致犊牛脑炎大肠杆菌Flic3和FimD蛋白的生物信息学分析及克隆表达

Bioinformatics analysis and cloning expression of Flic3 and FimD proteins of calf encephalitis Escherichia coli

为了对致犊牛脑炎大肠杆菌S9922的Flic3、FimD蛋白进行生物信息学分析,并进行诱导表达,试验利用在线分析软件对Flic3、FimD蛋白进行生物信息学分析,利用PCR技术扩增Flic3和FimD基因,构建重组质粒pET32a-Flic3和pET32a-FimD,转化BL21(DE3)大肠杆菌感受态进行原核表达,并进行SDS-PAGE分析和Western-blot检测。结果表明:Flic3蛋白的原子组成为C_(1381)H_(2284)N_(412)O_(496)S_(5),理论等电点为5.08,含有2个糖基化位点,α-螺旋、β-转角、β-片层和无规则卷曲分别占48.24%、6.39%、18.85%、26.52%,有13个可能的抗原表位。FimD蛋白的原子组成为C_(1828)H_(2798)N_(510)O_(588)S_(9),理论等电点为6.16,含有8个糖基化位点,α-螺旋、β-转角、β-片层和无规则卷曲分别占6.54%、5.50%、32.94%、56.02%,有15个潜在的抗原表位。Flic3和FimD蛋白都是亲水、稳定蛋白,都不含跨膜区和信号肽。SDS-PAGE分析结果显示,重组蛋白Flic3大小为51 ku,重组蛋白FimD大小为59 ku,皆以包涵体形式表达。Western-blot检测后目的条带与预期一致,具有良好的免疫原性。说明Flic3和FimD蛋白原核表达成功,可用于制备致犊牛脑炎大肠杆菌疫苗。

In order to carry out bioinformatics analysis and inducing expression of Flic3 and FimD proteins of calf encephalitisEscherichia coli S9922,in this experiment the online analysis software was used to carry out bioinformatics analysis of Flic3 and FimD proteins,and PCR method was used to amplify Flic3 and FimD genes for the construction of recombinant plasmids pET32a-Flic3 and pET32a-FimD,which were transformed into BL21(DE3)Escherichia colifor prokaryotic expression;and SDS-PAGE analysis and Western-blot detection were carried out.The results showed that the atomic composition of Flic3 protein was C_(1381)H_(2284)N_(412)O_(496)S_(5),and the theoretical isoelectric point was 5.08;it contained two glycosylation sites,and theα-helix,β-turn,β-lamella and random coils accounted for 48.24%,6.39%,18.85%and 26.52%respectively,and there were 13 possible epitopes.The atomic composition of FimD protein was C_(1828)H_(2798)N_(510)O_(588)S_(9),and the theoretical isoelectric point was 6.16;it contained 8 glycosylation sites,and theα-helix,β-turn,β-lamella and random coils accounted for 6.54%,5.50%,32.94%and 56.02%respectively,and there were 15 possible epitopes.Both Flic3 and FimD proteins were hydrophilic and stable proteins,and did not contain transmembrane regions and signal peptides.SDS-PAGE results showed that the size of Flic3 protein was 51 ku and the size of FimD protein was 59 ku,both of which were expressed as inclusion bodies.After western-blot,the target band was consistent with the expectation,which showed good immunogenicity.The structure and physicochemical properties of Flic3 and FimD proteins were analyzed and prokaryotic expression was carried out,which laid the foundation for screening the protective antigens of calf encephalitisEscherichia coli.