骆驼重组SpaA-N单域抗体的制备与性质研究  

Preparation and property study of the recombinant Camel anti-SpaA-N single domain antibodies

作　　者：芦清霞[1] 庆格乐图[1] 杨春静[1] 夏雪琴[1] 李江伟[1] 

机构地区：[1]新疆大学生命科学与技术学院新疆生物资源基因工程重点实验室,新疆乌鲁木齐830046

出　　处：《细胞与分子免疫学杂志》2012年第1期55-58,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(31060132)

摘　　要：目的:获得特异识别SpaA-N的单域抗体。方法:用His-SpaA-N重组抗原从新疆双峰驼单域抗体噬菌体展示文库中,筛选SpaA-N的结合子。经测序后亚克隆至pET30a并在E.coli BL21高表达,用镍离子亲和层析柱纯化。ELISA分析重组单域抗体的热稳定性,Western blot检测结合特异性。结果:经His-SpaA-N筛选富集后,筛选得到2个目的克隆。构建至pET30a,PCR和酶切鉴定目的基因大小与预计相符。SDS-PAGE显示,Mr 29 000和23 000有特异性目的条带。ELISA检测显示,抗SpaA-N的VHH对SpaA-N重组蛋白具有很好的结合活性;VHH热变性后,经室温复性均可以恢复其抗原结合活性。Western blot显示,重组VHH在Mr 66 000处可以识别丹毒丝菌中存在的表面蛋白。结论:获得了具有热稳定性和特异结合SpaA-N的单域抗体,为进一步研究spaA抗原在丹毒丝菌感染免疫中的作用提供了基础。AIM： In order to obtain single domain anti- body against surface protective antigen A （SpaA）of Erysip- elothrix rhusiopathiae. METHODS： The SpaA-N recombinant protein was used to screen binders from Bactrian camel VHH phage display library. After sequencing, the interested VHH gene fragments were subcloned into pET-30a vector to overexpress the protein in E. coil BL21. The binding specificity of the recombinant VHH with SpaA-N was determined by Western blotting. The thermal stability of single-domain antibody was evaluated by ELISA. RESULTS： By enrichment of screening, 2 clones were selected. Recombinant single domain antibodies purified by Ni-ion affinity chromatography showed a single band at M, 29 000, 23 000 on SDS-PAGE. ELISA results showed that VHH can bind its antigen specifically. After thermal denaturation, VHH can restore the antigen binding ability after refolding. Western blotting results showed that the recombinant VHH specific bind surface pro- tective antigen of Erysipelothrix rhusiopathiae at Mr 66 000. Two VHH single domain antibodies with high thermal stability and good antigen binding specificity were identified by screening Bactrian camel VHH phage display library. CONCLUSION： Two single domain antibodies that specifically aggulated SpaA-N is obtained, which provide the basis for further study in the immune role of single domain antibody against Erysipelothrix rhusiopathiae infection.

关 键 词：单域抗体 SpaA—N 亲和淘洗 热稳定性 骆驼 

分 类 号：Q789[生物学—分子生物学]