新疆双峰驼抗血清中重链抗体的分离鉴定

【目的】初步探讨新疆双峰驼Camelus bactrianus免疫系统中缺失轻链的重链抗体(heavy chain antibody,HCAb)是否具有常规抗体的基本抗原结合功能。【方法】采用本实验室构建的丹毒丝菌表面蛋白A原核表达载体pGEX4T-1-spaA-N,进行诱导表达,纯化获得重组抗原GST-SpaA-N,免疫双峰驼制备抗血清,经Protein A和Protein G亲和层析从抗血清中分离重链抗体及常规抗体,Western blotting鉴定重链抗体的抗原结合特性,间接ELISA比较重链抗体与传统抗体的抗原结合活性。【结果】经过Protein A/G纯化得到了IgG2。对各组分进行分析初步表明重链抗体约占血清IgG的60%—80%。采用GST-SpaA-N免疫双峰驼可以诱导高滴度的IgG2型重链抗体,在5次免疫后,骆驼抗spaA-N特异的重链抗体IgG2多抗血清的效价为1﹕51200。Western blotting结果显示分离的多克隆重链抗体可特异结合SpaA天然抗原,且ELISA分析结果表明重链抗体与常规抗体具有相同的抗原结合活性。【结论】首次从双峰驼抗血清中成功分离获得具有与常规抗体相同生物学功能的多克隆重链抗体,提示重链抗体可能在骆驼免疫防御中发挥重要作用。

Isolation and Identification of Heavy Chain Antibodies Derived from Immunized Xinjiang Camelus Bactrianus Antisera

【Objective】 To investigate whether HCAbs (heavy chain antibodies ) discovered in Camelus bactrianus consist of only two heavy chains possess the same biological activity as conventional antibodies. 【Method】 A recombinant expression vector pGEX4T-1-spaA-N which constructed previously and contained insert gene derived from surface protective antigen of Erysipeolothrix rhusiopathiae was induced and the recombinant GST-spaA-N was expressed. One bactrian camel was immunized with purified recombinant antigen GST-SpaA-N, and the anti-SpaA-N IgGs fractions were obtained via protein G and protein A chromatography. The antigen specificity and binding activity of polyclonal HCAbs were analyzed with western blotting and ELISA.【Result】 IgG2 HCAb was successfully isolated from camel sera. The Protein A/G eluted fraction revealed that HCAbs might on the percentages of 60%-80% of total IgG. The immunized camel was at least induced high titer IgG2 by SpaA-N, reached 1:51 200 on day 70. The isolated polyclonal IgG2 HCAb could bind SpaA specifically in western blotting. The result of ELISA showed the IgG2 HCAbs bear the same biological antigen binding activity as conventional ones. 【Conclusion】 The polyclonal HCAbs with the same antigen binding activity was isolated from a bactrian camel sera for the first time, indicating its important role in camel immune protection.