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目的:对恶性疟原虫pfs25的多拷贝基因重组菌株在毕赤酵母中进行表达分析,研究基因拷贝数对Pfs25蛋白表达量的影响。方法:构建重组质粒pAO815-αpfs25,利用BglⅡ-BamHⅠ同尾酶的特点,将基因表达框AOX1-αpfs25-AOX1(TT)插入到单拷贝表达质粒,依次重复,构建多拷贝重组质粒pAO815-(αpfs25)n,线性化后电转化毕赤酵母GS115,用MD平板筛选并进行表达分析。结果:构建得到1、2、3、4、5、6、7、8、10、12和14个pfs25基因拷贝的重组菌株,8拷贝pfs25基因的重组菌株表达量最高。结论:成功得到了pfs25基因的多拷贝表达重组菌株,经分析,多拷贝pfs25基因的目的蛋白表达量与其拷贝数并不呈线性正相关。
OBJECTIVE: To analyze the expression of Pfs25 gene in Pichia pastoris by analyzing the expression of Pfs25 gene in Pichia pastoris. METHODS: The recombinant plasmid pAO815-αpfs25 was constructed and inserted into the single copy expression plasmid AOX1-αpfs25-AOX1 (TT) according to the characteristics of the BglⅡ-BamHⅠ homologous to the same enzyme. The multiple copies of the recombinant plasmid pAO815- (αpfs25 ) n. After linearization, Pichia pastoris GS115 was electroporated and screened by MD plate and analyzed by expression. Results: The recombinant strains with 1, 2, 3, 4, 5, 6, 7, 8, 10, 12 and 14 copies of pfs25 gene were constructed and the recombinant strains with 8 copies of pfs25 gene were the highest. Conclusion: The multi-copy expression recombinant pfs25 gene was successfully obtained. The expression of the target protein of the multi-copy pfs25 gene was not positively correlated with its copy number.