| 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。 |
| 关键词: 家蝇抗菌肽(MDAP-2） 毕赤酵母 分泌表达 抑菌活性 |
| DOI： |
| 投稿时间：2015-07-23修订日期：2015-09-24 |
| 基金项目: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） |
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| Eukaryotic Expression of a Novel Antimicrobial Peptide Gene (MDAP-2) from Musca domestica Larvae and Its Antibacterial Activity |
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| (College of Animal Acience and Technology) |
| Abstract: |
| 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. |
| Key words: Musca domestica MDAP-2 Pichia pastoris secretory expression antibacterial activity |
