342bp人骨形态发生蛋白2成熟肽基因的克隆表达  

Cloning and pronucleus expression of the 342bp cDNA gene of hBMP2 mature peptide

作　　者：俞莉敏[1] 杨昀焯[1] 吴镇权[1] 李继云[1] 

机构地区：[1]北京大学深圳医院,广东深圳518036

出　　处：《现代中西医结合杂志》2008年第21期3250-3252,共3页Modern Journal of Integrated Traditional Chinese and Western Medicine

摘　　要：目的探讨克隆人骨形态发生蛋白2(hBMP2)成熟肽的最精简的基因序列并原核表达方法。方法剔除所有信号肽和中间前肽的非必需基因序列,以RT-PCR方法克隆扩增出hBMP2的342 bp的成熟肽基因片段,将其与原核表达载体PBV220酶切后相连接并测序,构建PBV220-hBMP2的真核基因原核表达系统,在DH5α大肠杆菌下扩增并温度诱导表达hBMP2蛋白质,SDS-PAGE检测表达蛋白。结果RT-PCR扩增出hBMP2的成熟肽基因342 bp片段与Genbank公布的序列完全一致,且经DNA电泳和蛋白电泳鉴定,构建PBV220-hBMP2原核表达系统可导入DH5α大肠杆菌,并在温度诱导下表达出约16 kD的蛋白条带,诱导培养4 h后的蛋白表达量达到最高峰。结论成功扩增342 bp的成熟肽hBMP2基因片段能在原核大肠杆菌上有效表达生产hBMP2蛋白质。Objective It is to approach the method of amplify and clone the 342bp cDNA gene of human bone morpho- genetic protein - 2 （ h.BMP2 ） mature peptide and then express pronuclearly. Methods After all the unnecessary gene sequence of signal peptide and middle prepeptide was eliminated, only the 342 bp mature peptide gene of hBMP2 was amplified by RT - PCR. Then the gene was sequenced and was cloned into PBV220 vector to construct PBV220 - hBMP2 pronuclear expression system for eukaryon gene. The recombinant plasmid PBV220 - h.BMP2 was transformed into E. COLI DH5a and incubated at 42 ℃ to express encoded protein- hBMP2, and then the expressed protein was analyzed with SDS- PAGE. Resuits The 342bp mature peptide gene of hBMP2 was amplified and sequenced exactly in comparison with the reported sequence in Genebank. With the identification of DNA electrophoresis and SDS- PAGE, PBV220 - hBMP2 pronuclear expression system could be constructed and transformed into E. COLI DH5α successfully. After incubated at 42 ℃, it could express about 16 kD hBMP2 protein, and the expression amount of protein achieved most at 4 - hour induction. Conclusion The 342 bp mature peptide gene of h.BMP2 can be cloned successfully and expressed effectively in E. COLI DH5α.

关 键 词：人骨形态发生蛋白 基因克隆 基因表达 大肠杆菌 

分 类 号：R596.1[医药卫生—内科学]