一种新型家蝇幼虫抗菌肽基因(MDAP-2)的真核表达及抑菌活性检测  

Eukaryotic Expression of a Novel Antimicrobial Peptide Gene(MDAP-2) from Musca domestica Larvae and Its Antibacterial Activity

作　　者：李文婷[1] 闫恕[1] 张丹丹[1] 贾博岩[1] 唐艳[1] 孔令聪[1] 裴志花[1] 刘树明[1] 马红霞[1,2] 

机构地区：[1]吉林农业大学动物科学技术学院,长春130118 [2]动物生产及产品质量安全教育部重点实验室吉林农业大学,长春130118

出　　处：《中国兽药杂志》2015年第11期11-16,共6页Chinese Journal of Veterinary Drug

基　　金：国家自然科学基金资助项目(31140026;31572574;31502121);吉林省世行贷款农产品质量安全项目(2011-Y05)

摘　　要：利用毕赤酵母表达系统表达新型家蝇抗菌肽MDAP-2基因(Musca domestica antimicrobial peptide,MDAP-2),为其作为新型抗菌物质应用于临床提供实验依据。根据已有的MDAP-2的氨基酸序列,采用PCR技术扩增获得MDAP-2基因,构建真核重组表达质粒pPIC9K-MDAP-2,将线性化的重组质粒电击转入毕赤酵母(P.pastoris)感受态GS115细胞内,将PCR鉴定为阳性的重组酵母菌进行甲醇诱导表达,并采用Tricine-SDS-PGAE检测表达产物的大小,经镍柱亲和层析纯化获得纯度较高的重组蛋白,采用管碟法检测重组蛋白的生物学活性。结果显示,重组质粒pPIC9K-MDAP-2获得高效表达,蛋白分子量为7.8 kD,管碟法检测重组蛋白对大肠杆菌与沙门氏菌均具有体外抑菌活性。MDAP-2基因的成功表达为进一步研究表达产物的生物学及免疫学活性奠定基础。Pichia pastoris expression system was applied to express a novel antimicrobial peptide MDAP- 2 gene from Musca domestica,which provide experimental evidence for MDAP- 2 used as a new antimicrobial substance in clinical application.Based on the amino acid sequence of antibiotical peptide MDAP-2,which was previously identified,polymerase chain reaction（PCR） technique was performed to amplified the MDAP-2 gene,and eukaryotic recombinant expression plasmid pPIC9K-MDAP —2 was constructed,subsequently the recombinant plasmid was digested used to transformed the Pichia pastoris（GS115 cells） through electroporation.The positive recombinant pPIC9k-MDAP-2 which detected by PCR induced by addition of methanol,using tricine- SDS-PAGE detected the weight of recombinant MDAP- 2 protein.The recombinant MDAP- 2 was purified using Ni — NTA HisTrap FF crude column chromatography and the bacteriostatic activity of the recombinant purified MDAP- 2 protein was assessed by tube plate method.The test results showed that the molecular weight of recombinant MDAP — 2 protein was about 7.8 kD,MDAP- 2 had in vitro antibacterial activity against Escherichia coli and Salmonella pullorum.The MDAP- 2 gene was expressed successfully in Pichia pastoris expression system which provide reference for further research in the biological and immunologic activities of MDAP- 2.

关 键 词：家蝇抗菌肽(MDAP-2) 毕赤酵母 分泌表达 抑菌活性 

分 类 号：S852.6[农业科学—基础兽医学]