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- 2017
Apidaecin型抗菌肽在毕赤酵母菌中的基因工程表达
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Abstract:
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
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