中国组织工程研究

中国组织工程研究 ›› 2012, Vol. 16 ›› Issue (15): 2699-2703.doi: 10.3969/j.issn.1673-8225.2012.15.009

• 组织构建基础实验 basic experiments in tissue construction • 上一篇    下一篇

肺表面活性物质相关蛋白C原核表达载体的构建、表达及纯化★

王  静，杜  江，周细中，刘  茹，沈  蔚，王  斌   

  1. 南方医科大学珠江医院儿科，广东省广州市  510282
  • 收稿日期:2012-01-01 修回日期:2012-03-07 出版日期:2012-04-08 发布日期:2012-04-08
  • 通讯作者: 王斌，博士，硕士生导师，主任医师，南方医科大学珠江医院儿科，广东省广州市 510282
  • 作者简介:王静★，女，1985年生，山西省文水县人，汉族，南方医科大学在读硕士，医师。wuanjunwj@163.com

Construction, expression and purification of a prokaryotic expression plasmid carrying pulmonary surfactant protein C  

Wang Jing, Du Jiang, Zhou Xi-zhong, Liu Ru, Shen Wei, Wang Bin   

  1. Department of Pediatrics, Zhujiang Hospital of Southern Medical University, Guangzhou  510282, Guangdong Province, China
  • Received:2012-01-01 Revised:2012-03-07 Online:2012-04-08 Published:2012-04-08
  • Contact: author: Wang Bin, Doctor, Master’s supervisor, Chief physician, Department of Pediatrics, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, Guangdong Province, China
  • About author:Wang Jing★, Studying for master’s degree, Physician, Department of Pediatrics, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, Guangdong Province, China wuanjunwj@163.com

PDF

377

被引次数

11

摘要：

背景：研究表明肺表面活性物质蛋白基因缺陷导致肺表面活性物质蛋白的结构发生变化。早期检测肺表面活性物质的含量对于预测肺部疾病的发生意义重大。
目的：克隆人肺表面活性物质相关蛋白C(surfactant associated protein C, SP-C)基因，构建原核表达载体PET-28a/SP-C，并纯化SP-C蛋白。
方法：提取正常人肺组织总RNA，RT-PCR技术获得SP-C cDNA序列，纯化后的SP-C基因插入至中间载体PMD-18T，得到重组质粒PMD-18T-SP-C，重组质粒经过Bam HⅠ和Hind Ⅲ双酶切后纯化回收得到具有黏性末端的SP-C cDNA，将质粒PET-28a同样经过双酶切后纯化回收得到与SP-C cDNA具有相同黏性末端的质粒片段，将具有黏性末端的SP-C cDNA与PET-28a定向连接后得到重组质粒PET-28a/SP-C。然后将鉴定正确的PET-28a/SP-C重组质粒转入BL21中诱导表达。
结果与结论：酶切鉴定及核苷酸序列测序证实扩增的SP-C cDNA及其重组质粒经过Bam HⅠ和Hind Ⅲ双酶切鉴定后，在5 000~7 500 bp和250~1 000 bp处可检测到2条条带。核苷酸序列测序结果证实，质粒中插入基因长597 bp，为一开放阅读框架，与GeneBank中公布的人SP-C cDNA序列相符。Western-blot检测结果显示，纯化后的SP-C蛋白在相对分子质量约27 000处出现1条新生条带，与预期的大小一致。结果证实，实验成功克隆人SP-C基因并插入至质粒PET-28a中，构建了PET-28a/SP-C重组质粒，将其体外转化至BL21后可以表达SP-C蛋白。

关键词: 肺表面活性物质相关蛋白C, 原核表达载体, 克隆, 转化, 组织构建

Abstract:

BACKGROUND: Studies have suggested that gene defection leads to the structural change of pulmonary surfactant protein C (SP-C). Early detection of pulmonary surfactants is of great significance for prediction of occurrence of lung diseases.
OBJECTIVE: To clone human pulmonary SP-C, to construct an prokaryotic expression vector PET28a/SP-C, and to obtain purified SP-C.
METHODS: The total RNA of normal lung tissue was extracted, and SP-C cDNA was then obtained by reverse transcription-polymerase chain reaction. The purified product of SP-C cDNA was then inserted into PMD-18T vector to obtain recombinant PMD-18T/SP-C. The recombinant plasmid PMD-18T/SP-C was cut by restriction enzymes Bam HⅠ/Hind Ⅲ and then purified to become SP-C cDNA with viscous ends, as was PET28a to become a linear plasmid fragment with the same viscous ends as SP-C cDNA. The SP-C cDNA was combined with the PET-28a that had been cut by the enzymes to construct the recombinant plasmid PET-28a/SP-C. The correct PET-28a/SP-C was transformed into BL21 to induce expression.
RESULTS AND CONCLUSION: Two bands were detected at 5 000-7 500 bp and 250-1 000 bp after SP-C cDNA and its recombinant plasmid were identified after Bam HⅠ and Hind Ⅲ double digestion. The fragment length of the inserted gene was 597 bp, in consistence with the SP-C cDNA sequence announced by GeneBank. Western-blot analysis showed that the purified SP-C protein had a new band with the expected size at a relative molecular mass of about 27 000. These findings indicate that human SP-C cDNA can be correctly cloned into the PET-28a, with construction of the recombinant plasmid PET-28a/SP-C, and SP-C protein may be expressed in BL21.

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