家蝇幼虫溶菌酶1基因的克隆与真核表达及表达产物抑菌活性的检测

Cloning,eukaryotic expression and antibacterial activity detection of the lysozyme-1 gene from Musca domesticalarvae

采用PCR扩增获得家蝇溶菌酶1基因(Musca domesticalysozyme-1,MdL-Ⅰ),构建真核重组表达质粒pPIC9K-MdL-Ⅰ。将线性化的重组质粒电击转入巴斯德毕赤酵母感受态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-Tris-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-MdL-Ⅰ获得高效表达,蛋白质的分子质量为14.2ku,管碟法检测重组蛋白对大肠杆菌、雏沙门菌均具有体外抑菌活性。本研究为利用毕赤酵母表达系统规模化生产具有抑菌活性的MdL-Ⅰ蛋白奠定了基础。

The lysozyme-1gene（MdL-Ⅰ）from Musca domestica larvae was amplified by polymerase chain reaction,and the eukaryotic recombinant expression plasmid pPIC9K-MdL-Ⅰ was constructed.Subsequently,the digested recombinant plasmid was transform into Pichia pastoris（GS115cells）through electroporation.The positive recombinant pPIC9K-MdL-Ⅰ detected by PCR was induced by addition of methanol.The molecular weight of recombinant MdL-Ⅰ protein was detected by using Tricine-Tris-SDS-PAGE.The recombinant MdL-Ⅰ was purified using Ni-NTA HisTrap FF crude column chromatography and the bacteriostatic activity of the purified recombinant MdL-Ⅰ protein was assessed by tube plate method.The test results showed that the molecular weight of recombinant MdL-Ⅰ protein was about 14.2ku in size,MdL-Ⅰ had in vitro antibacterial activity against Escherichia coli and Salmonella pullorum.The study result laid the foundation for using Pichiaexpression system to mass production of MdL-Ⅰ protein with antibacterial activity.