大鼠5-脂肪氧合酶cDNA的重组腺病毒制备及在大鼠血管外膜成纤维细胞中的表达  

Construction of recombinant adenoviral plasmid by homologous recombination in bacteria and expression of 5-lipoxygenase in rat adventitial fibroblasts

作　　者：董晓[1,2] 柯尊平[3] 王俊峰[1] 周明[1] 谭利[1] 党书毅[1] 

机构地区：[1]湖北医药学院附属太和医院心内科,湖北省十堰市442000 [2]湖北医药学院胚胎干细胞研究湖北省重点实验室,湖北省十堰市442000 [3]湖北医药学院计算机实验教学中心,湖北省十堰市442000

出　　处：《岭南心血管病杂志》2016年第2期201-206,共6页South China Journal of Cardiovascular Diseases

基　　金：湖北省自然科学基金青年项目(项目编号:2015CFB211);湖北省教育厅科学技术研究项目(项目编号:Q20132104);湖北医药学院附属太和医院国家级项目培育基金(项目编号:2013PY01)

摘　　要：目的利用细菌内同源重组法快速构建携带大鼠5-脂肪氧合酶(5-lipoxygenase,5-LO)cDNA的重组腺病毒质粒和制备表达大鼠5-LO的重组腺病毒,并在大鼠原代血管外膜成纤维细胞中实现过表达。方法聚合酶链反应(polymerase chain reaction,PCR)扩增大鼠5-LOcDNA片段,亚克隆至腺病毒穿梭质粒中,构建成转移质粒pshuttle-CMV-5LO;用PmeI线性化的pshuttle-CMV-5LO转化含腺病毒骨架质粒pAdEasy-1的感受态BJ5183,通过同源重组获得阳性重组质粒pAdEasy-1-5LO。重组质粒经鉴定后用Pad酶切,转入HEK293细胞,包装成重组腺病毒Ad-1-5LO,经纯化,鉴定,滴度测定,转染体外培养的大鼠原代血管外膜成纤维细胞,Western blot检测腺病毒载体在体外的过表达。结果 5-LOcDNA成功地插入到穿梭质粒中,pshuttle-CMV-5LO与pAdEasy-1在BJ5183中成功发生了同源重组,得到了重组腺病毒质粒pAdEasy-1-5LO,重组腺病毒经PCR和酶切鉴定表明携带有目的基因5-LOcDNA,滴度约为1.0×10^(15)pfu/L,转染大鼠原代血管外膜成纤维细胞后经Western blot检测,细胞内5-LO的表达明显增加。结论细菌内同源重组法构建腺病毒载体具有高效、省时、省力的特点,制备出的高滴度重组腺病毒Ad-1-5LO实现了在血管外膜成纤维细胞中的过表达,为研究5-LO途径在血管重构中的作用奠定了基础。Objectives To construct recombinant adenoviral plasmid containing rat 5-1ipoxygenase （5-LO） cDNA using homologous recombination in bacteria and to express 5-LO in rat adventitial fibroblasts. Methods 5-LO cDNA was amplified from recombinant pBluescript-SLO plasmid using special primers containing SalI and XholI restriction enzyme sites. The polymerase chain reaction （PCR） product was subcloned into the shuttle vector to generate transferred plasmid pShuttle-CMV-SLO. The vector pShuttle-CMV-SLO was linearized with Pme I and transformed into ultracompetent B J5183 bacteria containing pAdEasy- 1. Positive clone of homologous recombination （pAdEasy- 1-SLO） was identified by PCR, enzyme digestion and DNA sequencing. The positive recombinant adenoviral plasmid was digested with PacI and transfected HEK293 cells with Lipofectamine 2000 to package recombinant adenovirus particles （Ad-1-SLO）. The recombinant adenovirus was purified with density gradient centrifugation of cesium chloride. PCR was used to characterize the recombinant adenovirus expressing 5-LO. The titer and purity of the recombinant adenovirus were determined by ultraviolet spectroscopy. Rat adventitial fibroblasts were infected with the recombinant adenovirus and the expression of 5-LO in adventitial fibroblasts was detected by Western blot. Results 5-LO cDNA was successfully inserted into the shuttle vector. Homologous recombination occurred between pShuttle-CMV-5LO and pAdEasy-1 within B J5183 to generate pAdEasy-1-5LO. The recombinant adenovirus was confirmed to be successfully constructed with PCR. The titer of the purified Ad-1-5LO was 1.0×10^15 pfu/L and the infection efficiency was detected by 5-LO expression in rat adventitial fibroblasts. Conclusions The construction of adenovirns plasmid by homologous recombination in bacteria is quick and easy to perform. The preparation of recombinant adenovirus Ad-1-SLO with high titer provides a basis for further studies of 5-LO in vascular remodeling.

关 键 词：5-脂肪氧合酶 白三烯 腺病毒载体 同源重组 克隆 

分 类 号：R33[医药卫生—人体生理学]