重组猪β防御素2在毕赤酵母中的表达及其鉴定  

Expression and characterization of recombinant porcine β-defensin-2 gene in Pichia pastoris

作　　者：解民[1] 仲飞[1] 李秀锦[1] 吴凌娟[1] 李凌[1] 李巍[1] 

机构地区：[1]河北农业大学动物科技学院,河北保定071001

出　　处：《中国兽医学报》2010年第8期1028-1031,共4页Chinese Journal of Veterinary Science

基　　金：河北省科技支撑计划资助项目(07220401D);河北农业大学校长基金资助项目(XZJJ2005-06)

摘　　要：根据GenBank猪β防御素2(pBD-2)的氨基酸序列,参照酵母偏爱的密码子,设计无信号肽序列的pBD-2的基因编码序列,并由公司合成。将合成的基因通过SnaBⅠ和NotⅠ位点插入到酵母表达载体pPIC9K的α因子信号肽序列下游,构建成pBD-2基因的分泌表达载体pPIC9K/pBD-2。利用电穿孔法将经SalⅠ线性化的pPIC9K/pBD-2质粒导入毕赤酵母GS115中,通过G418加压筛选得到His+Mut+表型的高拷贝转化子。经PCR检测证明pBD-2基因在毕赤酵母染色体上得到整合。阳性重组酵母经摇瓶发酵培养和甲醇诱导,用SDS-PAGE方法在发酵上清中检测到重组pBD-2的存在,说明构建重组酵母菌能够分泌性表达pBD-2。琼脂孔穴扩散法检测结果显示,pBD-2对金黄色葡萄球菌有较好的抑菌活性。Porcine β-defensin-2（pBD-2） gene encoding sequence without signal peptide was designed and synthesized according to amino acid sequence in GenBank and the biased codon usage of Pichia pastoris.The synthesized gene was inserted into pPIC9K plasmid downstream of α factor leader sequence at SnaB Ⅰ and Not Ⅰ sites to construct the recombinant secreting expression vector pPIC9K/pBD-2.The recombinant plasmids linearized by SalⅠwere transformed into yeast GS115 by electroporation.The recombinant yeast encoding pBD-2 was identified by PCR.The transformants（with His＋,Mut＋ phenotype） containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418.The results showed that pBD-2 gene was integrated into yeast genome.The pBD-2 protein was detected in culture supernatant by the fermentation in flask and induced by methanol,indicating that pBD-2 gene could secreting expression in Pichia pastorisst.Agarose diffusion assay showed that the pBD-2 has an antibacterial activity against Staphylococcus aureus.

关 键 词：猪β防御素2 毕赤酵母 分泌表达 抗菌活性 

分 类 号：S855.12[农业科学—临床兽医学]