荧光假单胞菌GcM5-1A溶血素的基因克隆及其活性研究

发布时间：2018-03-21 11:24

本文选题：荧光假单胞菌　切入点：松材线虫　出处：《青岛大学学报(自然科学版)》2017年01期 　论文类型：期刊论文

【摘要】：利用基因克隆技术探究了松材线虫携带的荧光假单胞菌(Pseudomonas fluorescens)GcM5-1A是否含有溶血素基因以及溶血素活性。根据荧光假单胞菌疑似溶血素的基因序列设计一对引物,通过PCR技术扩增得到疑似溶血素基因Hly。将该基因克隆到表达载体pET-15b上,构建了pET-15b-Hly。重组质粒pET-15b-Hly转化E.coli BL21(DE3)构建了工程菌,工程菌经过异丙基-β-D硫代半乳糖苷(IPTG)诱导后,SDS-PAGE分析表明,疑似溶血素在大肠杆菌中得到了表达。利用Ni-Sepharose 6(FF)亲和树脂纯化了重组蛋白,溶血实验显示,纯化的重组蛋白可使鸡的红细胞发生溶血,证实本研究所克隆基因确实编码溶血素,利用黑松切根苗的生测表明,重组荧光假单胞菌溶血素对黑松幼苗有致萎活性,这为深入研究松材线虫携带细菌在松材线虫病病理进程中的作用打下了基础。
[Abstract]:Whether Pseudomonas fluorescens)GcM5-1A carried by Pseudomonas fluorescens)GcM5-1A contained hemolysin gene and hemolysin activity was studied by gene cloning technique. A pair of primers were designed according to the gene sequence of suspected hemolysin of Pseudomonas fluorescens)GcM5-1A. The suspected hemolysin gene Hly. was amplified by PCR technique. The gene was cloned into the expression vector pET-15b and transformed into E. coli BL21 (DE3) by recombinant plasmid pET-15b-Hly. The recombinant strain was induced by isopropyl- 尾 -D-thiogalactoside (IPTG) and analyzed by SDS-PAGE. Suspected hemolysin was expressed in E. coli. Recombinant protein was purified by Ni-Sepharose 6 FFF affinity resin. Hemolysis test showed that the purified recombinant protein could cause hemolysis of chicken erythrocytes, which confirmed that the cloned gene of this study does encode hemolysin. The bioassay of root-cutting seedlings of Pinus tabulaeformis showed that the recombinant Pseudomonas fluorescein hemolysin had wilting activity on Pinus nigra seedlings, which laid a foundation for further study on the role of bacteria carried by pine wood nematode in the pathological process of pine wood nematode disease.
【作者单位】：青岛大学生命科学学院;
【基金】：山东省重点研发计划项目(批准号:2016GNC110024)资助
【分类号】：S763

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