Apidaecin型抗菌肽在毕赤酵母菌中的基因工程表达  

Expression and Identification of Apidaecin in Pichia Pastoris

作　　者：施文[1] 马瑞[1] 周建业[2] 陈莉娅[1,3] 马媛媛 黄慧敏[1] 张小凤[1] 易根云[1] 李志强[2] 

机构地区：[1]兰州大学口腔医学院,甘肃兰州730000 [2]西北民族大学甘肃省口腔疾病研究重点实验室培育基地西北民族大学口腔医学国家民委重点实验室西北民族大学口腔医学院,甘肃兰州730000 [3]兰州大学附属第一医院,甘肃兰州730000 [4]陇南市第一人民医院,甘肃陇南742500

出　　处：《口腔医学研究》2017年第5期471-474,共4页Journal of Oral Science Research

基　　金：国家自然科学基金项目(编号:31360124;31560159)

摘　　要：目的;利用基因工程的方法,在毕赤酵母菌(Pichia patoris,P.pastoris)中成功表达Apidaecin型抗菌肽。方法:利用PCR技术在Apidaecin基因N端插入EcoR I酶切位点、GST标签(并连接DDDDK肠激酶位点),且在C端插入终止密码子和Not I酶切位点,后将上述片段扩增后与表达载体pPICZαA连接,构建重组表达载体pPICZαA-GST-Apidaecin,并测序鉴定;将pPICZαA-GST-Apidaecin重组质粒电转至P.pastoris X33中并进行发酵表达;表达产物经GST亲和层析纯化后进行SDS-PAGE凝胶电泳鉴定,EK肠激酶切除GST标签后进行抑菌实验。结果:SDS-PAGE凝胶电泳结果显示GST-Apidaecin融合蛋白表达的产物条带位于分子量约为28KD处,与理论分子量一致;抑菌实验表明,用EK肠激酶切除GST融合标签后,Apidaecin型抗菌肽对大肠杆菌有明显的抑菌活性。结论:Apidaecin型抗菌肽在P.pastoris菌中得到成功表达并具有抑菌活性。Objective：To express biologically active Apidaecin in pichia pastoris（P.pastoris）and detect its antibacterial activity.Methods：The EcoR I restriction sites and GST tag which was connected with DDDDK enterokinase site were inserted to the N-terminus of the Apidaecin gene by polymerase chain reaction（PCR）.A termination codon and Not I restriction sites were inserted at the C-terminus.The amplified fragments were connected to the expression vector pPICZαA and the recombinant expression vector pPICZαA-GST-Apidaecin was established and the sequence was identified.The pPICZαA-GST-Apidaecin was electrotransfected into P.pastoris X33 and was fermented.The expression products were purified and the molecular weight was appraised by SDS-PAGE gel electrophoresis.The antibacterial activity was tested after the GST was excised by EK kinase.Results：SDSPAGE gel electrophoresis showed that the molecular weight of the product was about 28 KD which was identical with the theoretical molecular weight.After removal of GST fusion tag by enterokinase,the antibacterial activity of Apidaecin to E.coli BL21 was significant.Conclusion：The antimicrobial peptides of Apidaecin typecan successfully expressed in P.pastoris and had significant antibacterial activity.

关 键 词：抗菌肽 Apidaecin 毕赤酵母菌 

分 类 号：Q78[生物学—分子生物学]