pEGFP-N1-TNFα表达载体的构建与鉴定

Construction and identification of pEGFP-N1-TNFα expression vector

目的构建pEGFP-N1-TNFα重组质粒并鉴定,为转导CIK细胞并定位表达奠定基础。方法以pMD19Sim-ple T-TNFα为模板,PCR扩增TNFαCDS区片段,将PCR产物进行15g/L琼脂糖凝胶电泳,并回收TNFα条带,用BglⅡ和HindⅢ将TNFα的PCR片段及目的载体pEGFP-N1双酶切,然后进行连接。连接产物转化至DH5α中。挑取克隆,提质粒,进行鉴定。结果 PCR扩增片段、双酶切出现722bp大小的目的基因条带与预期结果相符;插入片断测序结果与合成的寡核苷酸序列一致。结论成功构建了重组质粒pEGFP-N1-TNFα,为下一步用纳米材料转染CIK细胞的研究奠定了基础。

Objective To construct and indentify a recombinant pEGFP-N1-TNFa expression plasmid so as to lay a foundation for transfecting CIK cells and localize the expression. Methods pMD19 Simple T-TNFa was taken as template, the fragment Supported by the Technological Plan of Social Development of Yunnan Province （Fundamental Research Program, No. 2009ZCllgM）, the Health and Science Research Institutes Foundation of Yunnan Province （No. 2011WS0068）, and the Technological Plan of Social Development of Yunnan Province （Key Fundamental Research Program, No. 2009CC026） of CDS region of TNFe was amplified by PCR, and then the products of PCR amplification underwent agarose gel electrophoresis. The fragment TNF was excised, and then fragment TNFa and aimed vector were excised by Bgl H and Hind m double digestion. The excised products were connected; the product was then transformed into DH5a. The positive clones were selected, from which plasmid DNA was abstracted and identified by sequencing. Results The size of PCR products and double digested section were 722 bp, which was in accordance with the expected results; sequence analysis of inserted fragment revealed the same sequence as synthesize oligonucleotides. Gorlclusion The pEGFP-NI-TNF expression vector had been successfully constructed, which laid a foundation for future study on nanomaterial-mediated transfection into CIK cells.