诺如病毒P2结构域的原核表达及其抗血清制备

Prokaryotic expression of norovirus P2 domain and preparation of its antiserum

目的应用原核表达系统表达并纯化5种基因型(GⅠ.1、GⅡ.2、GⅡ.3、GⅡ.4、GⅡ.17)诺如病毒(norovirus,NV)的P2结构域,并制备其抗血清。方法利用生物信息学方法分析不同基因型NV P2序列保守性,合成P2 DNA序列,亚克隆至pET24a载体,构建重组原核表达质粒,转化大肠埃希菌BL21(DE3)感受态细胞,IPTG诱导表达。表达的重组P2蛋白纯化后免疫30只雌性BALB/c小鼠,制备抗血清。通过ELISA法检测抗血清的滴度和交叉反应,Western blot法检测抗血清的特异性,点杂交法分析抗血清对野生病毒的识别与结合能力。结果表达的重组P2蛋白相对分子质量约15000,纯度均达90%以上,GⅠ.1、GⅡ.2、GⅡ.3、GⅡ.4、GⅡ.17型P2蛋白表达量分别为60、26.4、19.8、16.3和33.3 mg/L。针对5种基因型NV P2抗原的抗血清滴度均达1∶128000以上,均能很好地识别自身相应的P2抗原,但不与不相关P2抗原相互作用,均不与重组人A组轮状病毒VP7抗原和重组幽门螺杆菌尿酶抗原发生交叉反应,且对野生型NV具有较好的识别与结合能力。结论利用原核表达系统成功表达并纯化了我国流行的5种基因型NV P2抗原,制备的抗血清滴度较高,特异性良好,为NV快速、现场诊断技术的建立奠定了基础。

Objective To express and purify the P2 domain of 5 genotypes(G Ⅰ.1,G Ⅱ.2,G Ⅱ.3,G I.4,and G Ⅱ.17)of norovirus(NV)in prokaryotic expression system and prepare their antisera.Methods The bioinformatics method was used to analyze the sequence conservation of P2 domains of different genotypes of NV.Then the P2 DNA sequence was synthesized and subcloned into pET24a vector to construct the recombinant prokaryotic expression plasmid,which was transformed into E.coli BL21(DE3)competent cells and induced by IPTG.30 female BALB/c mice were immunized with the purified recombinant P2 protein to prepare antiserum.The titer and cross reaction of antiserum were detected by ELISA,the specificity was detected by Western blot,and the recognition and binding ability to wild-type NV were analyzed by dot-blot.Results The expressed recombinant P2 proteins showed purity of over 90%with the relative molecular mass of about 15000.The expression levels of P2 protein of G Ⅰ.1,G Ⅱ.2,G Ⅱ.3,G I.4,and G Ⅱ.17 were 60,26.4,19.8,16.3 and 33.3 mg/L,respectively.The antiserum titers against five genotypes of NV P2 antigens were all over 1:128000,which well recognized their corresponding P2 antigens,while showed no interaction with unrelated P2 antigens and no cross-reaction with recombinant human group A rotavirus VP7 antigen and recombinant Helicobacter pylori uroenzyme antigen,and had good recognition and binding ability to wild-type NV.Conclusion Five genotypes of NV P2 antigens epidemic in China were successfully expressed and purified by prokaryotic expression system.The prepared antisera had high titer and good specificity,which laid a foundation of the establishment of rapid and on-site diagnosis technology of NV.