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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。
The complete cDNA of Hsp70 encoding tilapia was obtained by reverse transcription of blood mRNA of Nile tilapia, and the eukaryotic expression vector pPIC9K / rtHsp70 of tilapia was constructed by recombination. The heat shock protein of recombinant tilapia was expressed in Pichia pastoris KM71 70 (rtHsp70). The supernatant was analyzed by SDS-PAGE and Western blot. The target protein was purified by affinity chromatography and HiTrap Desalting pre-column desalting. The results showed that the gene cloned into pMD vector was identical to the complete coding sequence of the target gene. SDS-PAGE and Western blot analysis showed that the size of the protein band in the supernatant was consistent with the relative molecular mass of the target protein Approximately 2 mg of purified protein was obtained per liter of supernatant. The rtHsp70 Pichia pastoris obtained in this study and the purified rtHsp70 laid the foundation for the later rtHsp70-peptide vaccine research.