摘要
目的:以不同形式的A型肉毒毒素或类毒素为靶抗原,对已经构建的人源天然ScFv噬菌体抗体库进行特异性抗体的筛选及特异性抗体的序列分析。方法:制备3种不同形式的A型肉毒毒素或类毒素,抗原梯度包被免疫管,对人源天然ScFv噬菌体抗体库进行筛选,经过3轮"吸附-洗脱-扩增"淘选富集,制备单克隆噬菌体抗体颗粒,用ELISA验证ScFv的结合活性,并对获得的阳性克隆进行基因序列分析。结果:用3种不同形式的抗原进行筛选,各挑180个克隆进行单克隆噬菌体抗体颗粒的ELISA分析,其中以A型肉毒类毒素筛选获得52个阳性克隆,阳性率为28.8%,随机挑取12个克隆测序,结果表明7个ScFv序列正确且各不相同;以纯化后A型肉毒神经毒素(BONT/A)筛选获得18个阳性克隆,阳性率为10%,测序结果表明获得的14个ScFv序列正确且各不不同;以原核表达的A型肉毒毒素重链C段筛选获得122个阳性克隆,阳性率为68%,随机挑取55个克隆测序,结果表明30个正确的ScFv序列中14个系列完全相同,16个序列各不相同;结论:应用固相筛选的方法,从人源天然ScFv噬菌体抗体库可以获得肉毒毒素特异性抗体,为抗肉毒毒素马血清的更新替换提供了研究依据与应用基础。
Objective：To obtain specific ScFv from human nave ScFv phage display library with different format type A botulinum toxin or toxoid.Methods：The human nave ScFv phage display library was planned for three rounds with different format antigens.The antigen binding activities of random clones were tested by ELISA in order to select specific antibodies.The sequence of part positive clones were analyzed.Results：Random 180 clones which were picked respectively from three different format antigens were analyzed.For type A botulinum toxoid,52 positive clones were gotten.The positive rate was about 28.8%.Sequence analysis showed 7/12 sequences were correct and different.For BONT/A,18 positive clones were gotten.The positive rate was about 10%.Sequence analysis showed 14/18 sequences were correct and different.For BONT/A-Hc,122 positive clones were gotten.The positive rate was about 68%.Sequence analysis showed 30/55 sequences were correct.14/30 sequences were same and 16/30 sequences were different.Conclusion：We can get specific antibodies against type A botulinum toxin or toxoid from human nave ScFv phage display library with immobilized antigen.These results are basement for getting better quality therapeutic antibodies against type A botulinum toxin.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2011年第12期1093-1096,1100,共5页
Chinese Journal of Immunology
关键词
噬菌体抗体库
A型肉毒毒素
筛选
Phage antibody library
Type A botulinum toxin
Screening
