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目的在毕赤酵母(pichiapastoris)中高效表达人热休克蛋白70(hHSP70),制备具有天然与hHSP70相同结构和活性的重组hHSP70(rhHSP70)。方法用PCR法自人DNA文库中克隆hHSP70的DNA,构建真核表达载体pPICZα/hHSP70。经核酸测序确证后,电转化毕赤酵母菌株X33。用PCR法筛选Zeocin抗性的转染阳性克隆,Westernblot法鉴定甲醇诱导的培养上清液中的rhHSP70,筛选高表达工程菌。结果经PCR法克隆的hHSP70DNA序列与GeneBank登录的cDNA序列一致。将hHSP70DNA定向插入pPICZα,构建pPICZα/hHSP70真核表达载体并经电转化获得Zeocin抗性的转染阳性克隆。SDSPAGE和Westernblot分析显示了甲醇诱导的培养基上清中含有rhHSP70,分子量72kD。氨基酸序列分析证实rhHSP70氨基端15个氨基酸与天然rhHSP70完全一致。rhHSP70表达量高达250mg·L-1。结论获得高效表达rhHSP70的毕赤酵母工程菌。
Aim To express human hsp70 (hHSP70) efficiently in pichia pastoris, a recombinant hHSP70 (rhHSP70) with the same structure and activity as native to hHSP70 was prepared. Methods The DNA of hHSP70 was cloned from human DNA library by PCR and the eukaryotic expression vector pPICZα / hHSP70 was constructed. After confirmed by nucleic acid sequencing, Pichia pastoris strain X33 was electroporated. Zeocin-resistant transfection positive clones were screened by PCR, and rhHSP70 in methanol-induced culture supernatant was identified by Western blot. Results The cDNA sequence of hHSP70 cloned by PCR was identical with the cDNA sequence registered by GeneBank. HHSP70DNA was inserted into pPICZαto construct eukaryotic expression vector pPICZα / hHSP70 and electroporated into Zeocin-resistant transfection positive clones. SDSPAGE and Western blot analysis showed that the methanol-induced medium contained rhHSP70 with a molecular weight of 72 kD. Amino acid sequence analysis confirmed that the amino-terminal 15 amino acids of rhHSP70 were completely identical to native rhHSP70. rhHSP70 expression up to 250mg · L-1. Conclusion Pichia pastoris engineered bacteria highly expressing rhHSP70 were obtained.