重组人血管内皮生长因子165b在毕赤酵母中的表达及纯化  

Expression of vascular endothelial growth factor 165b in Pichia pastoris and purification of expressed product

作　　者：戴惠云[1] 朱瑞宇[1] 雷楗勇[1] 侯颖[1] 陈蕴[1] 金坚[1] 

机构地区：[1]江南大学药学院,江苏无锡214122

出　　处：《中国生物制品学杂志》2013年第7期966-968,973,共4页Chinese Journal of Biologicals

摘　　要：目的在毕赤酵母中表达重组人血管内皮生长因子(recombinant human vascular endothelial growth factor,rhVEGF)165b(rhVEGF-165b),并进行纯化。方法 PCR扩增hVEGF165b基因,插入毕赤酵母表达载体pPIC9k,构建重组表达质粒pPIC9k-VEGF165b,SalⅠ酶切线性化后,电击转化毕赤酵母GS115,甲醇诱导表达。表达产物经Ni-NTA sephrose镍柱纯化后,进行Western blot鉴定。结果重组表达质粒pPIC9k-VEGF165b经双酶切和测序,证明构建正确;表达的rhVEGF165b蛋白相对分子质量约为23 000,纯化后纯度达90%以上,具有人VEGF的抗原性。结论成功在毕赤酵母中表达了rhVEGF165b蛋白,纯化的蛋白纯度较高,为进一步研究其生物学功能奠定了基础。Objective To express human vascular endothelial growth factor 165b（VEGF165b） in Pichia pastoris and purify the expressed product.Methods VEGF165b gene was amplified by PCR and cloned into vector pPIC9k.The constructed recombinant plasmid pPIC9k-VEGF165b was linearized with Sal I and transformed into P.pastoris strain GS115 by electroporation for expression under induction of methanol.The expressed product was purified by Ni-NTA sephrose column and identified by Western blot.Results Recombinant plasmid pPIC9k-VEGF165b was constructed correctly as proved by restriction analysis and sequencing.The expressed rhVEGF165b,with a relative molecular mass of about 23 000,reached a purity of more than 90% and exhibited the antigenicity of VEGF.Conclusion The rhVEGF165b protein was successfully expressed in P.pastoris and reached a high purity after purification,which laid a foundation of further study on its biological function.

关 键 词：血管内皮生长因子165b 毕赤酵母 表达 纯化 

分 类 号：Q786[生物学—分子生物学]