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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 angiostatin restin (hRS), express in E.coli and determine its antiangiogenic activity. Methods: The hRS gene was amplified from human placenta tissue by RT-PCR and recombined into pGEM T vector and sequenced. The prokaryotic expression vector pGEX hRS was constructed and the fusion protein GST hRS was expressed. After affinity purification and thrombolysis, the chick embryo chorioallantoic membrane assay was used to detect the anti-angiogenic activity of the fusion protein. Results: The RT PCR product was 5 64 bp. The sequencing result was consistent with the C terminal sequence of collagen XV (COL15A1) in Genbank. However, the synonymous mutation of serine was induced at position 21 (TCT → TCG) and the position 82 (ACA → TCA) Serine to threonine. After induction of expression of human GST hRS fusion protein by thrombin, the molecular weight of 20kD, with anti-angiogenic activity. Conclusion: The successful cloning and expression of hRS laid the experimental foundation for the study of antiangiogenic therapy for solid tumors.