诺如病毒NV衣壳蛋白VP1表达纯化及多克隆抗体的制备

Expression and purification of Norovirus capsid protein VP1 and preparation of the polyclonal antibodies

目的获得纯化的诺如病毒(NV)衣壳蛋白VP1,免疫动物制备多克隆抗体。方法提取粪便样品中诺如病毒RNA,逆转录得到cDNA文库,通过PCR扩增获取VP1基因序列,构建到大肠埃希菌原核表达系统中诱导表达重组VP1蛋白。使用镍柱亲和层析法对重组蛋白进行纯化,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和考马斯亮蓝法(BSA)对重组蛋白的纯度与浓度进行分析,以重组的VP1蛋白为抗原,免疫雄性SPF级SD大鼠获得多抗血清,用ELISA测定抗体效价、Western blot检测抗体特异性。结果成功地构建出重组表达载体VP1-pET28a,并将其在大肠埃希菌BL21(DE3)中稳定地表达诱导重组蛋白。ELISA测其多抗血清的平均效价为1∶200 000,Western blot检测抗体在原核和真核特异性很高。结论本实验成功地利用原核表达系统表达诺如病毒衣壳蛋白VP1,为进一步研究诺如病毒的诊断和疫苗开发提供了条件。

Objective To obtain purified Norovirus capsid protein VP1,and prepare the polyclonal antibodies against VP1.Methods The RNA of Norovirus strain preserved in laboratory was extracted and reverse transcribed.VP1 gene sequence was obtained by PCR amplification and constructed in the prokaryotic expression system of E.coli to induce the expression of recombinant VP1 protein.The recombinant protein was purified by using nickel column affinity chromatography,and analyzed with SDS-PAGE and BSA methods.With the recombinant VP1 protein as the antigen,male SD rats（SPF）were immune to get the antiserum,which was detected for the titer by using ELISA,and for the specificity by using Western blot.Results The recombinant expression vector VP1-pET28 awas successfully constructed,and stably expressed in E.coli BL21（DE3）.The average titer of the polyclonal antibody was 1∶200 000 by ELISA.Western blot confirmed high prokaryotic and eukaryotic specificity of the antibodies.Conclusion With the success of the prokaryotic expression system of Norovirus capsid protein VP1,the study provided a basis for further study on the diagnosis of Norovirus and development of the vaccine.