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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 recombinant human vascular endothelial growth factor (rhVEGF) 165b (rhVEGF-165b) in Pichia pastoris and purify it. Methods The hVEGF165b gene was amplified by PCR and inserted into the expression vector pPIC9k of Pichia pastoris. The recombinant plasmid pPIC9k-VEGF165b was constructed. The recombinant plasmid pPIC9k-VEGF165b was digested by SalⅠ and transformed into Pichia pastoris GS115 by electroporation. The expressed product was purified by Ni-NTA sephrose nickel column and identified by Western blot. Results The recombinant plasmid pPIC9k-VEGF165b was confirmed by double enzyme digestion and sequencing. The relative molecular mass of the expressed rhVEGF165b protein was about 23 000, and its purity was over 90% after purification. It has the antigenicity of human VEGF. Conclusion The recombinant protein of rhVEGF165b was successfully expressed in Pichia pastoris. The purity of the purified protein was high, which laid the foundation for further study of its biological functions.