Apidaecin型抗菌肽在毕赤酵母菌中的基因工程表达

doi:10.13701/j.cnki.kqyxyj.2017.05.002

口腔医学研究 ›› 2017, Vol. 33 ›› Issue (5): 471-474.DOI: 10.13701/j.cnki.kqyxyj.2017.05.002

Apidaecin型抗菌肽在毕赤酵母菌中的基因工程表达

施文¹,马瑞¹,周建业^2*,陈莉娅^1,3*,马媛媛⁴,黄慧敏¹,张小凤¹,易根云¹,李志强²

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

收稿日期:2016-11-30 出版日期:2017-05-20 发布日期:2017-05-26
通讯作者: 周建业,E-mail:zhoujianye@gmail.com
陈莉娅:E-mail:chenchy@lzu.edu.cn
作者简介:施文(1990~ ),男,湖北武汉人,硕士在读,主要从事口腔内科及基因工程研究。
基金资助:
国家自然科学基金项目(编号:31360124,31560159)

Expression and Identification of Apidaecin in Pichia Pastoris.

SHI Wen¹, MA Rui¹, ZHOU Jian-ye^2*, CHEN Li-ya^1,3*, MA Yuan-yuan⁴, HUANG Hui-min¹, ZHANG xiao-feng¹, YI Geng-yun¹, LI Zhi-qiang².

1. School of Stomatology, Lanzhou University, Lanzhou 730000, China; 2. Key Laboratory of Oral Diseases of Gansu Province, Northwest University for Nationalities, Key Laboratory of Stomatology of State Ethnic Affairs Commission, Northwest University for Nationalities, School of Stomatology, Northwest University for Nationalities, Lanzhou 730000, China; 3. The First Hospital of Lanzhou University, Lanzhou 730000, China; 4. The First Hospital of Longnan, Longnan 742500, China.

Received:2016-11-30 Online:2017-05-20 Published:2017-05-26

摘要/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。

关键词: 抗菌肽, Apidaecin, 毕赤酵母菌

Abstract: 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: SDS-PAGE gel electrophoresis showed that the molecular weight of the product was about 28KD 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.

中图分类号:

R780.1

施文,马瑞,周建业,陈莉娅,马媛媛,黄慧敏,张小凤,易根云,李志强. Apidaecin型抗菌肽在毕赤酵母菌中的基因工程表达[J]. 口腔医学研究, 2017, 33(5): 471-474.

SHI Wen, MA Rui, ZHOU Jian-ye, CHEN Li-ya, MA Yuan-yuan, HUANG Hui-min, ZHANG xiao-feng, YI Geng-yun, LI Zhi-qiang.. Expression and Identification of Apidaecin in Pichia Pastoris.[J]. Journal of Oral Science Research, 2017, 33(5): 471-474.

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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.

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