枯草芽孢杆菌SX3411羊毛硫细菌素Subtilomycin结构基因subA的原核中串联表达和抗体制备

Prokaryotic Tandem Expression and Antibody Preparation of Subtilomycin Structural Gene subA from Bacillus subtilis SX3411

为了提高枯草芽孢杆菌SX3411菌株羊毛硫细菌素Subtilomycin前体基因subA表达产物的抗原性,采用化学合成的方法合成了SubA的3次重复串联体3×subA基因,构建了该基因的原核表达载体并转入大肠杆菌中。结果表明,3×subA基因在大肠杆菌中获得了融合表达。利用SDS-PAGE纯化大肠杆菌中融合表达的3×SubA,免疫家兔后制备抗体anti-3×SubA,该抗体经检测有较高的滴度。通过Western Blotting杂交,检测了Subtilomycin前体多肽SubA的胞内表达特性。结果表明,枯草芽孢杆菌SX3411在30℃培养的条件下进行不同时间取样检测,枯草芽孢杆菌3411约在培养的第10,12小时胞内有较多的SubA表达。综上所述,通过串联表达Subtilomycin前体基因subA所获得的融合表达产物3×SubA具有较好的抗原性,以此获得的抗体anti-3×SubA完全可以用于枯草芽孢杆菌SX3411中Subtilomycin前体检测和分析。

In order to improve the antigenicity of the expression product of Subtilomycin precursor gene of Bacillus subtilis SX3411,a three-times repeated tandem 3×subA gene was synthesized by chemical synthesis method.The prokaryotic expression vector of 3×subA was constructed and transferred into Escherichia coli.The results showed that the 3×subA gene was expressed in E.coli.SDS-PAGE was used to purify the E.coli expressed SubA.After immunizing rabbits,the antibody anti-3×SubA was prepared and had a high titer.The intracellular expression of Subtilomycin precursor polypeptide subA was detected by Western Blotting.The results showed that B.subtilis SX3411 was cultured at 30℃ for different time,and there were more subA expressions in the cells of B.subtilis 3411 at about the 10th and 12th hour.In conclusion,the fusion expression product 3×SubA obtained by tandem expression of the Subtilomycin precursor gene subA has good antigenicity,and the obtained antibody anti-3×SubA can be used for the detection and analysis of Subtilomycin precursors in B subtilis SX3411.