根据毕赤酵母Pichia pastoris表达系统对于密码子的偏嗜性,将去除信号肽的猪α干扰素(PoIFN-α)基因重新设计改造并合成一段新的核苷酸序列,置于毕赤酵母菌α因子分泌信号的DNA序列后,构建成pPICZαC-PoIFN-α分泌型重组表达载体,电转化进入野生型毕赤酵母菌X-33中,经ZeocinTM抗性筛选后获得大量多拷贝重组子,SDSPAGE和Wester-blot分析结果表明,所获得的重组子能够分泌表达出相对分子质量约为19 000的PoIFN-α特异蛋白,其有效蛋白表达量较高达54.105 mg/L.经Vero-VSV系统测定,PoIFN-α效价达到5.87×107 U/L.试验将PoIFN-α成熟肽全基因密码子进行改造,并实现了其在毕赤酵母菌表达系统中的高效分泌表达.%According to the codon metatropism of Pichia pastoris, the porcine interferon alpha (PoIFN-α)gene, in which the sequence encoding signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae,was cloned into P. pastoris expression vector pPICZαC. The recombinant plasmid pPICZαCPoIFN-α was then transformed into P. pastoris X-33 cells by electroporation, and stable multicopy recombinant P. pastris strains were selected by ZeocinTM resistance. A great quantity of multiple-inserted recombinants were obtained. SDS-PAGE and Western-blot assays of culture broth from a methanol-induced expression strain demonstrated that recombinant PoIFN-α, about 19 000 proteins,were secreted into the culture medium at the concentration of 54. 105 mg/L. The potency of PoIFN-α was up to 5.87 × 107 U/L by Vero-VSV systems measurement. This experiment changed the sequence coding of PoIFN-α by the codon metatropism of P. pastoris and the results proved that this change was feasible.
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