黄粉虫抗菌肽在大肠杆菌中表达条件优化及活性分析  

Expression optimization and characterization of Tenebrio molitor antimicrobiol peptides TmAMP1m in Escherichia coli

作　　者：热依汗古丽·阿里木[1] 毛新芳[1] 刘忠渊[1] 

机构地区：[1]新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室,新疆乌鲁木齐830046

出　　处：《生物工程学报》2013年第6期836-847,共12页Chinese Journal of Biotechnology

基　　金：新疆维吾尔自治区科技支疆项目(No.201291304);新疆生物资源基因工程重点实验室开放课题(No.XJDX0201-2012-01)资助~~

摘　　要：为了提高黄粉虫抗菌肽基因tmAMP1m在大肠杆菌中的表达量,研究了培养温度、诱导时间及IPTG浓度等不同条件对HIS-TmAMP1m融合蛋白表达量和活性的影响。通过Tricine-SDS-PAGE分析确定最佳表达条件,同时,通过琼脂孔穴扩散法检测其抑菌活性。结果表明,含有重组质粒的大肠杆菌在37℃,使用终浓度为0.1 mmol/L IPTG培养4 h时,融合蛋白表达量较高,可占细菌总蛋白40%以上,抗菌活性最好。用Ni2+亲和层析纯化获得较纯的融合蛋白,Western blotting分析表明其能与His单克隆抗体起特异性反应。诱导表达的融合蛋白对宿主菌生长产生一定程度抑制。融合蛋白经100℃煮沸10 h,在20℃反复冻融10次,与强酸强碱缓冲液、不同的有机溶剂和蛋白酶混合后都具有极强的稳定性,仍然表现出良好的抗菌活性。此外,最小抑菌浓度(MIC)测定结果表明,融合蛋白对5种菌具有良好的抗菌活性。研究结果为昆虫抗菌肽推广应用和进一步研究奠定了基础。To improve the expression level of tmAMPlm gene from Tenebrio molitor in Escherichia coli, we studied the effects of expression level and activity of the fusion protein HIS-TmAMPlm by conditions, such as culture temperature, inducing time and the final concentration of inductor Isopropyl beta-D-thiogalactopyranoside（IPTG）. We analyzed the optimum expression conditions by Tricine-SDS-PAGE electrophoresis, meanwhile, detected its antibacterial activity by using agarose cavity diffusion method. The results suggest that when inducing the recombinant plasmid with a final IPTG concentration of 0.1 mmol/L at 37 ℃ for 4 h, there was the highest expression level of fusion protein HIS-TmAMPlm in Escherichia coli. Under these conditions, the expression of fusion protein accounted for 40% of the total cell lysate with the best antibacterial activity. We purified the fusion protein HIS-TmAMPlm with nickel-nitrilotriacetic acid （Ni-NTA） metal-affinity chromatography matrices. Western blotting analysis indicates that the His monoclonal antibody could be specifically bound to fusion protein HIS-TmAMPlm. After expression by inducing, the fusion protein could inhibit the growth of host cell transformed by pET3Oa-tmAMPlm. The fusion protein HIS-TmAMPlm had better stability and remained higher antibacterial activities when incubated at 100 ℃ for 10 h, repeated freeze thawing at -20 ℃, dissolved in strong acid and alkali, or treated by organic solvents and protease. Moreover, the minimum inhibitory concentration results demonstrated that the fusion protein HIS-TmAMPlm has a good antibacterial activity against Staphylococcus aureus, Staphylococcus sp., Corynebacterium glutamicum, Bacillus thuringiensis, Corynebacterium sp. This study laid the foundation to promote the application of insect antimicrobial peptides and further research.

关 键 词：抗菌肽 条件优化 蛋白纯化 抗菌活性 

分 类 号：Q78[生物学—分子生物学]