摘要
目的 :为了解人的TGF β1基因的功能及生物学活性 ,制备具有生物学活性的TGF β蛋白。方法 :用基因重组技术构建pQE30 TGF β1原核表达载体 ,并在M1 5大肠杆菌中进行表达 ,利用Ni NTA琼脂柱纯化TGF β1单体 ,通过复性得到双体蛋白 ,利用MTT方法对复性的蛋白进行活性测定。结果 :经酶切鉴定及测序结果证明 pQE30载体上成功地插入了TGF β1成熟肽基因片段。pQE30 TGF β1原核表达载体在大肠杆菌中得到了高效表达 ,表达量约占全菌蛋白的 2 0 % ,表达得TGF β1单体蛋白经纯化后进行SDS PAGE电泳显示均得到了一条蛋白带 ,分子量为 1 5KD左右。MTT测活证明TGF β1单体蛋白经复性后具有良好的活性。结论 :pQE30 TGF β1原核表达载体的构建及重组TGF β1蛋白的制备 ,为深入了解TGF β1的生物学功能奠定了基础。
Objective:To further understand the function and biological activity of TGF-β, and to provide the production of biologically active TGF-β protein. Method:The TGF-β cDNA was cloned into a procaryotic expression vector…… pQE30 and the monomeric form of the recombinant TGF-β1 expressed in E.coli M 15 ; then Ni-NTA agarose column were used to purify the TGF-β protein. The monomeric protein was refolded and in vitro activity test was assayed by MTT. Result:With digestion of the enzymes and sequencing, we identified that the TGF-β1 matured peptide gene was inserted into procaryotic expression vector pQE30. After purified,the expression product of the plasmid in E.coli showed a single protein on SDS-PAGE, and its expression level was about 20% of the total bacterial protein. The dimeric protein, which is biologically active, was obtained by refolding. Conclusion:The construction of the recombinant plasmid and the preparation of the active protein of TGF-β1 have laid a solid foundation for further studying the function of TGF-β1.
出处
《中国矫形外科杂志》
CAS
CSCD
2003年第21期1490-1492,共3页
Orthopedic Journal of China
基金
国家 973课题资助项目 (G1 9990 542 0 4 )
