犬博卡病毒非结构基因NP1多克隆抗体的制备与鉴定

目的: 制备犬博卡病毒(MVC)的非结构基因NP1的兔多克隆抗体,并进行特异性鉴定。方法: 以MVC的感染性克隆pI-MVC为模板,构建NP1基因的原核表达质粒pGEX-4T-3-NP1,转化大肠杆菌BL21 后,在异巯基-1-硫代-β呋喃半乳糖(IPTG) 诱导下,表达谷胱甘肽S-转移酶(GST)-NP1蛋白。用亲和层析纯化的融合蛋白免疫大白兔,制备NP1抗血清并用ELISA法测定抗血清效价。采用Western blotting和免疫荧光法检测NP1抗血清的特性。结果: 成功构建MVC NP1基因的原核表达质粒pGEX-4T-3-NP1,在大肠杆菌中获得高效表达。表达产物经亲和层析纯化后得到高纯度的NP1融合蛋白,以其免疫大白兔获得NP1多抗血清,ELISA法检测效价达到1:400 000。Western blotting法及免疫荧光检测,所得抗血清具有较高的效价及特异性。 结论: 本研究利用原核表达的MVC GST-NP1 融合蛋白制备的NP1抗血清效价高、特异性高,为进一步深入研究NP1在MVC感染及致病分子机制中的作用奠定了基础。

Preparation of polyclonal antibody against non-structural NP1 of minute virus of canine and its property identification

Objective To prepare the polyclonal antibody against non-structural NP1 of minute virus of canine (MVC), and to identify its properties. Methods The target NP1 gene of MVC was amplified from MVC infection clone (pI-MVC),and was inserted into the pGEX-4T-3 vector to form the recombinant plasmid. Then the prokaryotic expression vector of pGEX-4T-3-NP1 was transform into E.coli (BL21),and the GST-NP1 fusion protein was induced under the optimized induction of isopropyl β-D-1-thiogalactopyranoside(IPTG).The recombinant proteins were purified using affinity chromatography.The purified fusion protein was inoculated into the adult rabbits to develop antiserum.After the titer of the antiserum was detected by ELISA,Western blotting and immunofluorescence methods were performed to evaluate the features of the prepared antiserum. Results The prokaryotic expression vector pGEX-4T-3-NP1 of MVC NP1 gene was successfully constructed.The soluble recombinant protein was highly expressed in E.coli BL21,and then it was purified and inoculated into the adult rabbits to obtain high titer antiserum.The ELISA result showed that the titer of the antiserum was 1:400 000.The Western blotting and immunofluorescence results showed that the specificity of the prepared antiserum was perfect. Conclusion The antiserum of NP1 prepared from MVC GST-NP1 fusion protein has shown a high titer and high specificity against NP1 proteins,which can be used for further study of NP1 in the molecular mechanisms of MVC infection.