家蚕和果蝇的抗菌肽cecropin B基因的原核表达及抑菌活性分析

Prokaryotic Expression and Bacteriostatic Activity Analysis of Antibacterial Peptide Cecropin B Genes from Bombyx mori and Drosophila melanogaster

动物抗菌肽天蚕素(cecropin)因具有抗菌谱广、活性较强、不易产生耐药性等特点而成为新型抗菌剂开发的材料。为了探讨通过原核表达的途径高效获取天然cecropin,以重叠PCR方法分别人工合成家蚕(Bombyx mori)和果蝇(Drosophilamelanogaster)的抗菌肽cecropin B基因(CecB2)并克隆至T载体,测序确认后分别克隆至pET-32a载体,转化至E.coli BL21(DE3)宿主菌进行表达。通过SDS-PAGE和Western blotting检测到2种目的蛋白均得到表达,但表达量存在差异,Bradford法测定纯化后的BmCecB2融合蛋白和DmCecB2融合蛋白的质量浓度分别为0.6、2.0 mg/mL。经Ni-NTA亲和层析纯化和透析处理后的2种产物的抗菌活性存在明显差异:DmCecB2和BmCecB2的质量浓度为10 mg/mL时,对E.coli DH5α的抑菌圈直径分别为15、8 mm左右,最小抑菌浓度分别为0.05、0.10 mg/mL。以上结果说明DmCecbB2的原核表达效率及表达产物的抑菌活性均明显高于BmCecB2。用生物信息学方法分析家蚕和果蝇的CecB2蛋白在结构上存在差异,这可能是导致2种产物活性不同的主要原因。

Cecropin,one of the animal antibacterial peptides,has become a material for developing novel antibacterial agents owing to its wide antimicrobial spectrum,strong activity,and uneasiness to incur drug resistance.In order to explore the way of obtaining natural cecropin protein through efficient expression in prokaryotes,the antibacterial peptide cecropin B gene（CecB2） of Bombyx mori and Drosophila melanogaster was artificially synthesized respectively by means of overlapping PCR,which was subsequently cloned into T vector respectively.After verification by sequencing,these genes were further cloned into pET-32a vector and transformed into E.coli BL21（DE3） for expression in the host bacteria.SDS-PAGE and Western blotting detected that the two target proteins were both expressed,but their expression levels were different.Determination of protein concentration by Bradford method showed that mass concentration of Bombyx mori cecropin B（BmCecB2） and Drosophila melanogaster cecropin B（DmCecB2） fusion proteins was 0.6 and 2.0 mg/mL respectively.After purification by Ni-NTA affinity chromatography and dialysis treatment,antibacterial activity of the two products was also obviously different.At mass concentration of 10 mg/mL,the expression products of both DmCecB2 and BmCecB2 had bacteriostatic circle of 15 and 8 mm in diameter respectively against E.coli DH5α.And the minimum inhibitory concentration of DmCecB2 and BmCecB2 was 0.05 and 0.10 mg/mL respectively.The above results showed that the prokaryotic expression efficiency and antibacterial activity of the expressed product of DmCecB2 was obviously higher than those of BmCecB2.Bioinformatic analysis revealed that structural difference of BmCecB2 and DmCecB2 proteins is the main cause of variation in their antibacterial activity.