基于多拷贝鳜鱼β-防御素基因的重组毕赤酵母菌株构建  

Construction of Recombinant Pichia pastoris Strains Based on Multi-Copy Siniperca chuatsβ-Defensin Gene

作　　者：吕星星 陶妍[1,2] 谢晶 钱韻芳[1,2] LYU Xing-xing;TAO Yan;XIE Jing;QIAN Yun-fang(Coll,of Food,Sci.&Technol.,Shanghai Ocean Uni.,Shanghai 201306;Shanghai Engirt.Res.Ctr.of Aqua-Product Processing&Preservation,Shanghai 201306)

机构地区：[1]上海海洋大学食品学院,上海201306 [2]上海水产品加工及贮藏工程技术研究中心,上海201306

出　　处：《微生物学杂志》2021年第1期16-24,共9页Journal of Microbiology

基　　金：国家“十三五”重点研发计划资助项目(2016YFD0400106)。

摘　　要：鳜鱼β-防御素(Siniperca chuatsiβ-defensin,ScBD)作为一种重要的蛋白质免疫因子,在其抵御病原微生物侵染的过程中起到了重要的作用,被认为是开发天然抗菌剂的理想候选者。ScBD的初级结构含信号肽和成熟肽,后者由43个氨基酸残基组成,决定了其生物学活性。通过同源建模法预测了ScBD的空间结构,显示其具有β-片层、伸展链和无规则卷曲等构象单元;3对特有的二硫键作为维持其空间结构的关键性次级键,保障了ScBD具有稳定的生物学活性。为了解决前期研究中重组ScBD的低表达量问题、探索目的基因拷贝数对转录水平的影响,使用同尾酶法构建了ScBD基因的二拷贝和四拷贝表达盒载体pPICZαA-ScBDn(n=2或4),分别电转至毕赤酵母X 33后成功筛选到各种重组菌株。qRT-PCR测定结果表明:含一拷贝、二拷贝和四拷贝表达盒载体的重组菌株的基因组中ScBD基因的拷贝数分别为1.19×10^(5)、2.56×10^(5)和5.29×10^(5),证明载体所含的表达盒数目与嵌合进毕赤酵母基因组中的ScBD基因拷贝数之间存在正相关关系,也由此证明了多拷贝表达盒载体的构建是提高目的基因拷贝数和转录水平的有效方法。结果为进一步确定ScBD基因拷贝数与蛋白质表达量之间的关系提供参考。Siniperca chuatsiβ-defensin(ScBD),as an important protein immune factor,plays an important role in the process of resisting infection of pathogenic microorganisms to S.chuatsi,also it is considered to be an ideal candidate for the development of natural antibacterial agents.The primary structure of ScBD contains signal peptide and mature peptide,the latter is composed of 43 amino acid residues,responsible for its biological activity.In this study,the spatial structure of ScBD was predicted through homologous modeling method,showing that it included some conformational elements,possessingβ-sheet,extension chain and random coil etc.Three pairs of unique disulfide bond as crucial secondary bond maintained the stability of spatial structure and ensured the ScBD to have biological activity.In order to solve the low expression problem of the recombinant ScBD,and explore the effect of target gene copy number on transcription level,the present study constructed recombinant vectors containing double-copy and quadruple-copy expression cassettes(pPICZαA-ScBDn(n=2 or 4))adopted isocaudomers method;respectively after transferred them electrically into competent Pichia pastoris X-33,the recombinant strains were screened out successfully.The results based on the real-time quantitative PCR(qRT-PCR)showed that the ScBD gene copy numbers in recombinant genenome contained single-copy,double-copy and quadruple-copy expression cassettes vectors were 1.19×10^(5),2.56×10^(5),and 5.29×10^(5) respectively,had proved that there existed a positive correlation between the expression cassette number and the ScBD gene copy number;it also proved that the construction of multi-copy expression cassettes vector was an effective method to increase the copy number of target gene transcription level.The results of this study have laid a good foundation for further determining of the relationship between ScBD gene copy number and protein expression level.

关 键 词：鳜鱼β-防御素 空间结构 多拷贝表达盒 毕赤酵母 重组菌株 

分 类 号：Q93-33[生物学—微生物学]