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

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。

Abstract:

To improve the expression level of tmAMP1m gene from Tenebrio molitor in Escherichia coli, we studied the effects of expression level and activity of the fusion protein HIS-TmAMP1m 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-TmAMP1m 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-TmAMP1m 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-TmAMP1m. After expression by inducing, the fusion protein could inhibit the growth of host cell transformed by pET30a-tmAMP1m. The fusion protein HIS-TmAMP1m 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-TmAMP1m 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.