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目的通过杆状病毒表达系统,获取高纯度GⅡ.3型诺如病毒重组衣壳蛋白,为制备抗GⅡ.3型诺如病毒单克隆和多克隆抗体提供免疫原。方法将GⅡ.3型诺如病毒衣壳蛋白基因片段修饰后插入p HTA表达载体中,经测序鉴定,将鉴定正确的重组质粒转化到MAX Efficiency~DH10Bac~(TM)感受态细胞中,获取表达杆粒并转染SF9细胞,表达GⅡ.3型诺如病毒重组衣壳蛋白。重组蛋白用Ni-NTA His蛋白亲和柱纯化,十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白免疫印迹(Western blot)方法鉴定。结果 SDS-PAGE和Western blot结果表达了分子量约为60 k Da的重组蛋白,动物试验表明重组蛋白具有较好的免疫原性,为制备抗GⅡ.3型诺如病毒单克隆抗体和多克隆抗体、建立相应的免疫学检测方法奠定了基础。结论构建了GⅡ.3型诺如病毒衣壳蛋白表达载体,并获得GⅡ.3型诺如病毒重组衣壳蛋白。
OBJECTIVE To obtain the recombinant capsid protein of high purity GⅡ.3 Norovirus by baculovirus expression system and to provide immunogen for the preparation of monoclonal and polyclonal antibodies against GⅡ.3 Norovirus. Methods The genotypes of Norovirus capsid protein of GⅡ.3 were inserted into p HTA expression vector and identified by sequencing. The correct recombinant plasmids were transformed into MAX Efficiency ~ DH10Bac ~ (TM) competent cells to obtain Stem cells were transfected and transfected into SF9 cells to express GII .3 Norovirus recombinant capsid protein. Recombinant proteins were purified by Ni-NTA His affinity column, SDS-PAGE and Western blot. Results The SDS-PAGE and Western blot results showed that the recombinant protein had a molecular weight of about 60 kDa. Animal experiments showed that the recombinant protein had good immunogenicity. To prepare monoclonal antibodies against GⅡ.3 Norovirus and polyclonal antibodies , The establishment of the corresponding immunological detection methods laid the foundation. Conclusions A Norovirus type Ⅱ coat protein expression vector was constructed and a Norovirus type Ⅱ coat protein was obtained.