摘要
【目的】原核表达空肠弯曲菌细胞致死性肿胀毒素B蛋白(CdtB),制备其单克隆抗体(mAb),并研究mAb抗毒性作用。【方法】扩增空肠弯曲菌cdtB基因并将其构建到pET-30a(+)和pGEX-6p-1表达载体,以原核表达的GST-CdtB蛋白为免疫原,应用杂交瘤技术进行细胞融合;采用间接ELISA方法测定细胞上清和mAb腹水效价,Dot-ELISA、Western blot分析mAb特异性,并以CaCo-2和HD-11细胞为模型,鉴定mAb抗毒性能力。【结果】成功构建重组原核表达质粒pET-30a(+)-cdtB和pGEX-6p-1-cdtB,并融合表达rHis-CdtB和rGST-CdtB蛋白。获得5株稳定分泌CdtB抗体的杂交瘤细胞株,分别命名为1F3,1F5,2E4,2E11,2F2。抗体Ig类和亚类检测显示2E11 Ig亚类为IgG2b,其他4株均为IgG1。抗体效价高达1:(1×108)。Dot-ELISA试验表明5株mAb均能与空肠弯曲菌标准株发生特异性反应,与非空肠弯曲菌呈阴性反应;Western blot法分析表明5株mAb均能与纯化蛋白rGST-CdtB有良好的反应性。基于CaCo-2细胞的黏附和侵袭实验表明mAb能显著降低细菌的黏附和侵袭能力(P<0.01)。【结论】成功制备了针对空肠弯曲菌CdtB蛋白的mAb。为进一步研究空肠弯曲菌致病机制,以及为研制治疗性类药物奠定了基础。
[ Objective] To express Campylobacter jejuni cytolethal distending toxin B protein （CdtB） in a prokaryote to prepare monoclonal antibodies （mAbs） against the protein, and to study their antitoxic effects. [ Methods] The C. jejuni cdtB gene was amplified and inserted into the expression plasmids pET-30a（ ＋ ） and pGEX-6p-1. The purified rGST-CdtB protein was used as the immunogen to screen hybridoma cells for mAbs against the protein. The mAb titers were determined with an indirect enzyme-linked immunosorbent assay （ELISA）, and their specificity with a Dot-ELISA and western blotting analysis. We determined the antitoxic properties of the mAbs in CaCo-2 and HD-11 cells. [ Results] Recombinant expression plasmids pET-30a （ ＋ ）-cdtB and pGEX-6p-1-cdtB were successfully constructed, and fusion proteins rHis-CdtB and rGST-CdtB expressed, respectively. Five hybridoma cell lines, designated 1F3, 1F5, 2E4, 2E11, and 2F2, were screened for the stable secretion of mAbs against CdtB. The immunoglobulin subclass of 2El 1 was IgG2b and that of the other mAbs was IgG1. The mAb titers in the ascites fluids were 1：1×10^8 on indirect ELISA. Dot- ELISA demonstrated that the five mAbs reacted specifically with C. jejuni. Western blotting analysis confirmed that the five mAbs reacted well with the rGST-CdtB fusion protein. The mAbs significantly reduced the adhesion and invasion capacities of the bacterium in CaCo-2 cells （P 〈 0.01 ）. [ Conclusion] The successful preparation of five mAbs specific for the CdtB protein will allow further study of the biological characteristics of CdtB and the pathogenesis of C. jejuni.
出处
《微生物学报》
CAS
CSCD
北大核心
2014年第8期950-955,共6页
Acta Microbiologica Sinica
基金
国家自然科学基金(31372449)
国家“863计划”(2012AA101601)
国家科技支撑计划(2014BAD13B02)~~
