新疆双峰驼纳米抗体、重链抗体和常规抗体的热稳定性比较

【目的】 比较纳米抗体、HCAbs与常规抗体之间在温度稳定性方面的差异,为研究HCAb的功能特性和骆驼适应极端环境的免疫特点提供参考。【方法】 从溶菌酶、蒜氨酸酶和丹毒丝菌表面抗原A 3种抗原免疫的新疆双峰驼血清中,采用Protein A和Protein G亲和色谱纯化IgG1、IgG2和IgG3 3种亚型抗体,并在大肠杆菌BL21(DE3)菌中表达和纯化3种纳米抗体。采用ELISA方法,测定经过22~90℃一系列热处理后、平衡至室温的各抗体与相应抗原的结合活性,以剩余抗原结合活性的百分比作为衡量抗体温度稳定性的参数。【结果】 3种抗原免疫后均能在骆驼激发明显的抗体反应。采用Protein A/G亲和色谱,从血清中纯化获得分子量分别为50 KD+25 KD、 46 KD和43 KD的IgG1、IgG2和IgG3亚型抗体。骆驼IgG2和IgG3重链抗体比IgG1具有更高的温度稳定性,其中IgG3表现出比IgG2对温度更高耐受的趋势。从细菌表达纯化的3种纳米抗体都表现出比重链抗体和常规抗体更高的温度稳定性。【结论】 抗体结构形式和分子大小可能显著影响了双峰驼抗体对温度的耐受性。

Comparison Analysis of the Thermal Stabilities between Nanobodies,Heavy Chain Antibodies and Conventional Antibodies in Xinjiang Camelus bactrianus

【Objective】 To explain the unique porterty of nanobodies by comparison analysis of the thermal stabilities between Heavy Chain Antibodies (HCAbs) and Convention Antibodies (ConAbs) from Xinjiang Camelus bactrianus. 【Method】 IgG2 and IgG3 isotypic HCAbs and IgG1 isotypic ConAbs were isolated and purified from lysozyme, alliinase and spaA immunized camel serum by protein A and Protein G affinity chromatography. Nanobodies were obtained from our previous works and expressed in BL21 and purified by Ni-gel affinity chromatography. The thermal stabilities of different antibodies were expressed as residual antigen binding activities after series of heat treated and renatured to room temperature in ELISA. 【Results】 Three isotypic antibody of IgG1, IgG2 and IgG3 with M.W. of 50 KD+25 KD, 46 KD and 43 KD were purified from camel serum separately. Nanobody Lys-23, Spa-G26 and Ali-A4 were purified as well. The antigen binding ELISA results showed Camel IgG2 and IgG3 heavy chain antibodies had higher thermal stability than IgG1, in which IgG3 tended to be more tolerant to temperature than IgG2. The three camel nanobodies possessed the highest thermal stabilities when compared with their HCAbs and ConAbs partners. 【Conclusion】 The isotypic and molecular size of camel antibodies might significantly influence the thermal stabilities. Whether these tolerances are related to the immune characteristics of camels adapted to extreme environments is worthy of further discussion.