The full-length gE gene was amplified by PCR from viral DNA extracted from the isolates as described previously [23 (link)]. The PCR product was purified using the V-ELUTE Gel Mini Purification Kit (Beijing Zoman Biotechnology Co., Ltd., Beijing, China), then ligated into the pMDTM 18 T Vector Cloning Kit (Takara, Dalian, China), and finally transformed into TreliefTM 5α Chemically Competent Cells (Tsingke, Beijing, China). The positive monoclonal clones were verified by PCR and sequenced by Wuhan AuGCT DNA-SYN Biotechnology Co., Ltd. (Wuhan, China), in triplicate.
Twenty-eight PRV strains were retrieved from the NCBI database and served as the reference strains. The nucleotide sequences and the corresponding amino acid variations in the gE gene between PRV isolates sequenced in our study and the reference strains were analyzed using DNASTAR Lasergene.v7.1 (DNASTAR, Inc., Madison, WI, USA). The phylogenetic tree was constructed by the neighbor-joining method using the MEGA 7.0 software (www.megasoftware.net ) with a bootstrap of 1000 replicates [24 (link)].
Twenty-eight PRV strains were retrieved from the NCBI database and served as the reference strains. The nucleotide sequences and the corresponding amino acid variations in the gE gene between PRV isolates sequenced in our study and the reference strains were analyzed using DNASTAR Lasergene.v7.1 (DNASTAR, Inc., Madison, WI, USA). The phylogenetic tree was constructed by the neighbor-joining method using the MEGA 7.0 software (