炭疽芽孢杆菌新抗原NlpC的重组表达与免疫评价  

Recombinant Expression and Immunological Evaluation of a Novel Antigen NlpC from Bacillus anthracis

作　　者：胡凯 宰晓东 殷瑛 李汭桦 王美荣 李耀辉 张跃 李玉杰 辜彦霏 徐俊杰 HU Kai;ZAI Xiao-Dong;YIN Ying;LI Rui-Hua;WANG Mei-Rong;LI Yao-Hui;ZHANG Yue;LI Yu-Jie;GU Yan-Fei;XU Jun-Jie(Beijing Institute of Biotechnology,Beijing 100071,China)

机构地区：[1]军事医学研究院生物工程研究所,北京100071

出　　处：《生物技术通讯》2020年第6期627-633,共7页Letters in Biotechnology

基　　金：国家自然科学基金(31800770);国家科技重大专项(2016ZX10004001)。

摘　　要：目的:构建炭疽芽孢杆菌新抗原NlpC的原核表达质粒,制备NlpC重组蛋白并进行免疫评价,为新型炭疽亚单位疫苗研发提供参考。方法:运用生物信息学软件分析和预测NlpC蛋白的跨膜区、信号肽、疏水性、保守性等性质;PCR扩增nlpc基因片段,构建NlpC原核表达质粒,IPTG诱导表达,His亲和层析法纯化目的蛋白,SDS-PAGE与Western印迹鉴定纯化的蛋白;NlpC重组蛋白联合佐剂免疫小鼠后,ELISA法检测免疫血清的抗体效价,体外实验检测免疫血清抑制芽孢萌发功能,BALB/c小鼠免疫攻毒实验评价免疫保护效果。结果:生物信息学分析结果表明NlpC有信号肽,无跨膜区,亲水性好,具有较高的保守性;构建了NlpC原核表达质粒,经诱导表达与纯化获得了纯度高于95%的NlpC蛋白;免疫评价结果显示NlpC联合Al(OH)3+CpG佐剂可激发小鼠产生高水平的抗体反应,NlpC免疫血清可在体外抑制炭疽芽孢萌发,NlpC免疫组可显著延长炭疽杆菌芽孢攻毒后小鼠存活时间。结论:NlpC具有较好的免疫原性,是潜在的炭疽杆菌保护性抗原,可作为新型炭疽亚单位疫苗的候选组分。Objective:To provide reference for development of a new anthrax subunit vaccine,the prokaryotic expression plasmid of the novel antigen NlpC of Bacillus anthracis was constructed,the recombinant protein NlpC was prepared and the immunological evaluation was conducted.Methods:Bioinformatics software was used to ana⁃lyze and predict the transmembrane region,signal peptide,hydrophobicity,conservatism and other properties of pro⁃tein NlpC.The nlpc gene was amplified by PCR and cloned into prokaryotic expressing vector.The target protein was induced by IPTG and then was purified by His-tag affinity chromatography.The purified protein was identi⁃fied by SDS-PAGE and Western blotting.Level of NlpC-specific antibodies in immunized mice was detected by ELISA.The inhibitory effect of immune serum on spore germination was tested in vitro,and the immunological pro⁃tective effect was evaluated by the challenge experiment with BALB/c mice.Results:The results of bioinformatics analysis showed that NlpC was hydrophilic protein with signal peptide sites and no transmembrane region.The pro⁃karyotic expression plasmid of NlpC was constructed,and the purity of the protein NlpC was over 95%after ex⁃pression purification.The results of immunological evaluation showed that NlpC combined with adjuvant Al(OH)3 plus CpG could stimulate mice to produce high levels of antibody response.Serum from NlpC immunized mice could inhibit the germination of B.anthracis spores in vitro.NlpC immunized group could significantly prolong the survival time of mice after challenge with B.anthracis spores.Conclusion:NlpC is a potential protective antigen of B.anthracis,and can be used as a candidate component of the new anthrax subunit vaccine.

关 键 词：炭疽芽孢杆菌 NlpC蛋白 原核表达 免疫评价 芽孢萌发 

分 类 号：Q78[生物学—分子生物学] R392.1[医药卫生—免疫学]