毕赤酵母组成型表达载体的构建及报告蛋白表达评价  

Assessment of construction of a constitutive expression vector in Pichia pastoris using an NucA reporter gene

作　　者：张永正[1] 温俊柳[1] 王燕[1] 魏海婷[1] 涂健[1] 祁克宗[1] 

机构地区：[1]安徽农业大学研究生院,安徽合肥230036

出　　处：《中国生物制品学杂志》2013年第10期1379-1383,共5页Chinese Journal of Biologicals

基　　金：国家"863计划"(2006AA10Z320);安徽省教育厅研重点项目(KJ2011A122)

摘　　要：目的构建毕赤酵母(Pichia pastoris)组成型分泌表达载体,并表达报告蛋白葡萄球菌核酸酶(staphylococcus nuclease,SNase,NucA),以评价其表达外源蛋白的能力。方法从巴斯德毕赤酵母GS115基因组中PCR扩增毕赤酵母甘油醛-3-磷酸脱氢酶启动子(glyceraldehyde-3-phosphate dehydrogenase promoter,pGAP)片段,克隆至载体pPIC9K中,构建组成型分泌表达载体pGHKα;从金黄色葡萄球菌基因组中PCR扩增编码报告蛋白金黄色葡萄球菌NucA成熟肽的序列nucA,克隆至载体pGHKα中,构建重组表达质粒pGHKα-nucA,经SacⅠ和BglⅡ依次酶切线性化后,电转化至毕赤酵母GS115中;PCR及TB-D平板法筛选阳性重组酵母菌;Tricine-SDS-PAGE检测表达产物;琼脂扩散法检测重组酵母菌表达上清液的核酸酶活性。结果组成型分泌表达载体pGHKα经PCR鉴定,证明构建正确;重组表达质粒pGHKα-nucA经PCR及测序鉴定,证明nucA基因片段正确插入载体pGHKα中;重组酵母菌GS115/pGHKα-nucA经PCR及TB-D平板法检测证明构建成功;表达的目的蛋白相对分子质量约为17 000,表达量约占上清总蛋白的42%,且具有显著的核酸酶活性。结论成功构建了毕赤酵母组成型分泌表达载体pGHKα,其能正确表达报告蛋白NucA,且表达的蛋白能分泌到细胞外,表达效率高,为异源蛋白在毕赤酵母中的安全高效表达奠定了基础。Objective To construct a constitutive secretion expression vector and express staphylococcus nuclease （SNa- se, NucA ） as a report protein to evaluate its ability in expression of foreign protein in Pichia pastoris. Methods The glyceraldehyde-3-phosphate dehydrogenase promoter（pGAP） fragment was amplified from the genome of P. pastoris GS115 and cloned into vector pPIC9K to construct a constitutive secretion expression vector pGHKα. The nucA gene encoding NucA mature peptide was amplified from the genome of Staphylococcus aureus and inserted into vector pGHKct. The constructed recombinant plasmid pGHKct-nucA was linearized with Sac I and Bgl Ⅱ , then transformed to P. pastoris GSll5 by electrotransformation. Positive recombinants were screened by PCR and TB-D plate assay. The expressed product was identified by Tricine-SDS-PAGE. The nuclease activity of expression supernatant of recombinant P. pastoris was detected by metachromatic agar-diffusion assay. Results PCR proved that the constitutive secretion expression vector pGHKo~ was constructed correctly. PCR and sequencing of recombinant plasmid pGHKct-nucA proved that nucA gene was inserted into pGHKo~ correctly. PCR and TB-D plate assay proved that recombinant P. pastoris GS115/pGHKct-nucA was established successfully. The expressed recombinant protein, with a relative molecular mass of about 17 000, contained about 42% of total protein in supernatant, and showed obvious nuclease activity. Conclusion Constitutive secretion expression vector pGHKα was constructed successfully, and the report protein NucA was expressed highly in P. pastoris and secreted outside the cells. It laid a foundation of safe and high expression of interest protein in P. pastoris.

关 键 词：毕赤酵母 甘油醛-3-磷酸脱氢酶启动子 组成型表达 葡萄球菌核酸酶 

分 类 号：Q782[生物学—分子生物学] Q786