Expression and Functional Verification of Nano-antibody Against Porcine Pseudorabies virus gE Protein
GUO Yu-Kun1,2, MA Ying-Xian1,2, MING Sheng-Li1,2, GUO Rui-Zhen1,2, YANG Guo-Yu1,2, GUO Yu-Jie1,2,*
1 College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China; 2 Key Laboratory of Animal Biochemistry and Nutrition, Ministry of Agriculture and Rural Affairs, Henan Agricultural University, Zhengzhou 450002, China
Abstract：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 anti-porcine 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 Ni2+ 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 nano-antibody 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.
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