摘要
目的：克隆人内皮抑素基因， 获得初步表达产物，为进一步研究打下基础。方法：用RT-PCR方法从中国汉族人正常肝组织扩增出内皮抑素cDNA，T/A插入pUCm-T载体，测序，再插入表达载体 pKPL-3a，转化大肠杆菌 POP2136，42℃诱导表达，SDS-PAGE分析表达蛋白。结果：中国人内皮抑素cDNA开放阅读框架全长552bp，编码184个氨基酸， 其85位谷氨酸和98位天冬氨酸密码子的第三位碱基出现改变，但编码的氨基酸不变。结论：构建了中国人内皮抑素的克隆载体和表达载体，初步表达了约20kD人重组内皮抑素。
Objective: In order to pave a way to cure human tumors, we plan to construct expression vector of human endostatin and produce recombinant endostatin from E.coli. Methods: According to the sequence of XVIII collagen gene , cDNA of human endostatin was obtained from the normal liver tissue of a Chinese Han man by RT-PCR . Endostatin cDNA was cloned into PUCm-T vector by T/A cloning technique and sequenced . Then endostatin cDNA was inserted into the PKPL-3a expression vector and the recombined plasmid was transformed into the host strain E.coli POP2136. The endostatin expression was induced by raising the temperature to 42 ℃ and analyzed by SDS-PAGE. Results: Endostatin cDNA sequence encoded 184 amino acid residues. The variation in bases encoding Glu85 and Asp98 was discovered in Chinese. Recombinant human endostatin was expressed as inclusion body and the host expressed a 20kD protein which was confirmed with SDS-PAGE analysis. Conclusion: The cloning and expression vectors of human endostatin were successfully constructed in our laboratory. Expression vector expressed a 20 kD recombined human endostatin at 42℃.
出处
《重庆医科大学学报》
CAS
CSCD
2001年第2期119-121,共3页
Journal of Chongqing Medical University
