人骨形态发生蛋白－1（hBMP－1）成熟肽的基因克隆及其在大肠杆菌中的表达

采用ＰＣＲ方法从ＢＭＰ－１的ｃＤＮＡ中克隆了ＢＭＰ－１成熟肽的编码基因，删除了ＢＭＰ－１前体分子Ｎ端的信号肽序列和Ｃ端的其他序列。用ＨｉｎｄⅢ消化１．３Ｋｂ的ＰＣＲ产物，将０．８４和０．４６Ｋｂ两片段分别克隆到ｐＵＣ载体中进ＤＮＡ序列分析。分别酶切回收ＥｃｏＲ－ＨｉｎｄⅢ和ＨｉｎｄⅢ－ＢａｍＨⅠ两目的基因片段，与大肠杆菌表达载体ｐＢＶ－２２０进行退火连接，使插入基因受控于Ｐ＿ＲＰ＿Ｌ启动子。将含有ＢＭＰ－１成熟肽编码基因重组表达质粒ｐＢＶＢＭＰ－１转化大肠杆菌宿主细胞进行温度诱导表达。结果表明，经４２℃热诱导后，大肠杆菌能以包涵体形式表达ＢＭＰ－１成熟肽，其分子量约为５２ｋＤａ。

The Gene Cloning and Expression of Mature Peptide of Bone Morphogenetic Protein-1 in Escherichia coli

Bone morophogenetic protein-1(BMP-1) plays an important role in the bone formation and theinteractions with other BMPs. In this research,BMP-1 cDNA was modified by deleting most of C terminus,leader sequence and nontranslation region,and the gene coding mature peptide of N terminus of BMP-1 wascloned by polymerase chain reaction. The initiation codon ATG and termination codon TGA were added to5' and 3' termini of 1.3 kb coding gene respectively,which was ideritified by DNA sequencing.Inconstruction of expression plasmid of BMP-1,E. coli vector , pBV-220 ,was used.The sequenced codinggene was inserted into MCS of pBV-220 under the control of the promote P_RP_L. After temperatureinduction,E. coli containing pBVBMP-1 plasmid could expressed recombinant ptoduct in the form ofinclusion body. SDS-PAGE showed that the molecular weight of the recombinant mature peptide of BMP-1 was about 52 KDa,which was demonstrated by Western blot.