猪β-防御素cDNA在枯草芽孢杆菌中的高效表达

发布时间：2019-03-03 09:39

【摘要】：【目的】猪β-防御素(β-defensin)是猪自身分泌的一种能够杀菌、调节免疫功能的小分子抗菌肽,是猪生长繁殖不可缺少的蛋白质。【方法】根据NCBI数据库中的猪防御素cDNA序列,人工合成该基因序列,同时引入相关调控序列及适当酶切位点。酶切该人工合成的DNA后,连接到表达载体p HT43中,然后转化大肠杆菌(Escherichia coli)DH5α,获得的转化子用于筛选重组表达质粒。将获得的重组表达质粒p HT43-SS-BD转化到枯草芽孢杆菌(Bacillus subtilis)WB800N中,获得基因工程菌株。使用蔗糖诱导基因工程菌表达猪β-防御素,SDS-PAGE鉴定其分子量大小,抑菌试验测定其抑菌活性。【结果】SDS-PAGE电泳结果显示在蔗糖诱导后得到约5 KDa的目的蛋白条带;平板抑菌试验结果表明,蔗糖诱导后的上清液对革兰氏阳性菌和革兰氏阴性菌都有抑菌效果,而没加蔗糖的对照组上清液无抑菌效果。【结论】该研究实现猪β-防御素cDNA在枯草芽孢杆菌中的高效表达,为解决抗菌肽来源限制及枯草芽孢杆菌的生产应用奠定了技术基础。
[Abstract]:[objective] Porcine 尾-defensin (尾-defensin) is a small antibacterial peptide secreted by pig itself, which can kill bacteria and regulate immune function. [methods] according to the cDNA sequence of porcine defensin in NCBI database, the gene sequence was synthesized artificially, and the related regulatory sequence and appropriate enzyme site were introduced at the same time. The synthesized DNA was ligated into the expression vector p-HT43, and then transformed into E. coli (Escherichia coli) DH5 伪. The transformant was used to screen the recombinant expression plasmid. The recombinant expression plasmid p HT43-SS-BD was transformed into Bacillus subtilis (Bacillus subtilis) WB800N to obtain the genetic engineering strain. Porcine 尾-defensin was expressed by sucrose-induced gene engineering bacteria, its molecular weight was identified by SDS-PAGE, and its bacteriostatic activity was determined by bacteriostatic test. [results] SDS-PAGE electrophoresis showed that the target protein band was about 5 KDa after sucrose induction. The results of plate bacteriostatic test showed that the supernatant induced by sucrose had antibacterial effect on both Gram-positive bacteria and Gram-negative bacteria. [conclusion] the high expression of porcine 尾-defensin cDNA in Bacillus subtilis was achieved in this study. It lays a technical foundation for solving the limitation of antibacterial peptides and the production and application of Bacillus subtilis.
【作者单位】：成都大学;四川华德生物工程有限公司;
【基金】：成都市科技惠民技术研发项目(2015-HM01-00176-SF)
【分类号】：S828

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