真核表达载体pIRES2-EGFP-SP-B-C／T1580的构建及其在293T细胞中的表达  

Construction of eukaryotic cell expression vectors pIRES2-EGFP.SP-B.C/T 1580 and evaluation oftheir expressions in 293T cells

作　　者：徐洪涛[1] 杨慧[1] 常立文[1] 李文斌[1] 赵玲霞[1] 袁文浩[1] 刘伟[1] 蔡保欢[1] 王席娟[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院儿科,武汉430030

出　　处：《中国医师杂志》2012年第10期1338-1341,共4页Journal of Chinese Physician

基　　金：国家自然科学基金项目（30872795;81170001）

摘　　要：目的构建两种人肺表面活性物质蛋白B（surfactant protein B，SP．B）基因变异型的真核表达载体，研究SP-B基因多态性与支气管肺发育不良（bronchopulmonary dysplasia，BPD）的关系。方法应用基因重组技术，构建pIRES2-EGFP-SP-B-C1580和plRES2-EGFP-SP-B．T1580真核表达质粒，经测序鉴定后，采用脂质体法转染293T细胞系，荧光显微镜观察绿色荧光蛋白在293T细胞中的表达，逆转录一聚合酶链反应一限制性片段长度多态性（reverse transcription-polymerase chain reaction-re-striction fragment length polymorphism,RT-PCR-RFLP）检测SP-B-C/T1580mRNA的表达，Westernblot检测SP-B蛋白的表达。结果两个重组质粒含有完整sP-BcDNA，在1580位点碱基序列分别为C和T，转染293T细胞后有相应的mRNA及蛋白表达。结论成功构建了真核表达载体plRES2．EG。FP-SP-B-C／T1580，为揭示支气管肺发育不良的发生机制奠定基础。Objective To construct two kinds of eukaryotic cell expression vectors pIRES2-EGFP- SP-B-C/T 1580 and evaluate their expressions in 293T cells, for the further study of relationship between polymorphism of suffactant protein B （SP-B） gene and bronchopuhnonary dysplasia （BPD）. Methods The eukaryotic plRES2-EGFP-SP-B-C/T 1580 expression vectors were constructed by gene recombination, and identified by gene sequencing. The recombinant expression vectors were transfected into 293T cells by lipo- fectamine2000. The expression of green fluorescence protein in 293T cells was observed by fluorescence mi- croscopy. The mRNAs and proteins of SP-B-C/T 1580 were tested and identified by reverse transcription- polymerase chain reaction-restriction fragment length polymorphism（RT-PCR-RFLP） and western blot. Re- sults Two recombinant plasmids contained the complete eDNA of SP-B with the same sequence as in gene bank. The base of SP-B 1580 gene of plRES2-EGFP-SP-B-C 1580 was C, that of plRES2-EGFP-SP-B-T 1580 was T. After being transfected into 293T ceils, highly efficient expression of SP-B-C/T 1580 gene was detected at mRNA and protein levels. Conclusions The plRES2-EGFP-SP-B-C/T 1580 eukaryotie cell ex- pression vectors were successfully constructed.

关 键 词：支气管肺发育不良 遗传学 基因表达 遗传载体 肺表面活性物质相关蛋白质B 遗传学 

分 类 号：R363[医药卫生—病理学]