坦布苏病毒E蛋白和结构域Ⅱ的生物信息学分析及多克隆抗体制备OA

Multiple bioinformatics tools were used to predict the Tembusu virus(TMUV)envelope E protein and its domain Ⅱ(ED Ⅱ)the transmembrane domains.Recombinant expression plasmids were constructed,expressed and purified the recombinant E and ED Ⅱ proteins in a prokaryotic system.Mouse-derived polyclonal antibodies against the E and ED Ⅱproteins were prepared.An indirect ELISA method was established using a checkerboard titration to determine antibody titers,and the specificity of the antibodies was evaluated by Western blotting and indirect immunofluorescence assay(IFA).The results showed that:(1)Bioinformatics analysis revealed that the E protein consists of 501 amino acid residues,is a hydrophilic protein with two transmembrane regions and no signal peptide.A total of 50 phosphorylation sites and two N-glycosylation sites(154 and 314 aa)were predicted.In the secondary structure of the E protein,α-helix,extended strand,β-turn,and random coil account for 22.55%,31.14%,11.78%,and 34.53%,respectively.Tertiary structure prediction indicated that the E protein consists of three domains(ED Ⅰ,ED Ⅱ,and ED Ⅲ).The prokaryotic expression vectors pET-28a-E and pET-28a-ED-Ⅱ were successfully constructed,and the recombinant E and ED Ⅱ proteins were purified and verified.Mouse-derived polyclonal antibodies against these proteins exhibited high titers of up to 1:1638400 and 1:409600,respectively.Both Western blotting and IFA confirmed the excellent specificity of the antibodies.