山羊β-防御素表达载体的构建及产物的活性鉴定

doi:10.11924/j.issn.1000-6850.2014-0969

中国农学通报 ›› 2015, Vol. 31 ›› Issue (2): 40-44.doi: 10.11924/j.issn.1000-6850.2014-0969

所属专题：畜牧兽医

山羊β-防御素表达载体的构建及产物的活性鉴定

康建锋,刘桂琼,刘胜敏,姜勋平

(华中农业大学动物科技学院,武汉 430070）

收稿日期:2014-04-04 修回日期:2014-04-04 接受日期:2014-05-30 出版日期:2015-03-19 发布日期:2015-03-19
通讯作者: 康建锋
基金资助:
国家现代肉羊产业技术体系建设项目(CARS39-1A)

Expression Vector Construction and Antibacterial Activity Characterization of Caprine β-defensin

Kang Jianfeng, Liu Guiqiong, Liu Shengmin, Jiang Xunping

(College of Animal Sciences and Technology of Huazhong Agricultural University, WuHan 430070)

Received:2014-04-04 Revised:2014-04-04 Accepted:2014-05-30 Online:2015-03-19 Published:2015-03-19

摘要/Abstract

摘要： 对山羊GBD-1进行载体构建，转到毕赤酵母中高效表达并对表达产物的生物学活性进行鉴定，为进一步研究防御素抗菌机理打下基础。根据GenBank中山羊β-防御素的CDS区序列，设计含有特殊酶切位点的引物扩增到目的片段，将该片段克隆到真核表达载体pPICZαA中，构建重组表达质粒pPICZαA/GBD-1，电转化到毕赤酵母中进行诱导表达。测序结果表明扩增的目的片段为114 bp，编码38个氨基酸，经Tricine-SDS-PAGE电泳验证在4.3 KD处有目的蛋白；作抑菌实验发现，重组蛋白对大肠杆菌、金黄色葡萄球菌有明显的抑菌效果。山羊防御素成功地在毕赤酵母中诱导表达，为进一步研究GBD-1的蛋白纯化以及抗菌机理打下基础，并为利用基因工程方法生产防御素提供理论依据。

关键词: 秸秆还田, 秸秆还田

Abstract: This study was to construct the vector, shift in the Pichia pasroris eith high expression, and identify antibacterial activity characterization of the product, the results could help further research on the antibacterial mechanism. According to the CDS sequences for caprine β-defensin in GenBank, the author designed a pair of specific primers with the restriction enzyme cutting site to get the sequence, cloned it to the expression vector pPICZαA, contained the recombinant plasmid pPICZαA/GBD-1, and transformed into pichia pasroris by electroporation to induction expression. Sequence analysis showed that the amplicon 114 bp encoding a polypeptide of 38 amina acids, the molecular mass about 4.3 KD was expressed by using the Tricine-SDS-PAGE electrophoresis analysis, the recombinant protein had antibacterial activity and killed E.coli and Sta.aureus. The caprine β-defensin was successfully expressed in Pichia pastoris, which laid a foundation for further study of GBD-1 in the antibacterial mechanism and preparing the antibodies, and provide a theoretical basis for genetic engineering in the production of defensins.

康建锋,刘桂琼,刘胜敏,姜勋平. 山羊β-防御素表达载体的构建及产物的活性鉴定[J]. 中国农学通报, 2015, 31(2): 40-44.

Kang Jianfeng,Liu Guiqiong,Liu Shengmin and Jiang Xunping. Expression Vector Construction and Antibacterial Activity Characterization of Caprine β-defensin[J]. Chinese Agricultural Science Bulletin, 2015, 31(2): 40-44.

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山羊β-防御素表达载体的构建及产物的活性鉴定

Expression Vector Construction and Antibacterial Activity Characterization of Caprine β-defensin

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