结核分枝杆菌Ag85B、TB10.4及Ag85B-TB10.4融合基因的克隆表达及生物信息学分析

目的构建结核分枝杆菌Ag85B、TB10.4及Ag85B-TB10.4融合表达载体,并进行抗原的生物信息学分析,为候选疫苗筛选奠定基础。方法从结核分枝杆菌H37Rv菌株中分别扩增出Ag85B基因,TB10.4基因,Ag85B-TB10.4;将Ag85B克隆入pET-28a(+)表达载体中,将TB10.4和Ag85B-TB10.4分别克隆入pET-SUMO表达载体中。上述重组质粒转化入大肠埃希杆菌BL21菌中(DE3),经IPTG诱导表达,并对其进行生物信息学分析。结果 (1)成功将结核分枝杆菌Ag85B和TB10.4,Ag85B-TB10.4基因分别克隆入pET28a(+)和pET-SUMO表达载体中,经IPTG诱导蛋白表达后,对经超声裂解的菌液上清进行SDSPAGE电泳,表明获得了与预期蛋白大小一致的表达产物,重组融合蛋白pET-SUMO-Ag85B-TB10.4蛋白分子质量约为54kDa。(2)生物信息学分析显示Ag85B-TB10.4融合蛋白具有多个潜在抗原位点。结论成功构建了重组质粒pET-28a-Ag85B,pET-SUMO-TB10.4和pET-SUMO-Ag85B-TB10.4,并获得了Ag85B,TB10.4和Ag85B-TB10.4的可溶性原核表达产物;生物信息学分析在Ag85B与TB10.4蛋白加入一段蛋白linker后蛋白的亲水性增强,抗原决定簇面积增大,为后续的功能实验研究奠定了基础。

Cloning,Expression and Bioinformatics of the Ag85B,TB10. 4 and Ag85B-TB10. 4 Fusion Gene in Mycobacterium Tuberculosis

Objective To clone the Ag85B,TB10.4,Ag85B-TB10.4 fusion gene of Mycobacterium tuberculosis and to analyze the bioinformatics of the antigen,in order to lay the foundation for the candidate vaccine screening.Methods Ag85B,TB10.4and Ag85B-TB10.4 were amplified from the M.tuberculosis H37Rv genome by polymerase chain reaction(PCR).Ag85B was cloned into pET-28a(+) expression vector,TB10.4 and Ag85B-TB10.4 were cloned into pET-SUMO expression vector.All recombinant plasmids were transformed into Ecoli BL21(DE3),and the expression products induced by IPTG were analyzed through bioinformatics.Results The recombinant plasmids TB10.4,Ag85B and Ag85B-TB10.4 were constructed successfully.The SDS-PAGE results showed that the expression products were consistent with the expected results.The molecular weight of the fusion protein pET-SUMUO-Ag85B-TB10.4 was 54 KDa.Bioinformatics analysis showed that there was a plurality of potential antigenic sites in Ag85B-TB10.4 fusion protein.Conclusion The recombinant plasmids pET-28a-Ag85B,pET-SUMO-TB10.4 and pET-SUMO Ag85B-TB10.4 were constructed successfully,and the soluble prokaryotic expression products of TB10.4,Ag85B and Ag85B-TB10.4 were obtained.The hydrophilicity and antigenic determinant cluster area of Ag85B-TB10.4 fussion protein increased after added linker between Ag85B and TB10.4 by the bioinformatics analysis,which laid the foundation for subsequent functional experimental study.