摘要
目的 :构建血管基膜衍生多功能肽克隆和原核表达载体 ,并对血管基膜衍生多功能肽氨基酸序列进行空间结构分析预测。方法 :用人源性IgG3上游铰链区连接肽连接的Tumstatin的 2个功能区片段 ,即血管基膜衍生多功能肽 (VB MDMP) ,利用合成的 3条长引物片段 ,进行PCR扩增。克隆到 pUC19载体 ,酶切和测序鉴定。亚克隆构建原核表达载体pGEX 4T 1 VBMDMP ,IPTG诱导表达 ,聚丙烯酰胺凝胶电泳鉴定表达产物。用GlutathioneSepharose 4B层析柱进行了亲和层析鉴定。将VBMDMP序列输入计算机 ,利用Antheprot软件分析。 结果 :血管基膜衍生多功能肽 (VBMDMP)基因经限制性内切酶酶切和测序鉴定 ,其大小和核苷酸序列正确 ,与设计完全一致。获得了原核表达融合蛋白GST VBMDMP。Anthep rot分析显示 ,人IgG3上游铰链区连接后的Tumstatin的 2个功能区域能自由伸展。 结论 :成功构建了血管基膜衍生多功能肽克隆和原核表达载体 ,并对其进行了空间结构分析。
Objective: To construct cloning and prokaryotic expression vector of vascular basement membrane-derived multifunctional peptide and to analyze its space conformation. Methods: Vascular basement membrane-derived multifunctional peptide (VBMDMP) sequence obtained by polymerase chain reaction was cloned into vector pUC19. We also subcloned it into prokaryotic expression vector pGEX-4T-1. The recombinant was confirmed by restriction endonuclease digestion and auto-sequencer. pGEX-4T-1-VBMDMP was transformed into E. coli JM109 0.1 mmol/L IPTG can induce high expression of GST-VBMDMP. GST-VBMDMP was obtained by Glutathione Sepharose 4B columns.The space conformation of VBMDMP was predicted by using computer program Antheprot. Results: The recombinant VBMDMP gene confirmed by restriction endonuclease digestion and auto-sequencer was consistent with sequence we designed. The high expression of GST-VBMDMP was induced by 0.1 mmol/L IPTG in E.coli JM109. GST-VBMDMP was highly purified. The analysis of software Antheprot demonstrated that the two domains of tumstatin linked by upper hinge region of IgG3 can extend itself freely. Conclusion: The cloning and prokaryotic expression vector was successfully constructed.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
2003年第3期185-189,共5页
Chinese Journal of Cancer Biotherapy
基金
湖南省卫生厅重点科研项目 (2 0 0 1 Y0 5 5 )
湖南省教育厅科研项目 (99C2 0 2 )
