黑腹果蝇抗真菌肽Drs及其同系物基因的原核表达与蛋白纯化研究  

Research on Expression in E.coli and Protein Purification of the Antifungal Peptide Drs and Isoforms from Drosophila melanogaster

作　　者：杨婉莹[1] 温硕洋[2] 邓小娟[1] 叶明强[1] 夏庆友[3] 曹阳[1] 黄亚东 

机构地区：[1]华南农业大学动物科学学院蚕丝科学系,广州510642 [2]华南农业大学资源环境学院昆虫学系,广州510642 [3]西南大学蚕学与生物技术学院农业部蚕桑学重点实验室,重庆4007162 [4]曁南大学医药生物技术研究开发中心,广州510632

出　　处：《中国农业科学》2007年第2期391-398,共8页Scientia Agricultura Sinica

基　　金：国家重点基础研究发展计划(973计划)项目(2005CB121000);国家自然科学基金项目(30370716);广东省自然科学基金项目(010294)(032256)(04020553);广东省科学技术计划重点项目(2003C104042)

摘　　要：【目的】Drosomycin(Drs)是从黑腹果蝇(Drosophila melanogaster)中鉴定发现的对丝状真菌有广谱抗性的抗真菌肽因子,此外,在黑腹果蝇基因组中,还存在另外6条Drs同系物的基因序列,对它们推导的氨基酸序列具有与Drs相同的保守位点和CSαβ模体结构。为鉴定Drs及其6个同系物的抗性功能差异,研究其分子结构与功能的关系,需要对这些同系物基因进行克隆表达,获得有生物学活性的纯化样品。【方法】本文采用PCR分段互补合成的方法准确地获得了Drs同系物基因,并克隆至pET-3C载体上,转化到宿主菌E.coli Origami(DE3)中进行诱导表达,表达产物经阳离子CM SepharoseTM Fast Flow层析、凝胶过滤Sephacryl S-100 High Resolution层析和包涵体的复性及Sephadex G-25脱盐等纯化处理后,进行抑菌活性检测。【结果】经纯化的7个Drs同系物的短肽样品,除Drs-lI外,其它的Drs同系物样品对供试真菌水稻立枯丝核菌(Rhizoctonia solani)均有较强的抑制作用。【结论】表明本研究中使用的纯化及复性方法对具有多个二硫键的小分子多肽是比较有效的。[Objective] Drosomycin （Drs） is an insect antifungal peptide from Drosophila melanogaster which has broad activity against filamentous fungi. Besides Drs, there are six gene sequences of its isoforms in the genome of Drosophila melanogaster The deduced acid amino sequences of them have the same conservative sites and the CSafl motif. To identify the functional divergence of Drs and its six isoforms and to study the relation between their molecular structure and function, the genes of seven isoform were expressed and the expression products with biological activity were purified. [ Method ] In this paper, the gene sequences of Drs isoforms were synthesized by two steps of PCR, cloned to the pET-3C vector and expressed in the E.coli Origami （DE3） by the induction of IPTG. The expression products were purified by Cation exchange chromatography with CM SepharoseTM Fast Flow and gel filtration chromatography with Sephacryl S-100 High Resolution. The pooled samples were treated to retrieve the dimensional structure and then loaded to Sephadex G-25 to desalt. The samples were used to determine antifungal activity. [Result] The results showed that the purified peptides of Drs isoforms, not including Drs-lI, had obvious activity against the tested fungus, Rhizoctonia solani. [ Conclusion ] The method of purification and retrieving were effective for the small peptide with disulfide bonds.

关 键 词：黑腹果蝇 抗真菌肽 Drosomycin 同系物 表达 蛋白纯化 

分 类 号：Q78[生物学—分子生物学]