抗犬细小病毒纳米抗体的制备及其中和活性鉴定

doi:10.16431/j.cnki.1671-7236.2022.09.028

摘要/Abstract

摘要： 【目的】建立抗犬细小病毒（CPV）纳米抗体（Nb）库，从中筛选出具有中和活性的Nb。【方法】以高免比格犬脾脏组织cDNA进行重链可变区（VH）扩增，与pBSD载体连接，构建pBSD-Nb库。结合细菌展示、流式细胞仪检测，在pBSD-Nb库中筛选高亲和力的Nb，获得阳性克隆并测序。利用Primer Premier 5.0软件设计Nb基因的上、下游引物，将阳性克隆质粒中Nb基因片段进行PCR扩增。利用pET-27b表达目的蛋白，通过变性、复性手段纯化目的蛋白，用SDS-PAGE分析纯化后的蛋白。将Nb包被96孔板，采用ELISA法检测Nb对CPV的亲和力。通过病毒微量中和试验检测Nb抑制CPV感染猫肾细胞（F81）的情况。【结果】 PCR扩增结果显示，在384 bp处可见明显条带，与预期大小相符，证明pBSD-Nb库构建成功。细菌展示后，流式细胞术检测结果显示，有9株阳性峰偏移率高的Nb，分别命名为Nb1、Nb2、Nb3、Nb4、Nb5、Nb6、Nb7、Nb8和Nb9。PCR扩增9株Nb基因片段，获得384 bp的条带。SDS-PAGE结果显示，在约14 ku处有一条带，说明成功获得纯化蛋白。ELISA检测结果表明，9株Nb中有6株Nb对CPV的亲和力较强。体外病毒中和试验检测发现，1株Nb能够中和病毒，抑制F81细胞病变，中和病毒的有效浓度为0.05 mg/mL。【结论】本研究成功建立pBSD-Nb库，筛选出6株对CPV具有高亲和力的Nb，并成功获得1株具有中和活性的Nb，为CPV治疗性抗体制剂的开发提供了新的思路。

关键词: 犬细小病毒(CPV); 纳米抗体; 细菌展示; 抗体活性

Abstract: 【Objective】 The aim of the experiment was to establish a canine nanobody (Nb) library against Canine parvovirus(CPV) and screen out the Nb with neutralizing activity.【Method】 Heavy chain variable region (VH) amplification was performed using cDNA from spleen tissue of high free Beagle dogs, then connected VH to pBSD vector to establish pBSD-Nb library.The high-affinity nanobodies were screened out from pBSD-Nb library by bacterial display technology combined with flow cytometry detection, and then positive clones were obtained and sequenced.The upstream and downstream primers of Nb genes were designed by Primer Premier 5.0 software, then Nb gene fragments were amplified by PCR.The target protein was expressed by pET-27b and purified by denaturation and renaturation.The purified protein were analyzed by SDS-PAGE.Nb was coated with 96 well plate, and the affinity of Nb to CPV was detected by ELISA method.The inhibition of Nb on CPV infection of F81 cells was detected by in vitro virus neutralization test.【Result】 PCR amplification results showed that a obvious band at 384 bp was consistent with the expected size, which proved that the pBSD-Nb library was successfully constructed.After the bacteria were shown, the results of flow cytometry showed that 9 clones of Nb with high positive peak deviation were screened and named Nb1, Nb2, Nb3, Nb4, Nb5, Nb6, Nb7, Nb8 and Nb9, respectively.9 clones of Nb gene fragments were amplified by PCR, and a band of 384 bp was obtained.SDS-PAGE results showed that there was a band at about 14 ku, indicating that the purified protein was successfully obtained.ELISA results showed that 6 of the 9 Nb strains had strong affinity for CPV.In vitro virus neutralization test showed that 1 strain of Nb could neutralize the virus and inhibit F81 cytopathic changes at an effective concentration of 0.05 mg/mL.【Conclusion】 In this study, the pBSD-Nb library was successfully established, 6 clones of Nb with high affinity for CPV were screened, and 1 clone of Nb with neutralizing activity was successfully obtained, providing a new solution for the development of CPV the rapeutic antibody.

Key words: Canine parvovirus (CPV); nanobody; bacterial display; antibody activity

中图分类号:

S852.65

任泽恒, 皮雪磊, 夏安然, 孙悦, 胡昌辉, 任桂萍. 抗犬细小病毒纳米抗体的制备及其中和活性鉴定[J]. 中国畜牧兽医, 2022, 49(9): 3539-3548.

REN Zeheng, PI Xuelei, XIA Anran, SUN Yue, HU Changhui, REN Guiping. Preparation and Identification of Neutralization Activity of Nanobody Against Canine Parvovirus[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3539-3548.

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