人成骨蛋白-1成熟肽基因5’端的修饰及其在大肠杆菌中的表达

Modification of N-terminal of mature peptide of human osteogenic protein-1 and expression in Escharichia coli

目的：对人成骨蛋白－１（ＯＰ－１）成熟肽基因进行修饰、克隆并在大肠杆菌中表达、检测，为ＯＰ－１的进一步纯化、复性、诱骨活性的研究以及未来的临床应用奠定基础。方法：在不改变氨基酸的前提下，用ＰＣＲ的方法对ＯＰ－１的成熟肽Ｎ端序列加以修饰，将编码成熟肽的ｃＤＮＡ３’端０．４２ｋｂ基因片段克隆于温控型大肠杆菌表达载体ｐＢＶ２２０，受控于ＰＬＰＲ启动子。重组子以大肠杆菌ＤＨ５α为宿主细胞进行温度诱导表达。用鼠抗牛天然ＢＭＰｓ单抗对表达产物进行ＥＬＩＳＡ检测和ＷｅｓｔｅｒｎＢｌｏｔｉｎｇ确证。结果：工程菌经４２℃、５ｈ诱导后，在ＳＤＳ－ＰＡＧＥ上出现一条新的蛋白带，分子量在１．６×１０４ｕ左右，约占菌体总蛋白的１９．８％。主要以包涵体形式存在的表达产物经初步纯化后，可获得纯度较高的重组人成骨蛋白－１成熟肽（ｒｈＯＰ－１）。表达产物的ＥＬＩＳＡ检测和ＷｅｓｔｅｒｎＢｌｏｔｎｇ确证确均呈阳性反应。结论：表达产物即为目的蛋白，使ｈＯＰ－１成熟肽基因５’端的修饰及其在大肠杆菌中的表达在国内第一次获得成功。

Aim: Cloning and expressing of human osteogenic protein-1(OP-1). Methods: The 0.42kb DNA fragment encoding mature peptide, which was modified at 5' terminal without changing amino acid residue, were obtained with BamH I and EcoR I from the plasmid pGEM/OP-1m M. The fragment was inserted into expression vector pBV220 following ATG that was controlled by P LP R. The recombinant plasmid pBV220/OP-1m M was transformed into E. coli DH5 α and induced at 42℃ to express encoded proteins. Results: SDS-PAGE revealed a new foreign protein band near 1.61×10 4 after 5h induction. The recombinant mature peptide of hOP-1 in inclusion bodies made up 19.8% of total bacteria protein and was purified partially. Furthermore, by ELISA and Western Bloting, McAb aqainst bovine nature BMPs had postive reactions with the expressed peptide. Conclusions: It was proved that the gene products was rhOP-1, the modification of N-terminal of matare peptide of human osteogenic protein-1 and expression in Escharichia coli is the first success in our country.