Apidaecin型抗菌肽在毕赤酵母菌中的基因工程表达

发布时间：2018-11-18 21:34

【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。
[Abstract]:Objective: to express Apidaecin type antimicrobial peptides successfully in Pichia pastoris (Pichia patoris,P.pastoris) by genetic engineering. Methods: PCR technique was used to insert EcoR I restriction site at the N end of Apidaecin gene, GST tag (and ligation of DDDDK enterokinase site), and terminal codon and Not I restriction site to be inserted at C terminal. The above fragments were amplified and ligated with the expression vector pPICZ 伪 A. The recombinant expression vector pPICZ 伪 A-GST-Apidaecinwas constructed and sequenced. The recombinant plasmid of pPICZ 伪 A-GST-Apidaecin was transformed into P.pastoris X33 and expressed by fermentation, and the expressed product was purified by GST affinity chromatography and identified by SDS-PAGE gel electrophoresis. EK enterokinase was removed from GST label for bacteriostasis test. Results: the results of SDS-PAGE gel electrophoresis showed that the product bands expressed by GST-Apidaecin fusion protein were located at the molecular weight of 28KD, which was consistent with the theoretical molecular weight. The bacteriostatic test showed that Apidaecin antibacterial peptide had obvious bacteriostatic activity against Escherichia coli after GST fusion label was removed by EK enterokinase. Conclusion: Apidaecin type antimicrobial peptide was successfully expressed in P.pastoris strain and had bacteriostatic activity.
【作者单位】：兰州大学口腔医学院;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;兰州大学附属第一医院;陇南市第一人民医院;
【基金】：国家自然科学基金项目(编号:31360124,31560159)
【分类号】：Q78

【相似文献】