应用共同抗原的多克隆与单克隆抗体进行肠道病毒诊断的研究

目的制备肠道病毒可能的共同抗原多克隆与单克隆抗体,用于早期诊断肠道病毒。方法RT-PCR扩增VP1N端122氨基酸这一可能的共同抗原基因片段,连接至PET-30a表达载体,转化到大肠杆菌BL21(DE3)中进行体外表达,表达重组蛋白经Western-blot活性鉴定后用电洗脱法进行纯化。纯化蛋白免疫昆明鼠获取多克隆抗体,多克隆抗体用饱和硫铵沉淀法进行纯化。纯化蛋白免疫BALB/c鼠,用杂交瘤技术进行单克隆抗体制备,并用Protein A/G亲和柱进行纯化。制备的抗体用间接免疫荧光法(IFA)检测肠道病毒。结果重组蛋白经Western blot检测,能被国外商品化的肠道病毒组特异性的5-D8/1单克隆抗体、含脊髓灰质炎病毒抗体的人血清与分别代表肠道病毒A、B、C三组的单型血清所识别。制备其相应的多克隆和单克隆抗体,与5-D8/1单克隆抗体同时应用间接免疫荧光法(IFA)检测29株(27型)福建分离株病毒抗原,显示:制备的多抗或单抗的检出率比5-D8/1单抗高约10%~40%。结论结合福建省当地株制备肠道病毒共同抗原的抗体检测变异较大的当地株,更具优势。

Diagnosis of enteroviruses with polyclonal and monoclonal antibodies against a putative common epitope

According to the data of studies on enteroviruses,combined with those of the gene sequence analysis of the(isolates) from Fujian province,the gene fragment coding to the putative common epitope in the N-terminal 122 amino acid residues of VP1 was amplified by PCR in vitro with the Fujian isolates as templates.The PCR products were then cloned into expression vector pET30a and transformed to E.coli BL21(DE3).Meanwhile,the antigenicity of the recombinant protein was(identified) by Western blotting with the commercial enterovirus group-specific monoclonal antibody 5-D8/1,human sera containing antibodies against polio viruses and the monotypic sera of sheep representing A,B and D groups of enteroviruses.The corresponding polyclonal and monoclonal antibodies against this recombinant protein were prepared accordingly.The detection rate of 29 strains(27 serotypes) of the Fujian isolates evaluated by indirect immunofluorescence assay(IFA) was found to be 10%-40% higher than that detected by the monoclonal antibody 5-D8/1.It is concluded that the use of these polyclonal and monoclonal antibodies exhibits greater potential to detect the local strains of isolates with possible variations.