大鼠肺表面活性蛋白C基因的克隆及原核表达

临床儿科杂志 ›› 2014, Vol. 32 ›› Issue (2): 183-185.

大鼠肺表面活性蛋白C基因的克隆及原核表达

唐炜，卢红艳，高楚楚

江苏大学附属医院（江苏镇江　212000）

收稿日期:2013-08-30 出版日期:2014-02-15 发布日期:2014-02-15

Cloning and prokaryotic expression of rat surfactant protein C in E.coli　

TANG Wei, LU Hongyan, GAO Chuchu

(Department of Pediatrics, The Affiliated Hospital of Jiangsu University, Zhenjiang 212000, Jiangsu, China)

Received:2013-08-30 Online:2014-02-15 Published:2014-02-15

摘要/Abstract

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。

Abstract: 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 epithelial cell A549 and the impact of SP-C on proliferation, differentiation and apoptosis of AECIIs in future.

唐炜，卢红艳，高楚楚. 大鼠肺表面活性蛋白C基因的克隆及原核表达[J]. 临床儿科杂志, 2014, 32(2): 183-185.

TANG Wei,LU Hongyan,GAO Chuchu . Cloning and prokaryotic expression of rat surfactant protein C in E.coli　[J]. , 2014, 32(2): 183-185.

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