人血管抑制因子vasostatin片段在大肠杆菌中的融合表达、纯化及活性检测

Expression,purification and bioactivity assay of human vasostatin fragments in E.coli BL21(DE3)

目的：克隆表达人vasostatin片段,纯化目的蛋白并检查活性,以研究人血管抑制因子vasostatin片段的活性区域。方法：重组构建人vasostatin片段和谷胱甘肽S转移酶(GST)融合表达质粒,转化大肠杆菌BL21 (DE3),IPTG诱导表达。通过GST亲和凝胶纯化目的蛋白,用凝血酶(Thrombin)酶切纯化产物获得单体va- sostatin片段,并采用MTT法检测其抑制内皮细胞增殖活性。结果：表达产物主要以包涵体形式存在,改变诱导条件可增加可溶性表达量,包涵体形式的表达量约占菌体蛋白总量的55%。活性试验表明,各GST-vasostatin片段和vasoatatin片段具有程度不同的抑制bFGF诱导的内皮细胞增殖的活性,其中以片段vasostatin氨基酸135-164的抑制活性最为显著。结论：人vasostatin片段中氨基酸135-164是抑制内皮细胞增殖活性较强的片段。

Objective:To study the effective domain of human vasostatin fragment,purified protein of the fragments were produced,and its biological activity was tested.Methods:The recombinant plasmids harboring glutathione S-trans- ferase(GST)gene and different human vasostatin fragment DNAs were constructed and were transformed into E.coli BL21(DE3)respectively.The fusion proteins were expressed by IPTG induction,and were purified by GST affinity chro- matography.Vasostatin fragments were released from the fusion proteins by thrombin cleavages,and the biological activi- ty was tested with MTT method.Results:The fusion proteins were expressed in inclusion body form.Changing the in- duction condition could increase the soluble fusion proteins.The expression levels of target proteins in inclusion body reached up to 55% of the total proteins.Biological activity assay showed that purified vasostatin fragments could inhibit the proliferation of endothelial cells similar to bioactivity of vasostatin,and the vasoatatin fragments consisting of amino acids 135-164 showed an obvious advantage.Conclusion:The antiangiogenic activities of human vasostatin resided in a do- main is an accessible fragment between amino acids 135-164.