【摘要】 以尼罗罗非鱼血液mRNA反转录获得编码罗非鱼Hsp70完整cDNA,重组构建罗非鱼真核表达载体pPIC9K/rtHsp70;在毕赤酵母Pichia pastoris KM71中表达重组罗非鱼的热休克蛋白70(rtHsp70),对表达上清液进行SDS-PAGE及Western blot分析;采用亲和层析、HiTrap Desalting预装柱脱盐纯化目的蛋白。结果表明:克隆至pMD载体上的基因与目的基因完整编码区序列完全一致;诱导表达上清液经SDS-PAGE及Western blot分析,上清液中蛋白条带大小与目的蛋白相对分子质量大小一致;每升诱导上清液可以获得约2 mg纯化蛋白。本研究中获得的rtHsp70毕赤酵母工程菌及纯化的rtHsp70为后期rtHsp70-肽疫苗研究奠定了基础。更多还原

【Abstract】 The recombinant cDNA of Tilapia Hsp70 was amplified from blood mRNA and inserted into vector pMD18-T.Sequencing revealed that the cDNA was subcloned into expression vector pPIC9K.The recombinant vector was transformed into the yeast Pichia pastoris KM71 via electroporation after sequencing.The transforming positive clones were screened by PCR and rtHsp70 in culture supernatant induced by methanol was identified by Western blot.Protein purification by the affinity chromatograph and gel filtration showed the sequence of rtHsp70 DNA was correct.It was a protein with relative molecular mass of 70 000 in the culture supernatant by SDS-PAGE and Western blot analysis.Two 2 mg purified rtHsp70 can be obtained in per liter expression supernatant,and Pichia pastoris engineer bacteria of rtHsp70 expression and purified protein were gained.The study is paved the way for further study of using rtHsp70 for immunologic adjuvant in peptide-vaccine application.更多还原