PRRSV GP4蛋白的原核表达及单克隆抗体的制备

Prokaryotic Expression of PRRSV GP4 Protein and Preparation of Monoclonal Antibodies

猪繁殖与呼吸综合征病毒(PRRSV)给养猪业带来巨大经济损失,尽快建立该病毒的快速诊断方法尤为重要。PRRSV ORF4基因编码的结构蛋白GP4为病毒感染所必须。本研究为制备GP4单克隆抗体,选取ORF4优势序列优化合成,连入pET-32a原核表达载体,构建重组质粒pET-32a-ORF4;经双酶切验证后,转化至BL21(DE3)感受态细胞,IPTG诱导表达并纯化;经SDS-PAGE及Westernblot鉴定成功后免疫小鼠,制备筛选单克隆抗体,对制备的单克隆抗体进行验证。结果表明,GP4蛋白以可溶形式表达,大小为30 kDa;纯化蛋白免疫小鼠后,共筛选4株IgG亚型阳性杂交瘤细胞株;所制备单克隆抗体可与抗原特异性结合。本研究为PRRSV诊断方法的建立及后续研究奠定了基础。

Porcine reproductive and respiratory syndrome virus(PRRSV)has brought huge economic losses to the pig industry.Therefore,it is particularly important to detective a rapid diagnosis method for the virus as soon as possible.The structural protein GP4 encoded by theORF4gene of PRRSV is necessary for virus infection.In order to prepare GP4 monoclonal antibodies(MAbs),the ORF4 superior sequence was selected to optimize synthesis and then connected to pET-32a prokaryotic expression vector.The constructed recombinant plasmid pET-32a-ORF4 was verified through double enzyme digestion and transformed into BL21(DE3)competent cells for protein expression with IPTG induction.The expressed protein was purified and confirmed in SDS-PAGE and Western blotting.The GP4 protein was expressed in supernatant form and the size was about 30 kDa.The purified protein was used to immunize mice for preparation of MAbs.As result,a total of 4 IgG subtype-positive hybridomas were obtained and subcloned for production of specific MAbs.The availability of MAbs to GP4 protein laid the foundation for future research and development of PRRSV diagnostic methods.