.