人血小板衍生生长因子B在大肠杆菌中的表达及对骨组织修复的影响

Molecular cloning and expression in Escherichia coli of human platelet derived growth factor B chain mature peptide gene and its effects on bone tissue repair

目的:为研究人血小板衍生生长因子BB(PDGF-BB)在大肠杆菌中的表达及其在治疗骨折愈合、骨质疏松等疾病中的作用,克隆了PDGF-B的成熟肽基因并进行了序列分析。方法:从脐静脉内皮细胞提取总RNA,经反转录聚合酶链式反应(RT-PCR)扩增出包含全长编码人PDGF-B基因cDNA,再以此为模板,用特异引物进行PCR扩增出PDGF-B成熟肽基因;将目的基因连接到载体pGEM-T上,进行序列测定。之后将此片段克隆到表达载体pQE30,构建成表达质粒pQE30-PDGFB。结果:重组质粒经酶切及PCR鉴定,证实PDGF-B成熟肽基因克隆成功,序列正确。结论:PDGFB基因的成功克隆和原核表达载体的构建,为促进骨修复功能研究及基因工程生产奠定了基础。

AIM:To clone and sequence the human platelet derived growth factor B(PDGF B)chain mature peptide gene for the purpose of protein expression in Escherichia coli and exploring its effects on the diseases such as fracture healing and osteoprosis. METHODS:To amplify the PDGF B cDNA(721bp) from total RNA of human vascular endothelial cells with reverse transcription polymerase chain reaction(RT PCR),then the mature peptide fragment(328bp) was amplified from the RT PCR results with polymerase chain reaction(PCR)and was cloned into the vector pGEM T,sequence analysis was done subsequently. The mature peptide was cloned into the expression vector pQE 30 and the expression recombinant plasmid pQE 30 PDGFB was constructed. RESULTS:Identification by restriction enzyme digestion and PCRdemonstrated that the human PDGF B chain mature peptide gene was cloned successfully and the sequence was precise either. CONCLUSION:The successful clone of PDGFB chain gene and construction of prokaryotic expression vector lay the foundation for the further study of bone repair and reconstruction and protein expression of PDGF BB.