【摘要】 目的将甲型流感病毒M2蛋白胞外区(M2e)基因与人血清白蛋白(HSA)基因融合,以构建高效分泌表达的毕赤酵母菌株。方法通过RT-PCR扩增HSA基因,构建pPICZα-HSA质粒,将人工合成的M2e序列与pPICZα-HSA质粒分别经BstBI和KpnI酶切消化后连接,构建真核表达载体pPICZα-HSA/M2e,线性化后电转化毕赤酵母X-33。用PCR法筛选Zeocin抗性阳性的克隆,SDS-PAGE和Western印迹法筛选高表达HSA/M2e菌株。结果经RT-PCR法克隆的HSA基因序列与GenBank登录的cDNA序列一致,构建的pPICZα-HSA/M2e真核表达载体,经电转化获得Zeocin抗性阳性的克隆。SDS-PAGE和Western印迹分析证实了甲醇诱导的培养基上清中含有HSA/M2e,分子量约为68kD。结论成功构建了分泌表达重组HSA/M2e融合蛋白的毕赤酵母菌株,为进一步研究其生物学活性奠定了基础。更多还原

【Abstract】 Objective To fuse the gene of M2e with that of HSA in order to construct a P.pastoris yeast strain with high-efficient expression of HSA/M2e.Methods The gene of HSA was amplified by RT-PCR and the plasmid of pPICZα-HSA was constructed.M2e artificial synthesized was digested with Eco81I and KpnI,then ligated to the same sites of pPICZα-HSA and obtained the expression vector pPICZα-HSA/M2e.The recombinant vector was linearized and introduced into P.pastoris X-33 by electroporation.Pichia pastoris secreting HSA/M2e were obtained to extract genomic DNA of transformed X-33 and perform PCR.SDS-PAGE and Western blot was used to screen high expressed that HSA/M2e engineering bacteria and to identify HSA/M2e in culture supernatant induced by methanol.Results The sequence of HSA obtained by RT-PCR was identical with that published on GenBank.Recombinant expression vector was constructed.SDS-PAGE and Western blot analysis showed that HSA/M2e was successfully expressed in the culture medium with an apparent molecular weight of 68 kD.Conclusions Pichia pastoris yeast strain with high-efficient expression of HSA/M2e is successfully constructed,providing a basis for further study of its bioactivity.更多还原