羊种布鲁氏菌Tat系统底物蛋白ErfK的抗原性和免疫原性研究

Study on the Antigenicity and Immunogenicity of Erf K Protein of Substrate in the Twin-arginine Translocation System of Brucella melitensis

为分析羊种布鲁氏菌双精氨酸转运系统(twin-arginine translocation system,Tat system)毒力相关底物蛋白L,D-转肽酶ErfK诱导宿主产生的免疫应答情况,首先对布鲁氏菌M28毒株ErfK基因序列(WP_002963714.1)进行生物信息学分析,参考序列设计引物,构建表达载体pET-30a(+)-ErfK,经IPTG诱导表达、亲和层析和镍柱纯化获得目的蛋白;利用SDS-PAGE和Western blot对重组ErfK(rErfK)蛋白进行抗原性鉴定;将rErfK蛋白免疫小鼠,分别在免疫后15、30和45 d收集血清和脾脏,检测蛋白免疫后小鼠的细胞免疫应答和体液免疫应答情况。生物信息学预测结果显示,ErfK蛋白无跨膜结构,为亲水性蛋白,有多个T细胞和B细胞抗原表位;SDS-PAGE和Western blot均可获得25 kDa的蛋白条带,重组蛋白可与布鲁氏菌阳性血清发生反应,证明rErfK有良好的抗原性;小鼠免疫试验结果显示,rErfK组小鼠脾脏CD8^(+)T细胞含量相比PBS组显著上升(P<0.05),且脾脏Th1型细胞因子IL-2、TNF-α、IFN-γ和Th2型细胞因子IL-4转录水平均显著上升(P<0.05),此外,rErfK免疫也能极显著诱导小鼠血清中总IgG和特异性IgG的产生(P<0.01)。上述结果表明,经大肠杆菌表达的布鲁氏菌rErfK蛋白可诱导小鼠产生良好的体液免疫和细胞免疫应答。本研究为基于ErfK蛋白的布鲁氏菌病诊断试剂和亚单位疫苗研发提供了参考依据。

In order to analyze the immune response of hosts that were induced by virulence related substrate protein L,D-transpeptidase ErfK of twin-arginine translocation system(Tat system)of Brucella melitensis,bioinformatics analysis was conducted for ErfK gene sequence(WP_002963714.1)of Brucella melitensis M28 strain,based on which,primers were designed to construct an expression vector pET-30a(+)-ErfK,and target protein was obtained through IPTG induction,affinity chromatography and nickel column purification.The antigenicity of recombinant ErfK protein(rErfK)was identified using SDS-PAGE and Western blot.rErfK protein was immunized into mice,serum and spleen were collected on 15,30 and 45 days after immunization,and the cellular and humoral immune responses of the immunized mice were detected.The bioinformatics prediction results showed that ErfK protein was hydrophilic without transmembrane structure,and several T and B cell antigen epitopes existed.25kDa protein bands could be obtained by SDS-PAGE and Western blot,and the recombinant protein could react with Brucella positive serum,proving a good antigenicity.Immunity test of mice showed that,compared to the control group,the content of CD8^(+)T cells in spleens of immunized mice was significantly increased(P<0.05),so was the transcription level of Th1-type cytokines of IL-2,TNF-αand IFN-γas well as Th2-type cytokine of IL-4(P<0.05).Furthermore,the toal and specific IgG level in the serum of mice could be greatly induced by rErfK immunization(P<0.01).In conclusion,Brucella rErfK protein expressed by E.coli prokaryotic expression system could induce mice to produce good humoral and cellular immune response.The development of reagents and subunit vaccines based on ErfK protein for brucellosis was provided with a reference by the study.