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目的构建犬博卡病毒(CBV)结构蛋白VP2的兔抗VP2多克隆抗体并进行特异性鉴定。方法应用PCR方法扩增获得CBV结构蛋白VP2 C末端区域300个氨基酸所对应的基因片段。经双酶切和测序分析后,连接到原核表达载体p ET-32a(+),构建重组质粒p ET-32a(+)-VP2,转化至E.coli BL21中,异丙基硫代β-D-半乳糖苷(IPTG)诱导表达,表达产物SDS-PAGE分析鉴定;纯化融合蛋白后免疫新西兰家兔,制备多克隆抗体,检测抗体效价及特异性。结果通过酶切和测序证实成功构建原核表达载体p ET-32a(+)-VP2,转化后可以成功诱导并纯化出大小与预期一致的蛋白;制备的多克隆抗体经ELISA检测效价达到1∶6 400 000,Western blot法及免疫荧光染色鉴定抗体的特异性。结论成功制备了抗CBV结构蛋白VP2的多克隆抗体。
Objective To construct and characterize rabbit anti-VP2 polyclonal antibody against VP2 of canine bocavirus (CBV). Methods The gene fragment corresponding to 300 amino acids in the C-terminal region of CBV structural protein VP2 was amplified by PCR. After double digestion and sequencing analysis, the recombinant plasmid p ET-32a (+) - VP2 was ligated into prokaryotic expression vector p ET-32a (+), and transformed into E.coli BL21. D-galactoside (IPTG) induced expression, the expression product was identified by SDS-PAGE analysis; purified fusion protein was immunized New Zealand rabbits, polyclonal antibodies were prepared to detect antibody titer and specificity. Results The prokaryotic expression vector p ET-32a (+) - VP2 was successfully constructed by restriction enzyme digestion and sequencing. The expected size of the protein was successfully induced and purified after transfection. The titer of the prepared polyclonal antibody was 1:1 6 400 000, Western blot and immunofluorescence staining to identify the specificity of the antibody. Conclusion The polyclonal antibody against CBV structural protein VP2 was successfully prepared.