Selection and characterization of specific nanobody against bovine virus diarrhea virus (BVDV) E2 protein.

Tiansen Li,Hui Zhang,Peng Wu,Meiling Huang,Jinhua Ding,Jinliang Sheng,Hongran Xiao,Guoqi Zhang,Chuangfu Chen

doi:10.1371/journal.pone.0178469

Abstract

Bovine viral diarrhea-mucosal disease (BVD-MD) is caused by bovine viral diarrhea virus (BVDV), and results in abortion, stillbirth, and fetal malformation in cows. Here, we constructed the phage display vector pCANTAB 5E-VHH and then transformed it into Escherichia coli TG1-competent cells, to construct an initial anti-BVDV nanobody gene library. We obtained a BVDV-E2 antigen epitope bait protein by prokaryotic expression using the nucleotide sequence of the E2 gene of the BVDV-NADL strain published in GenBank. Phage display was used to screen the anti-BVDV nanobody gene library. We successfully constructed a high quality phage display nanobody library, with an initial library capacity of 4.32×105. After the rescue of helper phage, the titer of the phage display nanobody library was 1.3×1011. The BVDV-E2 protein was then expressed in Escherichia coli (DE3), and a 49.5 kDa band was observed with SDS-PAGE analysis that was consistent with the expected nanobody size. Thus, we were able to isolate one nanobody that exhibits high affinity and specificity against BVDV using phage display techniques. This isolated nanobody was then used in Enzyme Linked Immunosorbent Assay and qRT-PCR, and ELISA analyses of BVDV infection of MDBK cells indicated that the nanobodies exhibited good antiviral effect.

Highlights

Bovine viral diarrhea-mucosal disease (BVD-MD), known as bovine viral diarrhea-mucosal disease virus or mucosal virus, is a contagious disease that often occurs in domestic animals [1, 2]
After rescue of the helper phage, the phage titer of nanobody library was 1.3×1011 with 96% exhibiting the appropriate size of gene insert (Fig 1D), which suggests that the diversity of the antibody library was good
We have shown that nanobody screening through a phage display approach can aid in developing new vaccine technologies and mitigating bovine viral diarrhea virus (BVDV) infectivity

Summary

Introduction

Bovine viral diarrhea-mucosal disease (BVD-MD), known as bovine viral diarrhea-mucosal disease virus or mucosal virus, is a contagious disease that often occurs in domestic animals [1, 2]. BVD-MD is caused by BVDV viral infection from viruses in the Pestivirus genus of the Flaviviridae family. Infection presents many symptoms for livestock, including diarrhea, persistent infection, miscarriage, and fetal malformation[3]. The main pathological features are gastrointestinal mucosal inflammation, intestinal wall erosion and lymphoid tissue necrosis, weight loss and leukopenia. Diseased livestock are the main infection reservoir and transmission to healthy livestock can occur by direct or indirect contact[4].

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Conclusion