【摘要】 目的:实现大肠杆菌高效可溶表达人源抗菌肽LL-37。方法:LL-37基因克隆至原核载体pET32a,于大肠杆菌BL21(DE3)中诱导表达。运用相关生物信息学软件分析重组蛋白Trx-LL-37的理化性质、亲/疏水性、蛋白质二级结构及其可溶表达概率。实验还考察了不同诱导温度对重组蛋白可溶表达比例的影响。结果:生物信息学分析显示,Trx-LL-37分子量21.5kD,理论等电点6.3,物理性质稳定,二级结构简单,具有可溶表达倾向。重组蛋白最佳诱导温度为17℃,与37℃相比,可溶表达比例由37.2%提高至50.2%,并且总表达量也提高了5%左右。抑菌结果显示纯化产物对多种常见细菌的生长具有抑制作用。结论:可采用融合方式通过原核系统高效可溶表达LL-37,为LL-37的功能研究打下基础。更多还原

【Abstract】 Objective:The article was designed to highly express soluble human antibacterial peptide LL-37 in E.coli.Method:The DNA sequence encoding LL-37 was cloned into the pET32a vector and expressed in E.coli strain BL21(DE3).The physical-chemical properties,hydrophilicity or hydrophobicity,the secondary structure and the soluble expression probability of Trx-LL-37 were analyzed with bioinformatics analysis software.The effect of temperature on the proportion of soluble expression was also investigated in this experiment.Result:Bioinformatic analysis showed that,physical properties of Trx-LL-37 was stable,the secondary structure was simple,and Trx-LL-37 had the tendency to be expressed in a soluble way.Further optimization studies showed that the expression system was very efficient to produce soluble target protein at 17℃.Compared to 37℃,the percentage in total soluble protein of Trx-LL-37 was increased from 37.2% to 50.2%,and the expression level of Trx-LL-37 was increased by 5%.In terms of antibacterial activity,the purified product was able to inhibit the growth of common pathogens.Conclution:The results established that LL-37,human antibacterial peptide,was successfully expressed by the prokaryotic expression system in a soluble way at low induced temperature.更多还原