牛病毒性腹泻病毒E2蛋白纳米抗体筛选及反应原性检测

通过噬菌体展示技术筛选牛病毒性腹泻病毒(BVDV)重组E2蛋白特异性纳米抗体,验证纳米抗体反应原性。使用BVDV灭活疫苗免疫羊驼,分别在第0、21、49及70天采集全血,测得抗体效价后分离全血中淋巴细胞,提取总RNA,反转录后PCR扩增目的片段。目的片段和pCANTAB5E使用限制性内切酶酶切连接后转至TG1感受态细胞中,应用噬菌体展示技术构建VHH噬菌体展示文库。再经过3轮"吸附-洗脱-筛选"后得到与BVDV-E2结合的噬菌体,用ELISA鉴定其反应性。结果获得插入率为90.8%,库容为1.02×107 CFU/mL的文库。ELISA结果和序列分析显示,得到2条与E2蛋白具有良好反应性的纳米抗体且与VHH同源性较高的序列。研究结果为BVDV的防控和新型疫苗的研制奠定基础。

Screening of the E2 nanobody against bovine viral diarrhea virus and detection of its reactogenecity

The aim was to obtain the specific nanobodies against bovine viral diarrhea virus.Using BVDV-E2 recombinant protein to immunize the lymphocytes in the blood were separated.The phage display library was constructed by using phage display technology,and then the phages combined with BVDV-E2 protein were obtained through three successive bioscai sieves,the obtained VHH sequences were sequences and alignmented.The high affinity nanodody against BVDV-E2 was screened by ELISA,and then the affinity and activity of the nanobody were verified.The result showed that phage expression library with a insertion rate 90.8%,the database 1.02×107 CFU/mL was successfully constructed.Five BVDV-E2 positive clones were obtained by screening,these genes were clone into prokaryotic expression system,and the high purity BVDV-E2 nanobodies were obtained.Two different high affinity VHH genes were obtained by affinity identification.The high purity BVDV-E2 nanobodies could be combined with artificial antigen E2,and also be blocked by competitive nanobodies.This study laid a foundation for further study on assembly of BVDV kit and development of BVDV vaccine.