新疆家蚕抗菌肽在毕赤酵母中表达及活性研究

Molecular Cloning,Expression of Cecropin-XJ Gene from Silkworm and Antibacterial Activity in Pichia pastoris

目的:在毕赤酵母中表达新疆家蚕抗菌肽基因(Cecropin-XJ)并检测其活性。方法:根据作者实验室已克隆获得的新疆家蚕抗菌肽Cecropin-XJ基因设计引物,通过PCR方法扩增Cecropin-XJ,将PCR产物和表达载体pPIC9K用EcoRⅠ及NotⅠ双酶切,构建重组表达质粒pPIC9K-Cecropin-XJ,酶切及测序正确后,电转化到毕赤酵母GS115,对分泌表达的重组蛋白进行活性检测。结果:PCR扩增获得192 bp Cecropin-XJ,成功构建pPIC9K-Cecropin-XJ,优化诱导条件证明在pH 6的BMMY培养液中,0.5%甲醇诱导约48h后,获得的表达产物活性较强,对多种革兰氏阴性菌和阳性菌具有抗菌活性,在100℃条件下,其活性可维持100min以上。结论:新疆家蚕抗菌肽在毕赤酵母中分泌表达,为大规模发酵生产奠定了基础。

Objective：To understand Cecropin-XJ gene from silkworm better,gene cloning,expression in Pichia pastoris and biological activity detection of Cecropin-XJ was carried out in present study.Method：The gene Cecropin-XJ was amplified from the recombinant plasmid of pcDNA3-Cecropin-XJ by PCR,digested and inserted into yeast expression vector pPIC9K to construct recombinant expression plasmid pPIC9K-Cecropin-XJ.After the sequence was determined,the recombinant expression plasmid was transformed into Pichia pastoris GS115 by electroporation method.Antibacterial activity was detected.Result： The Cecropin-XJ gene of 191bp was amplified and recombinant plasmids pPIC9K-Cecropin-XJ was constructed.The pH value and methanol concentration was adjusted to optimize the expression condition.Antibacterial assays demonstrated that Cecropin-XJ had antibacterial property against Gram-positive as well as Gram-negative bacteria.In addition,the antibacterial peptide could remain its inhibition activity after treating for more than 100 min in boiled water.Conclusion： The successful cloning and expression of Cecropin-XJ in Pichia pastoris has laid a foundation for its further application.