鱼腥藻PCC7120染色体基因对asr0757/alr0758的表达优化及其鉴定  

Optimized expression of chromosomal genepair asr0757/alr0758 in Anabaena sp. PCC 7120 and its identification

作　　者：陈洁[1] 陈思礼[1] 吴汇兰 

机构地区：[1]中南民族大学生命科学学院,武汉430074

出　　处：《华中师范大学学报（自然科学版）》2016年第2期263-268,共6页Journal of Central China Normal University：Natural Sciences

基　　金：国家自然科学基金项目(31001099/C190101);中央高校自然科学基金项目(CJSl3003;CJS13004);中南民族大学微生物与生物转化重点实验室项目(XJS09002);"十二五"国家级中南民族大学民族药学实验教学中心建设项目

摘　　要：NCBI预测鱼腥藻PCC7120染色体上的基因对asr0757/alr0758可能构成一个毒素-抗毒素系统.为研究其是否属于毒素-抗毒素系统家族,分别构建这两个基因的表达载体.用1.0 mM IPTG诱导重组菌表达,优化表达条件,并对表达出的蛋白进行纯化与western blot检测.SDSPAGE结果显示:asr0757与alr0758在37℃下诱导6h后,均成功诱导表达出目的蛋白asr0757(13.5KD)和alr0758(17.9KD).抗毒素表达量较好,毒素alr0758表达量较少.通过设置诱导时间梯度和IPTG浓度梯度优化毒素基因表达条件,优化结果显示:毒素基因alr0758在28℃,IPTG浓度为0.4mM,诱导10h时有最大表达量.蛋白纯化与western blot结果证实诱导表达的蛋白为目的蛋白.Chromosomal gene pair asr0757/alr0758 in Anabaenasp.PCC 7120 was predicted to belong to the toxin-antitoxin system（TAS）with homology analysis by software NCBI.To verify this,expression vectors were constructed for the above two genes,respectively.The vectors were induced by 1.0mM isopropylβ-D-1-thiogalactopyranoside（IPTG）.Then,the inducing conditions were optimized,after which the proteins were purified and identified through western blot.Result of SDS-PAGE revealed that proteins of genes asr0757/alr0758 were expressed successfully after induced for 6h at 37℃.The expression level of asr0757（13.5kD）appeared to be higher than that of alr0758（17.9kD）.Therefore,induction time gradient and IPTG concentration were optimized to improve the expression level of alr0758.Results showed that gene alr0758 obtained relatively large expression quantity after 10 hinduction at 37℃ with 0.4mM IPTG.Protein purification and western blot results showed that the expression proteins were the target proteins.

关 键 词：鱼腥藻PCC7120 asr0757/alr0758 表达 优化 鉴定 

分 类 号：Q812[生物学—生物工程]