马属抗菌肽hepcidin在酵母系统中的表达与鉴定  

Expression and identification of equine Hepcidin in Pichia pastoris

作　　者：宋楠[1] 吴培星[2] 张继瑜[2] 徐玲 孙茜胜 李纯玲 伏小平[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州畜牧与兽药研究所,甘肃兰州730050 [3]北京养元兽药有限公司,北京101300 [4]北京天之泰生物科技有限公司,北京100085

出　　处：《甘肃农业大学学报》2011年第1期5-9,13,共6页Journal of Gansu Agricultural University

摘　　要：根据GenBank上马属抗菌肽hepcidin的氨基酸序列,设计并合成全长279bp的hepcidin基因,将该基因插入pPICZαA中构建真核表达载体,PCR鉴定的阳性质粒电转化毕赤酵母菌X-33,用不同浓度Zeocin筛选高拷贝阳性重组菌,甲醇诱导表达,表达上清液经超滤膜纯化后,利用SDS-PAGE电泳和抑菌试验进行鉴定.结果表明:PCR检测和基因测序表明hepcidin基因合成正确,成功构建真核表达载体pPICZαA-hepcidin;SDS-PAGE电泳显示,诱导表达hepcidin蛋白分子质量约10ku,超滤纯化后蛋白浓度达177.8mg·L-1;抑菌结果表明,该表达产物对金黄色葡萄球菌、枯草芽孢杆菌、无乳链球菌均有较好的抗菌活性.According to the sequences published in GenBank,a DNA sequences of equine hepcidin was designed and synthesized.Then,the target gene was cloned into the Pichia pastoris expression vector pPICZαA to construct the recombinant plasmid.After confirmed by PCR detection,positive plasmids were electroporated into the yeast strain X-33,the high copy inserts were selected by different dose of Zeocin.The expression level of the protein was detected by SDS-PAGE and the experiment of antimicrobial circles.The results showed that the analysis by SDS-PAGE indicated that the expression of hepcidin had a molecular weight of 10ku,and highest expression level of protein reach to 177.8mg/L.The recombinant hepcidin displayed significant antibacterial activity against Staphylococcus aureus,Bacillus subtilis and Streptococcus agalactiae.

关 键 词：抗菌肽hepcidin 毕赤酵母 表达 

分 类 号：S852.6[农业科学—基础兽医学]