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【目的】研究对虾白斑综合征病毒(White spot syndrome virus,WSSV)囊膜蛋白sVP53B克隆、表达、纯化及抗血清制备。【方法】根据WSSV囊膜蛋白基因序列,设计引物,PCR扩增出功能序列(Svp53B),构建到pET-16b载体后,转化至大肠杆菌Rosetta 2诱导表达,用SDS-PAGE、Western blotting检测优化表达。表达产物采用Ni-NTA琼脂糖磁珠进行纯化、割胶回收融合蛋白,以纯化的Svp53B-his为抗原,免疫兔子获得多克隆抗体,通过间接ELISA检测抗体的效价。【结果】构建重组质粒p ET-16b-Svp53B,在大肠杆菌Rosetta 2中以1 mmol/L IPTG诱导表达量最高,主要以包涵体形式表达。纯化包涵体蛋白免疫兔子,获得多克隆血清,效价达到1:150 000。【结论】原核表达并纯化得到高纯度的WSSV囊膜蛋白s VP53B,制备的兔源多克隆血清亲和力高、特异性好,这对后期进一步研究VP53B与经口侵染相关功能奠定了基础。
【Objective】 To investigate the cloning, expression, purification and antiserum preparation of sVP53B from White Spot Syndrome Virus (WSSV). 【Method】 According to the sequence of WSSV envelope protein, a functional fragment (Svp53B) was amplified by PCR and designed into pET-16b vector. The recombinant plasmid was transformed into E.coli Rosetta 2 and induced by SDS-PAGE and Western blotting expression. The expressed product was purified by Ni-NTA agarose beads, and the fusion protein was recovered by tapping. The purified Svp53B-his antigen was used to immunize rabbits to obtain polyclonal antibodies. The antibody titer was detected by indirect ELISA. 【Results】 The recombinant plasmid p ET-16b-Svp53B was induced by 1 mmol / L IPTG in Escherichia coli Rosetta 2 and expressed mainly in inclusion bodies. Rabbit was immunized with purified inclusion body protein to obtain polyclonal serum with a titer of 1: 150,000. 【Conclusion】 Prokaryotic expression and purification of highly purified WSSV capsid protein VP53B, prepared rabbit polyclonal serum with high affinity and specificity, which laid the foundation for further study on the function of VP53B and oral infection.