血管基膜衍生多功能肽基因克隆、表达及空间构象分析

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(VBMDMP)，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Glutathione Sepharose 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。Antheprot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

Cloning, Expression and Space Conformation Analysis of Vascular Basement Membrane-Derived Multifunctional Peptide

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.