【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.更多还原

【Abstract】 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.更多还原