人溶菌酶基因的克隆及其在毕赤酵母中的表达

目的：利用巴斯德华赤（Pichia pastoris）酵母真核表达系统，构建真核表达载体pPIC9K与人溶菌酶基因（humanlysozyme,hLY）的重组质粒pPIC9K-hLY，表达出具有活性的人溶菌酶。方法：此项研究在本实验室构建的人溶菌酶（hLY）基因与pUC19的重组质粒pUC19-hLY的基础上，通过设计一对引物，用多聚酶链式反应（PCR）扩增出人溶菌酶全基因片段后，将其克隆到巴斯德毕赤酶母分泌型表达载体pPIC9K中，构建重建质粒pPIC9K-hLY，经Bg1Ⅱ酶切线性化后，用电穿孔法将其转入毕赤酵母细胞内。通过G418筛选，选到的高拷贝转化子经PCR检测人溶菌基因与毕赤酵母染色体是否稳定整合，阳性克隆经甲醇诱导进行表达。结果：序列测定，经PCR扩增后的人溶菌酶基因与献发表的序列相比，缺失4个碱基。我们决定采用重组PCR法予以纠正，经序列测定结果正确。用PCR检测，人溶菌酶基因与毕赤酵母染色体稳定整合。用甲醇诱导表达并以SDS－PAGE分析，在Mr为15000左右出现一条蛋白带，表达量约占上清总蛋白的0．47。Western Blot证实表达产物具有天然人溶菌酶的抗原性。用黄色小球菌平板溶圈法鉴定表态产物具有人溶菌酶的活性。结论：成功的构建了重组质粒pPIC9K-hLY并在毕赤醇母中表达了人溶酶基因。

Cloning and expression of human lysozyme gene in Pichia pastoris

AIM To construct a recombinant plasmid between vector pPIC9K and human lysozyme gene. Active human lysozyme was expressed. METHODS Recombinant plasmid pUC19 hLY was amplified by PCR. It was cloned into expression plasmid pPICPK and it's lined fragment was transformed into electroporated Pichia pastoris strains. Recombinant of human lysozyme and Pichia pastoris was obtained. Was used G418 to screen positive clone, and PCK to identify integration of gene of human lysozyme into genome of Pichia pastoris. The positive clone was expressed in Pichia pastoris strains. RESULTS Sequence of human lysozyme we got different from published sequence. In oredr to get the correct sequence, we divise two primers to correct the mutant gene. The sequence was determined again. This time the result showed that the sequence was in accordance with the published sequence. Gene of human lysozyme have been integrated into genome of Pichia pastoris with PCR identified. The expression of human lysozyme was induced by Methylotrophic. An anticipated 15 000 protein band appeared on SDS PAGE gel and Western blot and amounted to 0.47 of the total screened protein. The expressed raw product exhibited the antigenicity and activity for human lysozyme. CONCLUSION Recombinant plasmid pPIC9K hLY has been successfully constructed and expressed in Pichia pastoris .