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| 抗堆型艾美耳球虫子孢子单链抗体的构建及表达 | |
| 引用本文： | 王承民,初冬,秦建华,刘丽艳,魏刚才,谢红兵,齐永华,王三虎,吴艳云,何宏轩.抗堆型艾美耳球虫子孢子单链抗体的构建及表达[J].河南农业大学学报,2007,41(2):207-211. |
| 作者姓名： | 王承民 初冬 秦建华 刘丽艳 魏刚才 谢红兵 齐永华 王三虎 吴艳云 何宏轩 |
| 作者单位： | 1. 河南科技学院细胞工程重点实验室,河南,新乡,453003 2. 国家林业局野生动物疫源疫病监测总站,辽宁,沈阳,110034 3. 河北农业大学动物科技学院,河北,保定,071001 4. 中国科学院动物研究所国家野生动物疫病研究中心,北京,100080 |
| 基金项目： | 河南科技学院重点基金资助项目(200313) |
| 摘 要： | 应用RT-PCR技术,从稳定分泌抗堆型艾美耳球虫子孢子抗原的单克隆抗体(McAb)的杂交瘤细胞Easp-3G3中扩增出抗体VH和VL基因,通过在VH和VL可变区基因之间引入连接肽(Gly4Ser)3,体外构建抗堆型艾美耳球虫的单链抗体基因,并将其克隆至表达载体pET28a( )载体中进行表达,用Ni-NTA的金属亲和层析法进行纯化,通过SDS-PAGE和ELISA测定其纯度和生物学活性.电泳结果分析表明重组蛋白的相对分子量为32kDa,纯度为94%;ELISA反应结果证明该单链抗体保持了单克隆抗体的生物学特性,并能与堆型艾美耳球虫子孢子抗原发生特异性反应. |
| 关 键 词： | 堆型艾美耳球虫 子孢子 单链抗体 基因克隆 表达 |
| 文章编号： | 1000-2340（2007）02-0207-05 |
| 收稿时间： | 2006-10-10 |
| 修稿时间： | 2006-10-10 |
Construction and Expression of ScFv against Eimeria acervulina Sporozoite |
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| WANG Cheng-min,CHU Dong,QIN Jian-hua,LIU Li-yan,WEI Gang-cai,XIE Hong-bing,QI Yong-hua,WANG San-hu,WU Yan-yun,HE Hong-xuan.Construction and Expression of ScFv against Eimeria acervulina Sporozoite[J].Journal of Henan Agricultural University,2007,41(2):207-211. | |
| Authors: | WANG Cheng-min CHU Dong QIN Jian-hua LIU Li-yan WEI Gang-cai XIE Hong-bing QI Yong-hua WANG San-hu WU Yan-yun HE Hong-xuan |
| Institution: | 1. Cell Engineering Key Lab,Henan Institute of Science and Technology,Xinxiang 453003,China; 2. State Research Center for Wildlife Animal Disease Control, Institute of Zoology of Chinese Academy of Sciences, Beijing 100080,China ; 3. College of Animal Science and Technology, Hebei Agricultural Aniversity, Baoding,071001 , China ; 4. General Station of Wildlife Pathogen-Origins and Epidemic Diseases Monitoring,State Forestry Administration, Shenyang 110034, China |
| Abstract: | VH and VL genes were amplified from hybridoma cell Easp-3G3,secreting monoclonal antibody Easp-3G3,by RT-PCR,and connected by linker(Gly4Ser)3 to form scFv gene,which was cloned into expression vector pET28a( ) and finally expressed in E.coli.The expressed product Easp-3G3-scFv was purified by metal affinity chromatography using Ni-NTA,its purity and biological activity were determined using SDS-PAGE and ELISA.SDS-PAGE analysis showed that the relative molecular weight of recombinant protein was 32 kDa with the purity of 94%.ELISA assay revealed that Easp-3G3-scFv retained the immunoactivity of parent mAb,being capable of binding specifically to Eimeria acervulina sporozoite antigen. |
| Keywords: | Eimeria acervulina sporozoite ScFv gene cloning expression |
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