pDC315-SPA-mCLCA3载体的构建及其在气道上皮细胞系的靶向表达分析  

Effect of pDC315-SPA-mCLCA3 targeting system on mCLCA3 gene expression in airway epithelial cell lines in vitro

作　　者：何丽[1,2] 梅丽[1] 马经平[2] 张惠兰[1] 张波[1] 熊维宁[1] 赵建平[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院呼吸内科,卫生部重点呼吸病研究室,湖北武汉430030 [2]荆州市中心医院呼吸内科,湖北荆州434020

出　　处：《中国病理生理杂志》2011年第6期1240-1244,共5页Chinese Journal of Pathophysiology

基　　金：武汉市科学技术计划资助项目(No.200850731357)

摘　　要：目的:构建腺病毒穿梭质粒(pDC315)-肺表面活性蛋白A基因启动子(SPA)-小鼠钙激活氯通道3(mCLCA3)融合基因表达载体,分析其在气道上皮细胞系的靶向表达。方法:PCR法从PGL-SPA和pCR-Blunt Ⅱ-TOPO载体中克隆全长2 948 bp的SPA-mCLCA3基因序列并将其亚克隆入pDC315-EGFP载体,构建pDC315-SPA-mCLCA3靶向表达载体。测序鉴定正确后分别转染气道上皮细胞系(H441细胞)和非气道上皮细胞系(HEK293细胞)。每种细胞分pDC315-EGFP组(EGFP阳性,mCLCA3阴性)和pDC315-SPA-mCLCA3组(mCLCA3阳性),分析mCLCA3在不同上皮细胞的表达水平。结果:测序结果证实成功构建含有气道上皮细胞特异性启动子SPA的mCLCA3重组质粒。靶向表达分析表明mCLCA3 mRNA在高表达SPA的H441细胞中表达显著高于其在HEK293细胞的表达(P<0.05);免疫细胞化学表明H441细胞pDC315-SPA-mCLCA3组中mCLCA3蛋白表达阳性而pDC315-EGFP组表达阴性,HEK293细胞两种质粒转染组mCLCA3蛋白均阴性。结论:成功构建气道上皮细胞靶向表达mCLCA3基因的重组腺病毒穿梭载体pDC315-SPA-mCLCA3,为后续构建腺病毒靶向载体Ad-SPA-mCLCA3及研究mCLCA3在气道上皮缺陷中的作用提供实验基础。AIM：To construct a recombinant adenovirus shuttle plasmid pDC315-SPA（gene promoter of pulmonary surfactant-associated protein A）-mCLCA3（mouse calcium-activated chloride channel 3） and to study the targeting expression of mCLCA3 in airway epithelial cells.METHODS： The full length of SPA-mCLCA3 gene was cloned by PCR from PGL-SPA and pCR-Blunt II-TOPO vectors,and subcloned into pDC315-EGFP to construct pDC315-SPA-mCLCA3 targeting expression vector.After identified by nucleotide sequencing analysis,the vector pDC315-SPA-mCLCA3 and control vector pDC315-EGFP （used for negative control as well as GFP positive control） were transfected into H441 cells and HEK293 cells with lipofectamine 2000.The mRNA expression of mCLCA3 in each cell line was determined by RT-PCR.The protein level of mCLCA3 in transfected cells was detected by immunocytochemistry.RESULTS： The recombinant plasmid containing SPA promoter and mCLCA3 fusion gene was confirmed by sequencing analysis.The expression of mCLCA3 mRNA was undetectable in the 2 cell lines transfected with pDC315-EGFP.In the cells transfected with pDC315-SPA-mCLCA3,mRNA expression of mCLCA3 was significantly higher in H441 cells than that in HEK293 cell line（P0.05）,indicating pDC315-SPA-mCLCA3 targeting expression vector had high transcriptional targeting activity in airway epithelial cells.The result of immunohistochemistry showed that the protein expression of mCLCA3 was not detected in the control cells while strong immunoreactivity was observed in H441 cells transfected with pDC315-SPA-mCLCA3.The protein expression of mCLCA3 was not detected in HEK293 cells with or without pDC315-SPA-mCLCA3 transfection.CONCLUSION： The recombinant adenovirus shuttle plasmid pDC315-SPA-mCLCA3 has the targeting expression specificity to airway epithelial cells.

关 键 词：肺表面活性物质相关蛋白A启动子 CLCA3蛋白 小鼠 气道上皮细胞 靶向表达 

分 类 号：R363[医药卫生—病理学]