抗猪伪狂犬病毒gE蛋白纳米抗体的表达及功能验证  

Expression and Functional Verification of Nano-antibody Against Porcine Pseudorabies virus gE Protein

作　　者：郭玉堃 马英先 明胜利 郭瑞珍 杨国宇 郭豫杰[1,2] GUO Yu-Kun;MA Ying-Xian;MING Sheng-Li;GUO Rui-Zhen;YANG Guo-Yu;GUO Yu-Jie(College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450002,China;Key Laboratory of Animal Biochemistry and Nutrition,Ministry of Agriculture and Rural Affairs,Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]河南农业大学动物医学院,郑州450002 [2]河南农业大学农业农村部动物生物化学与营养重点实验室,郑州450002

出　　处：《农业生物技术学报》2021年第10期2051-2060,共10页Journal of Agricultural Biotechnology

基　　金：国家重点研发计划项目(2017YFD0501100)。

摘　　要：伪狂犬病的暴发为其防控带来巨大挑战,建立一种新的特异性高与敏感性强的诊断方法,以及获得有病毒中和效果的抗体,对于该病的防控、治疗至关重要。本研究旨在原核表达出可溶性的抗猪(Sus scrofa)伪狂犬病毒(Pseudorabies virus, PRV) gE (envelope glycoprotein E)蛋白纳米抗体,并对抗猪伪狂犬病毒g E蛋白纳米抗体在猪伪狂犬病毒的诊断和治疗中的应用进行了初步探索。设计并合成了抗猪伪狂犬g E蛋白纳米抗体基因序列,命名为PRVgENb;将PRVgENb与pET21b载体构建后用大肠杆菌(Escherichia coli)表达系统进行融合蛋白的原核表达,经Ni^(2+)纯化后获得单一纳米抗体PRVgENb;将制备的纳米抗体PRVgENb进行HRP标记后,和PRV毒株用作直接ELISA (direct ELISA)试验和病毒中和试验。结果表明,原核表达与纯化后获得了纯度较高的融合蛋白PRVgENb;直接ELISA结果表明,在抗原浓度一定的条件下,纳米抗体PRVgENb能与特异性抗原在较低浓度条件下结合,具有较高活性,而在抗体浓度一定的条件下,其能敏感的识别低浓度到高浓度区间的特异性抗原;病毒中和试验结果表明,纳米抗体PRVgENb能抑制PRV的复制,有一定的中和活性。本研究建立了稳定获得纳米抗体PRVgENb的方法,并初步探索了其在直接ELISA中的使用,同时证明其能抑制PRV的复制。纳米抗体PRVgENb为PRV的诊断、治疗及后续研究奠定了实验基础。The outbreak of pseudorabies poses a huge challenge to its prevention and control. The establishment of a new diagnostic method with high specificity and sensitivity and the acquisition of antibodies with virus neutralizing effects are essential for the prevention, control and treatment of the disease.The purpose of this study was to express a soluble nano-antibody against the envelope glycoprotein E(gE)protein of porcine(Sus scrofa) Pseudorabies virus(PRV) in prokaryotic cells and to explore the application of this antibody in the diagnosis and treatment of porcine pseudorabies. In this study, the gene sequence of antiporcine pseudorabies gE protein antibody was designed and synthesized, which was named PRVgENb, and PRVgENb-pET21b vector was constructed. The fusion protein was expressed in Escherichia coli. After purification on a Ni^(2+)affinity chromatography column, the single nano-antibody PRVgENb was obtained,which was labeled with HRP for direct ELISA and PRV virus neutralization test. The results showed that the fusion protein PRVgENb had high purity after prokaryotic expression and purification. Direct ELISA results showed that under a certain concentration of antigen, a low concentration of nano-antibody PRVgENb bound to the specific antigen with high activity. While a certain concentration of antibody could sensitively recognize low to high concentrations of specific antigen. The results of the virus neutralization test showed that the nanoantibody PRVgENb inhibited the replication of PRV and had a certain neutralization activity. This experiment established a method to stably obtain the nanobody PRVgENb, and initially explored its use in direct ELISA,and proved that it can inhibit the replication of PRV. Nanobody PRVgENb lays an experimental foundation for the diagnosis, treatment and follow-up research of PRV.

关 键 词：猪伪狂犬病毒 gE蛋白 纳米抗体 直接ELISA 中和抗体 

分 类 号：S852.23[农业科学—基础兽医学]