牛支原体HB0801株VspX蛋白单克隆抗体的制备与鉴定

doi:10.16431/j.cnki.1671-7236.2017.03.035

《中国畜牧兽医》 ›› 2017, Vol. 44 ›› Issue (3): 868-878.doi: 10.16431/j.cnki.1671-7236.2017.03.035

牛支原体HB0801株VspX蛋白单克隆抗体的制备与鉴定

陈曦, 朱洪梅, 赵刚, 胡长敏, 陈颖钰, 郭爱珍

华中农业大学动物医学院, 农业微生物国家重点实验室, 武汉 430070

收稿日期:2016-08-20 出版日期:2017-03-20 发布日期:2017-03-21
通讯作者: 郭爱珍 E-mail:aizhen@mail.hzau.edu.cn
作者简介:陈曦(1974-),女,黑龙江汤原人,硕士,讲师,研究方向:牛支原体病,E-mail:chenxi@mail.hzau.edu.cn
基金资助:
国家自然科学基金（31302111、31272587）；中央高校基本科研业务费专项资金（2013QC001）

Preparation and Identification of the Monoclonal Antibodies against VspX Protein in Mycoplasma bovis strain HB0801

CHEN Xi, Zhu Hong-mei, ZHAO Gang, HU Chang-min, CHEN Ying-yu, GUO Ai-zhen

State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China

Received:2016-08-20 Online:2017-03-20 Published:2017-03-21

摘要/Abstract

摘要：

为获得牛支原体（Mycoplasma bovis，M.bovis）VspX蛋白单克隆抗体，将编码该蛋白的基因克隆、表达并纯化，作为免疫原，以QuickAntibody-Mouse 5W为免疫佐剂，免疫BALB/c小鼠。经3次免疫后，将小鼠脾细胞与SP2/0骨髓瘤细胞融合，经3次亚克隆筛选后，共获得5株能稳定分泌抗VspX蛋白抗体的杂交瘤细胞株，分别命名为1A8、3A3、3C12、3H9及4D11。亚型鉴定表明，3C12重链为IgG2b，其余4株为IgG1，轻链均为κ链。间接ELISA结果表明，5株细胞培养上清的抗体效价在1：1×10⁴~1：2×10⁵，腹水效价在1：1×10⁵~1：8×10⁵。选其中两株杂交瘤细胞株3H9和4D11的腹水纯化，进行亲和力测定，解离常数分别为6.3×10⁹和7.8×10⁹，属高亲和力抗体。Western blotting结果显示，5株单抗均能与牛支原体发生特异性反应，而单抗4D11与羊无乳支原体标准株PG2和丝状支原体丝状亚种标准株PG3均不反应。流式细胞术结果表明，单抗4D11与牛支原体表面的VspX的结合呈剂量依赖性。间接免疫荧光结果表明，单抗4D11可以识别黏附到胚胎牛肺细胞上的重组VspX蛋白。本试验成功制备的单克隆抗体为VspX蛋白功能的研究奠定基础。

关键词: 牛支原体; VspX蛋白; 单克隆抗体; 特异性

Abstract:

To obtain the monoclonal antibody (McAb) against VspX protein of Mycoplasma bovis (M. bovis),VspX gene was amplified, expressed and purified. Then, BALB/c mice were immunized subcutaneously three times with the purified recombinant VspX (rVspX) mixed with QuickAntibody-Mouse 5W adjuvant. Three days after the last injection, spleen cells were collected aseptically, and fused with SP2/0 myeloma cells in the presence of polyethylene glycol. By the clone selection, five stable hybridomas against VspX protein were obtained, separately named as 1A8, 3A3, 3C12, 3H9 and 4D11. Antibody titers in cell supernatant were from 1:1×10⁴ to 1:2×10⁵, while from 1:1×10⁵ to 1:8×10⁵ in ascites of mice by indirect ELISA. The subtypes were determined to be IgG1 and IgG2b class, and all light chains were κ chain. The affinity constant of McAb 3H9 and 4D11 were 6.3×10⁹ and 7.8×10⁹, respectively, and they belonged to high-affinity antibodies. Western blotting results showed that all of five McAbs could specifically react with M.bovis, however, McAb 4D11 could not react with Mycoplasma arginini PG2 and Mycoplasma mycoides subsp. PG3. Flow cytometry showed that McAb 4D11 reacted with surface VspX of M. bovis in a dose-dependent manner. Indirect immunofluorescence assay demonstrated that 4D11 McAb was able to detect rVspX protein binding to embryonic bovine lung cells. In the present study, McAbs against rVspX protein had been successfully prepared, which provided a basis for future researches about the function of VspX protein and the pathogenesis of M. bovis.

Key words: Mycoplasma bovis; VspX; monoclonal antibody; specificity

中图分类号:

S852.62

陈曦, 朱洪梅, 赵刚, 胡长敏, 陈颖钰, 郭爱珍. 牛支原体HB0801株VspX蛋白单克隆抗体的制备与鉴定[J]. 《中国畜牧兽医》, 2017, 44(3): 868-878.

CHEN Xi, Zhu Hong-mei, ZHAO Gang, HU Chang-min, CHEN Ying-yu, GUO Ai-zhen. Preparation and Identification of the Monoclonal Antibodies against VspX Protein in Mycoplasma bovis strain HB0801[J]. , 2017, 44(3): 868-878.

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牛支原体HB0801株VspX蛋白单克隆抗体的制备与鉴定

Preparation and Identification of the Monoclonal Antibodies against VspX Protein in Mycoplasma bovis strain HB0801

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