人C-型利钠肽-血清白蛋白融合蛋白质的构建及在毕赤酵母中的分泌表达

目的构建重组表达人C-型利尿钠肽(CNP)-人血清白蛋白(HSA)融合蛋白质的毕赤酵母。方法重叠PCR拼接CNP(400 bp)-HSA(1 800 bp)成融合基因,双酶切后克隆至表达载体pPIC9K中。电穿孔法转染毕赤酵母菌KM71,摇瓶培养分泌表达。结果融合基因约为2 200 bp,序列测定正确。SDS-PAGE分析相对分子质量约为80 000,Western blot和RIA鉴定显示其为CNP与HSA的杂合分子。结论实现了HSA-CNP融合蛋白质在毕赤酵母中的分泌表达。

Secretive expression of the fusion protein HSA-CNP in Pichia pastoris

Purpose To develop the recombinant expression of fusion protein human serum albumin(HSA)-type C natriutetic peptides(CNP)in Pichia pastoris.Methods The fusion gene of HSA(about 1 800 bp)-CNP(about 400 bp)was prepared with overlapping PCR to spang joint HSA genes and CNP genes.After digesting with EcoRⅠ and NotⅠ,the fusion gene was cloned into an expression vector pPIC9k.The plasmid of pPIC9K/HSA-GCSF was constructed and transformed into P.pastoris KM71 by electroporation.With the methanol solution as a inducer,the fusion protein was expressed from the recombinant P.pastoris KM71.Results The fusion gene of HSA-CNP was about 2 200 bp by electrophoresis and the nuclear acid sequence of the gene was correct.The fusion protein with about 80 000 that secreted in the recombinant P.pastoris KM71 was made of CNP and HSA by Western blot analysis or by the CNP radioimmunoassay.Conclusion The fusion protein HSA-CNP was successfully expressed in Pichia pastoris.