摘要
采用微波加热法提取金黄色葡萄球菌基因组DNA,根据NCBI上登录的肠毒素基因sek设计引物,PCR克隆selk基因,对selk基因测序并进行生物信息学分析,在此基础上构建重组表达质粒p ET-32a(+)-selk.测序结果表明本研究克隆得到具有正确编码序列的新型肠毒素selk基因;Protparam分析表明在一级结构水平上该蛋白具有较高的热稳定性;亲疏水性分析表明SEl K蛋白是一个亲水性较高的蛋白质;同源建模表明SEl K蛋白的domain B结构域缺乏传统肠毒素所具有的胱氨酸环,但SEl K蛋白domain B中β-折叠片数量比传统肠毒素更高.这些结果为进一步研究SEl K蛋白的结构与功能奠定基础.
In this study,S. aureus genomic DNA was isolated by microwave heating method. The PCR primers were designed based on the sek gene sequence published on the National Center for Biotechnology Information（ NCBI） website. The selk gene was amplified and sequenced; at the same time,the selk gene was subcloned into the p ET-32a（ ＋） vector to construct the expression plasmid p ET-32a（ ＋）-selk. Furthermore,the bioinformatic analysis of SEl K was done. The sequencing results showed that the recombinant expression plasmid p ET-32a（ ＋）-selk with correct open reading fragment was obtained. The protparam analysis showed that SEl K protein was characterized by high thermo-stability predicted from its primary structure. According to Kyte-Doolittle hydrophilicity plots,SEl K protein could be highly hydrophilic. By means of homology modeling,SEl K lacked a cystine loop structure in domain B and had more β-pleated sheet compared to classical staphylococcal enterotoxins. The results of this study could provide useful information for future research on SEl K protein structure and function.
出处
《西南民族大学学报（自然科学版）》
CAS
2016年第3期274-280,共7页
Journal of Southwest Minzu University(Natural Science Edition)
基金
国家自然科学基金项目(31371781)
四川省应用基础项目(2014JY0253)
教育部新世纪人才支持计划项目(NCET-11-0847)
关键词
金黄色葡萄球菌
肠毒素selk基因
载体构建
生物信息学分析
Staphylococcus aureus
staphylococcal enterotoxin selk gene
expression vector construction
bioinformatic analysis
