摘要
目的:用酵母表达系统表达具有生物活性的vasostatin,并检测其抗血管生成活性。方法:将vasostatin基因克隆入酵母诱导型表达载体pPIC9K中,经过SacⅠ线性化、电转化毕赤酵母蛋白酶缺陷菌株Pichiapastoris KM17、筛选阳性克隆、PCR鉴定和DNA序列分析,获得了vasostatin基因重组到酵母染色体中的阳性重组子,阳性重组子经甲醇诱导和分离纯化,获得了重组vasostatin蛋白,CAM实验检测重组蛋白抑制血管新生活性。结果:酵母表达的重组vasostatin产量达12 mg.L-1,CAM实验表明重组vasostatin能够显著抑制新生血管生成。结论:酵母表达系统能高效表达具有抑制新生血管生成活性的重组vasostatin蛋白。
Objective： Using yeast-expressed system to express soluble and biologically active recombinant vasostatin.Methods： Vasostatin gene was cloned into expression vector pPIC9K,then linearized by Sac Ⅰ enzyme,transformed into yeast Pichia pastoris KM17 by electroporation.The transformants with vasostatin gene integration the activity of recombinant protein was tested by CAM assay were selected by G418＋ selection,then confirmed PCR and DNA sequencing.The recombinant proteins were induced to express by methanol and purified.The activity of recombinant protein was tested by CAM assay.Results： The yeast-expressed recombinant vasostatin was with a yield of 12 mg·L-1.CAM assay showed that the recombinant protein obviously inhibited the angiogenesis.Conclusion： Yeast-expressed system was successful to express recombinant vasostatin having biological activity of anti-angiogenesis.
出处
《东南大学学报（医学版）》
CAS
2011年第5期692-695,共4页
Journal of Southeast University(Medical Science Edition)
基金
江苏省自然科学基金资助项目(BK2010046
B22010074
BY2009147)
"重大新药创制"科技重大专项(2009ZX09103-675
2009ZX09102-206)
教育部博士点基金资助项目(2011CB933502)
常州市科技局资助项目(CQ20100009
CN20100016
CZ20100008)
常州市武进区科技局资助项目(WG2009007
WS201004)
