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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。
OBJECTIVE: To obtain a single domain antibody that specifically recognizes SpaA-N. Methods: The His-SpaA-N recombinant antigen was screened from the bacteriophage phage display library of Bactrian camels in Xinjiang, and the binders of SpaA-N were screened. After sequencing, subcloned into pET30a and overexpressed in E. coli BL21 and purified by nickel ion affinity chromatography. ELISA analysis of the thermal stability of recombinant single domain antibodies, Western blot detection of binding specificity. Results: After His-SpaA-N screening and enrichment, two clones of interest were screened. Constructed into pET30a, PCR and restriction enzyme digestion identified the size of the gene in line with the expected. SDS-PAGE showed Mr 29 000 and 23 000 specific bands of interest. ELISA showed that Anti-SpaA-N VHH had good binding activity to SpaA-N recombinant protein. After heat-denatured VHH, its antigen binding activity could be restored by renaturation at room temperature. Western blot showed that recombinant VHH can recognize the surface proteins present in Erysipelothrix at Mr 66 000. Conclusion: The single-domain antibody with thermal stability and specific binding to SpaA-N was obtained, which provided a basis for further study on the role of spaA antigen in immune infection of Erysipelas.