摘要
目的运用基因重组的方法，对人转化生长因子β１（ｔｒａｎｓｆｏｒｍｉｎｇ ｇｒｏｗｔｈ ｆａｃｔｏｒ－β１）进行基因克隆，并在大肠杆菌中表达。同时观察其促进骨折愈合的作用。方法以 ｐＢＶ ２２０作为原核细胞表达载体，将 ＴＧＦ－ β１ｃＤＮＡ片段定向重组到 ｐＢＶ ２２０的多克隆位点上，构建原核细胞表达性质粒ｐＴＧＦ－ β１，并转化至大肠杆菌中进行温度诱导表达。制造兔桡骨骨折模型，经皮注射重组 ＴＧＦ－ β１，并与单纯挠骨骨折模型做对照。术后行Ｘ线和同位素骨显像检查。结果表达产物经ＳＤＳ－ＰＡＧＥ分析，ＴＧＦ－β１原核表达体系可高效表达 ＴＧＦ－ β１，其表达产物占菌体可溶性蛋白的 ２３％。用大鼠肾成纤维细胞（ＮＲＫ细胞）进行活性检测，该方法生产的 ＴＧＦ－ β１具有该蛋白的野生生物学活性。术后 Ｘ线检查，实验组骨折愈合较对照组平均超前２周；同位素核素骨显像检查，实验组与对照组差异有显著性意义（ Ｐ＜ ０ ０５）。结论 ＴＧＦ－β１原核表达体系的建立，为 ＴＧＦ－ β１的生产提供了高效的表达工程菌，其表达产物能明显促进骨折愈合。
Objective By means of the method of gene recombinant, human transforming growth factor(TGF) β_1 gene cloning was done by expressing the TGF -- β_1 in escherichia coli, its activity of enhancing fracture healing was determined. Methods The plasmid TGF -- β_1 was constructed by recovering TGF -- β_1 cDNA fragment and inserted directly into PBV 220, a prokaryotic expression vector. The recombinant plasmid was transformed into E. colt to establish the prokaryotic expression system and induced to express encoded protein. The recombinant TGF -- β_1 was injected into the established radius fracture in experimental group, and the radius fractures without TGF -- β_1 injection was used as the control group. Results Radiographic examination after operation showed that bone union occurred in 3 weeks on most of the experimental group, and bone union occurred in 5 weeks on most of the control group. There is a statistical difference between experimental and control group(P < 0. 05) in skeletal scintigraphy. Conclusion The establishment of this system provides an efficient expression cell line for producing TGF --β_1 and the recombinant TGF -- β_1 can enhance fracture healing.
出处
《中华骨科杂志》
CAS
CSCD
北大核心
1999年第12期735-738,共4页
Chinese Journal of Orthopaedics
关键词
转化生长因子Β
骨折愈合
重组蛋白质类
大肠杆菌
Transforming growth factor beta(TGF-β) Fracture healing Recombinant proteins Escherichia coli
