禽脑脊髓炎病毒VP1蛋白纳米抗体的筛选及活性检测

Screening and active detection of nanobody for the VP1 protein of avian encephalomyelitis virus

禽脑脊髓炎病毒(avian encephalomyelitis virus,AEV)VP1蛋白作为诱饵蛋白,采用酵母双杂交技术对非免疫双峰驼纳米抗体酵母文库进行筛选。随机选择15个用QDO/X/A平板筛选到的纳米抗体(Nanobody or VHH)阳性克隆,经过酵母共转验证、序列测定分析,最终选择在QDO/X/A平板显强阳性且序列正确的8株VHHs在E.coli BL21(DE3)中进行表达、纯化;间接ELISA结果表明,筛选出的8株纳米抗体均与AEV标准抗原具有反应原性,其中2株纳米抗体VHH4、VHH9的活性高于阳性对照。这一结果为AEV的检测与病原研究奠定了一定的基础。

This study aimed to obtain nanobodies （or variable domain of heavy chain antibodies, VHH）against avian encephalomyelitis virus （AEV）. the AEV VP1 protein as bait was applied to screen the non- immunized Camelus Bactrianus VHH yeast two-hybrid （Y2H） library according to the Clontech Mate Plate System User Manual. 15 positive clones on the QDO/X/A plate were randomly selected through co-transformation validation in Y2HGOLD yeast bacteria and se- quence analysis,8 strongly positive VHHs on the QDO/X/A plate and validity of sequence were finally expressed and purified. The Indirect ELISA result showed that selected 8 VHHs had a re- actogenicity with the standard AEV antigen. Among them, the activity of two VHHs （VHH4, VHH9） was higher than that of positive control. This study laid a foundation for the detection and pathogen research of AEV.