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目的构建环氧合酶-2L启动子(Cox-2L)及2型生长抑素受体(SSTR2)基因重组腺病毒载体,以研究SSTR2重新表达及腺病毒感染对胰腺癌细胞的影响。方法用逆转录-聚合酶链反应(RT-PCR)法从人正常胰腺组织中调取SSTR2基因,以HL60细胞基因组DNA为模板,以PCR法调取Cox-2L基因,以SSTR2及Cox-2L基因片段为模板,以PCR法扩增Cox-2L-SSTR2融合基因并连入TaKaRa公司pAxcwit腺病毒载体中,以电穿孔法转化E．Coli后挑选阳性克隆进行PCR及酶切鉴定。以pAxcwit-Cox-2L-SSTR2转染HEK293细胞获得重组腺病毒颗粒,进行PCR、酶切鉴定。结果调取并连接后的基因片段经测序证实为Cox-2L、SSTR2及Cox-2L-SSTR2融合基因,Cox-2L-SSTR2基因片段及PolyA克隆入T载体,经酶切并测序证实PMDl8-T-Cox-2L- SSTR2-PolyA构建成功。pAxcwit-Cox-2L-SSTR2-PolyA经酶切及PCR鉴定,Cox-2L-SSTR2-PolyA融合基因正确连入腺病毒载体。结论成功构建了Cox-2L-SSTR2基因重组腺病毒载体,为进一步研究SSTR2在胰腺癌细胞中的表达及作用奠定了基础。
Objective To construct the recombinant adenovirus vector of COX-2L and SSTR2 to study the effect of SSTR2 re-expression and adenovirus infection on pancreatic cancer cells. Methods The SSTR2 gene was extracted from human normal pancreatic tissue by reverse transcription polymerase chain reaction (RT-PCR). The genomic DNA of HL60 was used as a template to retrieve the Cox-2L gene by PCR. The SSTR2 and Cox-2L The gene fragment was used as a template to amplify the Cox-2L-SSTR2 fusion gene by PCR and ligated into TaKaRa pAxcwit adenovirus vector to transform E by electroporation. Coli positive clones were selected for PCR and restriction enzyme digestion. The recombinant adenovirus particles were transfected into HEK293 cells with pAxcwit-Cox-2L-SSTR2, and PCR and restriction enzyme digestion were performed. Results The fragment of Cox-2L, SSTR2 and Cox-2L-SSTR2 fusion gene, Cox-2L-SSTR2 gene fragment and PolyA cloned into T vector were confirmed by sequencing. The results of restriction endonuclease digestion and DNA sequencing confirmed that PMD18-T -Cox-2L-SSTR2-PolyA was successfully constructed. After digested with pAxcwit-Cox-2L-SSTR2-PolyA and identified by PCR, the Cox-2L-SSTR2-PolyA fusion gene was correctly inserted into the adenoviral vector. Conclusion The recombinant adenoviral vector carrying Cox-2L-SSTR2 gene was successfully constructed, which laid the foundation for further study of the expression and role of SSTR2 in pancreatic cancer cells.