新城疫病毒F蛋白纳米抗体的筛选及活性鉴定

doi:doi: 10.11843/j.issn.0366-6964.2016.08.015

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (8): 1645-1651.doi: doi: 10.11843/j.issn.0366-6964.2016.08.015

新城疫病毒F蛋白纳米抗体的筛选及活性鉴定

高小龙△，胡湘云△，付向晶，王燕平，仝丽娜，王海新，王兴龙，张淑霞，萧飒，杜恩岐^*，杨增岐^*

（西北农林科技大学动物医学院，杨凌 712100）

收稿日期:2016-02-29 出版日期:2016-08-23 发布日期:2016-09-08
通讯作者: 杨增岐，教授，E-mail：yzq8162@163.com
作者简介:高小龙（1988-），男，陕西宝鸡人，博士，主要从事动物传染病和基因工程抗体研究，E-mail：gaoxiaolong1017@163.com；△共同第一作者
基金资助:
国家自然科学基金（31272578）；西北农林科技大学引进人才科研启动基金（Z111021006）

Screening and Characterization of VHH against Newcastle Disease Virus Fusion Protein

GAO Xiao-long^△，HU Xiang-yun^△，FU Xiang-jing，WANG Yan-ping，TONG Li-na，WANG Hai-xin，WANG Xing-long，ZHANG Shu-xia，XIAO Sa，DU En-qi^*，YANG Zeng-qi^*

(College of Veterinary Medicine，Northwest A & F University，Yangling 712100，China)

Received:2016-02-29 Online:2016-08-23 Published:2016-09-08

摘要/Abstract

摘要：

旨在筛选新城疫病毒（NDV）F蛋白的纳米抗体，并对筛选的抗体活性进行初步鉴定。以NDV F蛋白中和表位构建诱饵，利用酵母双杂交技术对双峰驼天然重链抗体可变区（VHH）酵母双杂交文库进行筛选，获得4株VHH抗体序列。挑选其中2株最符合VHH特征的进行原核表达与纯化，并对2株VHH活性进行鉴定。ELISA结果显示，VHH1与VHH2与NDV病毒的反应性显著高于阴性对照（P<0.05），表明VHH与NDV病毒反应活性良好；Western blot结果显示，2株VHH可特异性识别F蛋白；细胞中和试验结果表明，2株VHH抗体均具有一定的中和活性。以上结果提示本研究成功筛选出2株活性较好的抗NDV F蛋白的VHH抗体，为VHH在NDV防控中的应用奠定了基础。

关键词: 新城疫病毒, F蛋白, VHH抗体, 酵母双杂交

Abstract:

This study aimed to obtain VHH (variable domains of camelid heavy-chain antibodies，VHH) against Newcastle disease virus (NDV) fusion protein (F protein).The synthesised DNA sequence encoding neutralizing epitope of F protein was used to construct bait plasmid.Then，the constructed bait was used to screen a naive Camelus Bactrianus VHH yeast two-hybrid library through yeast two-hybrid technology.After screening，four positive clones were primarily obtained and two of four VHHs with hallmark of VHH sequence were picked out for expression and purification.Subsequently，the biological activities of purified VHH were evaluated.Indirect ELISA results showed that the reactivity between VHH1 or VHH2 and NDV virion was significant higher than negative control.Western blot results suggested that VHH could specifically react with F protein.Neutralization assay further demonstrated that these two VHH could inhibit the replication of virulent NDV at some extent.These results indicate that we successfully obtained two VHH against NDV F protein，which laid the foundation for its application in ND control.

Key words: Newcastle disease virus, fusion protein, VHH, yeast two-hybrid

中图分类号:

高小龙，胡湘云，付向晶，王燕平，仝丽娜，王海新，王兴龙，张淑霞，萧飒，杜恩岐，杨增岐. 新城疫病毒F蛋白纳米抗体的筛选及活性鉴定[J]. 畜牧兽医学报, 2016, 47(8): 1645-1651.

GAO Xiao-long，HU Xiang-yun，FU Xiang-jing，WANG Yan-ping，TONG Li-na，WANG Hai-xin，WANG Xing-long，ZHANG Shu-xia，XIAO Sa，DU En-qi，YANG Zeng-qi. Screening and Characterization of VHH against Newcastle Disease Virus Fusion Protein[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(8): 1645-1651.

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新城疫病毒F蛋白纳米抗体的筛选及活性鉴定

Screening and Characterization of VHH against Newcastle Disease Virus Fusion Protein

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