摘要
本研究参考GenBank中蓝舌病毒16型(Bluetongue virus serotype 16,BTV-16)标准株RSAvvvv的序列,人工合成编码BTV-16 NS2蛋白的S8基因,将其亚克隆至原核表达载体pPRoEx-HTb中,构建重组表达质粒pPro-NS2,并转化大肠杆菌BL21进行IPTG诱导表达,并进行可溶性分析。SDS-PAGE结果显示,表达的rNS2蛋白主要以包涵体的形式存在于菌体中。利用组氨酸标签纯化树脂获得了高纯度的NS2蛋白,免疫新西兰白兔制备抗NS2蛋白的多克隆抗体。Western blot结果显示,制备的抗NS2蛋白多克隆抗体不仅可与重组表达的rNS2蛋白反应,而且可与BTV感染细胞后的天然NS2蛋白反应。间接免疫荧光结果显示,该抗体也可与BTV感染C6/36细胞中的天然NS2蛋白反应,且发现NS2大量分布于C6/36细胞的胞质中。本研究所制备的NS2蛋白的多克隆抗体具有很好的反应性和特异性,为进一步研究NS2蛋白的功能奠定了基础。
The S8 gene encoding for NS2 protein of Bluetongue virus serotype 16(BTV-16) was synthesized according to the sequence of the reference strain RSAvvvv in GenBank and then cloned into expression plasmid pProEx-HTb to construct the recombinant plasmid pPro-NS2. The resulting pPro-NS2 was transformed into E.coli BL21 for expression with induction of IPTG. The recombinant NS2 protein(rNS2) was expressed as inclusion bodies and purified with His-Tag purification resin. New Zealand white rabbits were immunized with rNS2 to generate the polyclonal antibodies. The specificity and reactivity of the antibodies were evaluated in Western blot and immunofluorescence assay(IFA). The NS2 antibodies reacted with the recombinant protein from E. coli as well as natural protein in BTVinfected cells in Western blot. In addition, the NS2 protein was distributed in the whole cytoplasm of BTV-infected C6/36 cells as shown in IFA. The availability of the NS2 antibodies would be useful for the analysis of the function of NS2 protein.
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