学术
2株重组弓形虫P30抗原单克隆抗体的制备和鉴定
王际平[1] 杨秋林[1] 蔡亮[1] 张如胜[1] 张愉快[1] 邱玉华[2]
[1]南华大学寄生虫教研室,湖南衡阳421001 [2]苏州大学免疫学研究室,江苏苏州215007
摘 要:
目的制备重组P30抗原单克隆抗体,研究其生物活性。方法重组质粒pET28b-P30经BL21（DE3）表达,纯化、复性后免疫BALB/c小鼠,运用杂交瘤技术制备单克隆抗体。ELISA筛选阳性克隆,经亚克隆建株。体内诱生腹水法制备抗体,测定其亚类、效价,Western blot分析其特异性,双单抗夹心-ELISA测定其敏感度。双单抗夹心-ELISA法检测弓形虫感染鼠血清中的CAg。结果筛选出2株抗重组P30的单克隆抗体9D7、2H9,抗体重链分别为IgG2a、IgG1,轻链均为κ链,Western blot显示2株单抗均能识别弓形虫速殖体天然P30抗原。双单抗夹心-ELISA检测抗原量为0.5μg/ml;同法检测鼠血清中的CAg,感染第4、6、8 d的阳性率分别为20%、60%、60%。结论制备的P30抗原单克隆抗体具有较高的敏感性和特异性,为其应用研究打下了基础。[著者文摘]
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文章出处:
《中国病原生物学杂志》-2007年2卷5期 -344-346页
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文献标识码:
A
文章编号:
1673-5234（2007）05-0344-03
Preparation and identification of two monoclonal antibodies against Toxoplasma gondii rP30
WANG Ji-ping ,YANG Qiu-lin ,CAI Liang , ZHANG Ru-sheng , ZHANG Yu-kuai , Qiu Yu-hua （1. Department of Parasitology, Nanhua University, Hengyang 421001, China ; 2. Lab of Immunology, Suzhou University）
Abstract:
Objective To prepare the monoclonal antibody（McAbs） against recombinant P30（rP30） of Toxoplasma gondii and analyze its biological characteristics. Methods After expressed in Escherichia coli （DE3） , recombinant protein P30 was purified, renatured and used to immunize mice. B lyphocytes hybridization technique was applied to prepare the aimed McAb. Positive clones was screened with ELISA, then subcloned to establish stable cell lines. Ascites were induced to produce the McAbs. The specificity of McAbs was identified by Western blot analysis. And Double-McAb-Sandwich ELISA was used to detecte the sensitivity of McAb and the T. gondii CAg in the infected mouse serum. Results Two monoclonal antibodies against T. gondii rP30 （9D7, 2H9） were obtained and they secret anti-P30 McAb continuosly and steadily. Their subclasses belong to IgG 1, IgG 2a respectively and the light stains, both of them are κ. The result of Western blot indicated that the McAbs can identify the P30 from lysed T. gondii tachyzoites. The sensibility was 0.5 μg/ml. The positive rate of CAg in mice serum was 20%, 60%, 60% on 4, 6, 8 days post-T, gondii infection, respectively. Conclusion The mouse anti-P30 McAbs are prepared successfully, which lay the foundation for further study on related applications.[著者文摘]
Key words:
Recombinant P30; B lyphocytes hybridization technique; monoclonal antibody; double-McAb-Sandwich-ELISA; circulating antigen
基金资助:
致谢：感谢苏州大学生物技术研究所邱玉华教授,孙中文博士和苏州第五人民医院朱翔博士的热情帮助和技术指导.
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