摘要
目的 :克隆人血管发生抑制因子restin(hRS) ,在E .coli中融合表达 ,并测定其抗血管活性。方法 :用RT PCR法从中国人胎盘组织中扩增hRS基因 ,重组入pGEM T载体中并测序鉴定 ,构建原核表达载体pGEX hRS ,表达融合蛋白GST hRS。融合蛋白经亲和纯化及凝血酶切后 ,采用鸡胚绒毛膜尿囊膜试验检测其抗血管生成活性。结果 :RT PCR产物为 5 64bp ,测序结果与Genbank中胶原XV(COL15A1)的C端序列一致 ,但在 2 1位 (TCT→TCG)引起丝氨酸的同义突变 ,82位 (ACA→TCA)引起丝氨酸突变为苏氨酸。诱导表达的人GST hRS融合蛋白经凝血酶切后 ,分子量为 2 0kD ,具有抗血管生成活性。结论 :hRS的成功克隆、表达为抗血管生成治疗实体瘤的研究奠定了实验基础。
Objective: To clone human angiogenesis inhibitor restin (hRS), express fusion protein in E.Coli and determine its biological activity. Methods: Restin gene was amplified by RT PCR from Chinese human placenta tissue, then inserted into plasmid vector pGEM T and sequenced. Prokaryotic expression vector pGEX hRS was constructed and fusion protein GST hRS was expressed. After the fusion protein purified by affinity chromatography and digested by thrombin, the anti angiogenic activity of restin was tested by chicken chorio allantoic assay. Results: RT PCR product is 564 bp, the result of DNA sequencing identified the PCR product with the cDNA encoded human restin (Genbank COL15A1), but the synonymous mutation in bases encoding Ser 21 (TCT→TCG) and mutation in Ser→Thr 82 (ACA→TCA) were also discovered. The expressed protein size was 20 kD after isolated fusion protein digested by thrombin, it appeared the expressed restin had the power to inhibit angiogenesis. Conclusion: The successful cloning and expression of human restin lay the foundation for the therapy of solid tumors.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2002年第3期194-197,共4页
Chinese Journal of Cancer Biotherapy
基金
重庆市科委基金资助
