GⅡ.4型人诺如病毒VP1蛋白单克隆抗体的制备及鉴定

Preparation and characterization of monoclonal antibody against major capsid protein VP1 of GⅡ.4 human norovirus

目的制备抗GⅡ.4型人诺如病毒(human norovirus,HuNoV)衣壳蛋白VP1单克隆抗体,并鉴定其生物学特性。方法PCR扩增GⅡ.4型HuNoV VP1基因序列,将其插入至原核表达载体pGEX-5X-1中,构建重组质粒p GEX-5X-1-VP1,转化至E.coli BL21,经IPTG诱导表达,并通过亲和层析纯化获得重组蛋白GST-VP1。利用杂交瘤技术将小鼠骨髓瘤细胞SP2/0与经GST-VP1抗原免疫的BALB/c小鼠的脾细胞进行融合,通过间接ELISA法筛选出阳性杂交瘤细胞株,制备单克隆抗体,并进行纯化。采用间接ELISA法、Biacore法和Western blot法分别检测单克隆抗体的效价、亲和力及特异性。结果经菌落PCR及测序鉴定,重组质粒pGEX-5X-1-VP1构建正确。重组蛋白GST-VP1相对分子质量约85000,大部分以可溶形式存在,纯化后浓度为1.100 mg/mL。筛选获得3株能分泌GⅡ.4型HuNoV VP1单克隆抗体的杂交瘤细胞株,命名为NoV C1、NoV D9和NoV H6,效价均可达1∶10^7以上,亲和力常数KD(M)分别为4.422×10^-7、4.303×10^-8、7.540×10^-7 mol/L,均可与HuNoV VP1天然蛋白及GST-VP1重组蛋白发生特异性结合。结论利用原核表达系统成功表达了GST-VP1蛋白,并通过杂交瘤技术制备了高效价的GⅡ.4型HuNoV VP1特异性单克隆抗体,为GⅡ.4型HuNoV免疫诊断和疫苗的研发奠定了基础。

Objective To prepare the monoclonal antibodies(McAbs)against major capsid protein VP1 of GⅡ.4 human norovirus(HuNoV)and analyze its biological characteristics.Methods The gene coding GⅡ.4 HuNoV VP1 was amplified by PCR and inserted into prokaryotic expression vector pGEX-5X-1.The constructed recombinant plasmid pGEX-5X-1-VP1 was transformed to E.coli BL21 for expression under induction of IPTG.The expressed recombinant protein GST-VP1was purified by affinity chromatography.Mouse myeloma SP2/0 cells were fused with the spleen cells of BALB/c mice immunized with GST-VP1 by using hybridoma technique,and the positive hybridoma cells were screened by indirect ELISA.The prepared McAbs were purified and tested for titer,affinity and specificity by indirect ELISA,Biacore method and Western blot respectively.Results Colony PCR and sequencing proved that recombinant plasmid pGEX-5X-1-VP1was constructed correctly.Recombinant GST-VP1 protein,with a relative molecular mass of about 85000,mainly existed in a soluble form,of which the concentration was 1.100 mg/mL after purification.Three hybridoma cell strains secreting McAbs against GⅡ.4 HuNoV VP1 were screened and named as NoV C1,NoV D9 and NoV H6 respectively,by which the titers of secreted McAbs were more than 1∶10^7,and the affinity constants KD(M)were 4.422×10^-7,4.303×10^-8and 7.540×10^-7mol/L respectively.All the McAbs showed specific binding to natural GⅡHuNoV VP1 and recombinant protein GST-VP1.Conclusion GST-VP1 was successfully expressed by using prokaryotic expression system,and high titer McAbs against GII.4 HuNoV VP1 were successfully prepared by using hybridoma technique,which laid a foundation of immunological diagnosis of GⅡ.4 HuNoV infection and vaccine development.