人TIMP—3cDNA的克隆,表达及其抗血管生成作用

从人新鲜的胎盘组织中提取总ＲＮＡ，以ＲＴ－ＰＣＲ法获取了人组织金属蛋白酶抑制－３成熟蛋白的ｃＤＮＡ。序列分析表明，ＴＩＭＰ－３成熟蛋白含有１８８个氨基酸残基，其中１２个半胱氨酸残基在ＴＩＭＰ家族中高度保守。将人ＴＩＭＰ－３ｃＤＮＡ插入含７启动子的质粒ｐＥＴ－２４构建表达质粒ｐＥＴ－ＴＩＭＰ３，转化大肠杆菌ＢＬ２１，筛选表达菌株ＢＬＴＩＭＰ３。

Cloning and Expression of the cDNA Encoding Human Tissue Inhibitor of Metalloproteinase 3 and Its Inhibition on Angiogenesis

We have isolated the cDNA encoding the mature tissue inhibitor of metalloproteinase 3 (TIMP 3) by using RT PCR method with total RNA extracted from human fresh placenta .The result of sequencing indicated that the mature TIMP 3 polypeptide contained 188 aa residues, including 12 conserved cysteinyl residues in the TIMP family. The expression plasmid pET TIMP 3 was constructed by inserting TIMP 3 cDNA into plasmid pET 24(a ) containing T7 promoter and transformed into E.coli BL21(DE3).An expression strain BL TIMP 3 was selected. SDS PAGE analysis revealed that the human TIMP 3 protein was highly expressed and accumulated up to above 30% of the total bacterial proteins in the form of inclusion body after induced by 1 mmol/L IPTG for 3-5 h.The partially purified and renatured TIMP 3 can significantly inhibit angiogenesis as shown by CAM assay.