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

doi:10.6048/j.issn.1001-4330.2020.02.013

Xinjiang Agricultural Sciences ›› 2020, Vol. 57 ›› Issue (2): 319-325.DOI: 10.6048/j.issn.1001-4330.2020.02.013

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

Kunduziayi Abudushalamu, JIANG Zhiqiang, LIU Yafei, XIONG Jingfan, Nasibai Abuduwahp, LI Jiangwei

College of Life Science and Technology, Xinjiang University/The 104th Regimental Farm of the 12th Agricultural Division, Xinjiang Production and Construction Corps, Urumqi 830046, China

Received:2019-10-04 Published:2020-03-02
Correspondence author: LI Jiangwei(1967-),male,Urumqi,professor,(E-mail)jwli67@sina.com
Supported by:
Supported by National Natural Science Foundation of China “Diversity Analysis and data Mining of Camel Antibody Library and Discovery of New Nano-Antibody Based on High-Throughput Sequencing” (31570935)

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

坤杜孜阿依·阿布都沙拉木, 姜志强, 刘娅菲, 熊静璠, 娜斯拜·阿卜杜瓦哈普, 李江伟

新疆大学生命科学与技术学院/省部共建新疆生物资源基因工程重点实验室,乌鲁木齐 830046

通讯作者: 李江伟(1967-),男,新疆乌鲁木齐人,教授,硕士生导师,研究方向为抗体工程及肿瘤免疫治疗,(E-mail)jwli67@sina.com
作者简介:坤杜孜阿依·阿布都沙拉木(1994-),女,新疆伊犁人,硕士研究生,研究方向为抗体工程,(E-mail)295335585@qq.com
基金资助:
国家自然科学基金面上项目 "基于高通量测序对骆驼抗体库的多样性分析和数据挖掘及新纳米抗体的发现"(31570935)

Abstract

Abstract: 【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.

Key words: heavy chain antibodies;nanobodies;Xinjiang Bactrian camel;thermal stabilities;ELISA

摘要： 【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。

关键词: 重链抗体, 纳米抗体, 新疆双峰驼, 温度稳定性, ELISA测定

CLC Number:

S852

Kunduziayi Abudushalamu, JIANG Zhiqiang, LIU Yafei, XIONG Jingfan, Nasibai Abuduwahp, LI Jiangwei. Comparison Analysis of the Thermal Stabilities between Nanobodies, Heavy Chain Antibodies and Conventional Antibodies in Xinjiang Camelus bactrianus[J]. Xinjiang Agricultural Sciences, 2020, 57(2): 319-325.

坤杜孜阿依·阿布都沙拉木, 姜志强, 刘娅菲, 熊静璠, 娜斯拜·阿卜杜瓦哈普, 李江伟. 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[J]. 新疆农业科学, 2020, 57(2): 319-325.

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

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

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