猪β防御素2成熟肽在酵母中的表达  

Expressing of porcine β-defensin-2 mature peptide in the yeast

作　　者：胡翰[1,2] 于宾宾[1,2] 何启盖[1,2] 

机构地区：[1]华中农业大学动物医学院 [2]农业微生物国家重点实验室,武汉430070

出　　处：《微生物学报》2011年第5期704-709,共6页Acta Microbiologica Sinica

基　　金：国家生猪现代产业技术体系(nycytx-009)~~

摘　　要：【目的】将猪β防御素2成熟肽基因片段正确整合到酵母基因组染色体上,从而得到稳定的猪β防御素2成熟肽的毕赤酵母表达株。实现猪β防御素2成熟肽的表达。【方法】首先参考酵母偏爱密码子,设计3段引物序列,利用PCR技术扩增得到β防御2成熟肽基因,构建了重组质粒pPIC9k-GST-pBD-2和pPIC9k-pBD-2。将线性化的重组质粒电转化到毕赤酵母KM71细胞中。最后筛选得到酵母阳性克隆,通过不断调节表达条件,实现猪β防御素2成熟肽的表达。【结果】将GST-pBD-2基因序列和pBD-2基因序列分别成功整合到酵母KM71基因组中,重组毕赤酵母工程菌构建成功;重组酵母蛋白GST-pBD-2和PBD-2都成功获得了表达;PBD-2成熟肽表达上清对猪霍乱沙门氏菌弱毒株C500有一定的抑制作用。【结论】获得表达pBD-2成熟肽的酵母菌株,本实验是用真核细胞表达pBD-2成熟肽的一次探索,为后续大量表达pBD-2成熟肽方法的研究打下了基础。[Objective]To construct the recombinant Pichia pastoris KM71 in which Porcine β-defensin-2（pBD-2）mature peptide could be stably expressed and the antimicrobial effect was evaluated.[Method]According to the requirement of yeast codon bias for protein expression,three pairs of primers were designed for development of SOE-PCR to amplify the genes coding pBD-2 mature peptide.The resultant genes were cloned into pPIC9k and pPIC9k-Gultathione S Transferase（GST）vectors to yield the recombinant expressing vector,pPIC9K-pBD-2 and pPIC9k-GST-pBD-2.The two recombinant plasmids were linearized,followed by transformation with Pichia pastoris KM71 cells to produce the positive clones of recombinant yeasts.The expression conditions were continuously optimized to produce mature peptide of pBD-2.[Result] Gene fragments for GST-fusion PBD-2 and pBD-2 alone were amplified and integrated into genomic chromosome of the yeast KM71,respectively,and the recombinant yeasts were obtained;GST-fusion pBD-2 peptide and singular pBD-2 peptide were successfully expressed.The mature peptide of pBD-2 showed a certain inhibition effect on the growth of the Salmonella choleraesuis C500-strain.[Conclusion]The pBD-2 was expressed successfully and casted a light for massive production of the pig defensin,a new antimicrobial agent.

关 键 词：猪β防御素2 成熟肽 真核表达 活性实验 

分 类 号：S852.4[农业科学—基础兽医学] S828[农业科学—兽医学]