中国农业科学院农业信息研究所 中国农学会
Agricultural Information Institute of CAAS China Association of Agricultural Science Societies
En
Ch
中国农业科学院农业信息研究所 中国农学会 主办

AI检索

订阅

返回顶部
当前位置: 首页 > 文章 > 肠出血性大肠杆菌VT1B亚单位的重组表达及其单克隆抗体的制备 中国兽医科学 2010 (5) 475-480
Position: Home > Articles > Recombinant expression of VT1B gene of enterohemorrhagic Escherichia coli and the preparation of monoclonal antibodies against the expressed recombinant protein Chinese Veterinary Science 2010 (5) 475-480

肠出血性大肠杆菌VT1B亚单位的重组表达及其单克隆抗体的制备

作  者：
洪杰华;李广兴;任晓峰;霍贵成;冯书章
单  位：
东北农业大学动物医学院;东北农业大学动物医学学院东北农业大学食品科学学院军事医学科学院军事兽医研究所;东北农业大学动物医学学院;军事医学科学院军事兽医研究所
关键词：
肠出血性大肠杆菌;VT1B;原核表达;单克隆抗体
摘  要：
为制备抗肠出血性大肠杆菌(EHEC)VT1B亚单位的单克隆抗体(McAb),以大肠杆菌EDL933的基因组DNA为模板扩增VT1B基因,纯化后经BamHⅠ和XhoⅠ酶切,连入表达载体pGEX-6P-1,构建重组表达质粒pGEX-VT1B,转化到大肠杆菌BL21中,经IPTG诱导,实现了融合蛋白的高效表达,表达量约占菌体总蛋白的27%。重组蛋白纯化后,免疫BALB/c小鼠制备单克隆抗体,获得3株融合细胞1G11、2H8、1B10,Western-blotting表明,此3株单抗能与VT1B蛋白特异性结合而与载体蛋白不反应。Ig亚类分别为IgG1、IgG2a和IgG2b,染色体数均大于100,腹水单抗ELISA抗体效价大于1∶256000,亲和常数分别为0.54×108M-1、0.95×108M-1、0.33×107M-1,间接ELISA叠加试验初步鉴定结果表明,这3株单抗针对的是同一抗原表位。此3株特异性单抗的获得为VT1毒素的检测奠定了基础。
译  名：
Recombinant expression of VT1B gene of enterohemorrhagic Escherichia coli and the preparation of monoclonal antibodies against the expressed recombinant protein
作  者：
HONG Jie-hua1,LI Guang-xing1,REN Xiao-feng1,HUO Gui-cheng2,FENG Shu-zhang3 (1.College of Veterinary Medicine,Northeast Agricultural University,Harbin 150030,China;2.College of Food Science,Northeast Agricultural University,Harbin 150030,China;3.Military Veterinary Institute,Academy of Military Medicine of PLA,Changchun 130062,China)
关键词：
enterohemorrhagic Escherichia coli;Vero toxinⅠB;prokaryotic expression;monoclonal antibody
摘  要：
In order to develop monoclonal antibodies(McAbs) against VT1B of enterohemorrhagic Escherichia coli(EHEC),a VT1B fragment was amplified from E.coli EDL933 by PCR and digested with the endonucleases BamHⅠ and XhoⅠ.After being purified,the fragment was inserted into the expression vector pGEX-6P-1.The recombinant plasmid was named pGEX-VT1B,and transformed into E.coli BL21.The recombinant protein was efficiently expressed in form of inclusion bodies,and made up to 27% of total cell proteins.The BALB/c mice were immunized with the purified recombinant protein to prepare monoclonal antibodies and three strains McAbs 1G11,2H8 and 1B10 were screened from the immunized mice.Western-blotting showed that all McAbs could react specifically against VT1B rather than the GST and were classified into IgG1,IgG2a and IgG2b respectively with the chromosome number of over 100.The indirect ELISA showed that the titer of ascites was over 256000 each and the affinity constant was 0.54×108M-1,0.95×108M-1 and 0.33×107M-1,respectively.The additional ELISA indicated that 1G11,2H8 and 1B10 recognized the same antigenic epitope.The preparation of 3 McAbs provided a basis for detecting Vero toxinⅠ.

相似文章

导出引用
计量
文章访问数: 5
HTML全文浏览量: 0
PDF下载量: 0

所属期刊

推荐期刊

友情链接Friendly link
农业信息研究所Agricultural information institute 农业智能知识服务平台Agricultural intelligent knowledge service platform 农业学术服务平台Agricultural Knowledge Service System 中国知网CNKI 维普网VIP 万方WANFANG DATA
联系我们Contact us

电话Tell：010-82109652-808

邮箱E-mail：agrijournal@caas.cn

服务Service
关于我们About us 意见建议Opinions and suggest
版权说明Copyright
单位：中国农业科学院农业信息研究所 中国农学会 © 2017中国农业科学院农业信息研究所 ICP备案号：京ICP备09089781号-46