摘要
目的构建高氧下早产大鼠肺表面活性蛋白C基因原核表达质粒,实现其在大肠杆菌中的表达。方法孕21 d SD早产大鼠,生后12 h暴露于85%高氧中,7 d后处死,取肺组织,提取RNA,并合成cDNA,进行PCR扩增,克隆进pMD18-T载体,经过酶切和测序验证,成功构建原核表达载体pET-28a(+)-sp-c,重组质粒在大肠杆菌BL21菌株中经IPTG诱导表达,SDS-PAGE及Western blotting分析检测表达产物。结果经酶切和测序验证,成功构建pET-28a(+)-sp-c质粒,高效表达出相对分子质量21 000的融合蛋白。结论在大肠杆菌中成功表达SP-C重组融合蛋白,有助于研究SP-C蛋白与内质网之间的作用机制、SP-C错折叠蛋白在肺上皮A549细胞中的表达以及对AECⅡs增殖、分化及凋亡的影响。
Objectives To construct prokaryotic expression plasmid of rat surfactant protein C （sp-c） gene under hyperoxia and expression in E.coli. Methods Twenty-one-day-old SD premature rats were exposed to 85%hyperoxia 12 hours after birth. The rats were executed after 7 days and their RNA were extracted from lung and cDNA was synthesized and amplified. And then the cDNA was cloned into pMD18-T vector and confirmed by enzyme digestion and sequencing. After the prokaryotic expression vector pET-28a（＋）-sp-c was constructed, the recombinant plasmid was induced by IPTG and expressed in E.coli BL21 strain. The fusion protein was analyzed by SDS-PAGE and Western blotting. Results The pET-28a（＋）-sp-c plasmid was constructed and the fusion protein with relative molecule mass of 21000 was highly expressed. Conclusions SP-C is successfully expressed in E. coli, which can be used to study the mechanism of action between SP-C and endoplasmic reticulum, the expression of SP-C in lung epi-thelial cell A549 and the impact of SP-C on proliferation, differentiation and apoptosis of AECIIs in future.
出处
《临床儿科杂志》
CAS
CSCD
北大核心
2014年第2期183-185,共3页
Journal of Clinical Pediatrics
基金
江苏省自然科学基金(No.BK2011485)
镇江市社会发展项目(No.SH2011022)
关键词
高氧
肺发育不良
大鼠
hyperoxia
bronchopulmonary dysplasia
rat
