猪繁殖与呼吸综合征病毒核衣壳蛋白的原核表达及其单克隆抗体的制备

Procaryotic Expression and Purification of the Recombinant Nucleocapsid Protein of PRRSV and Preparation of the Monoclonal Antibody

试验通过RT-PCR法扩增出猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)核衣壳蛋白(N蛋白)基因并克隆到原核表达载体pet-30a中,转入到BL21感受态细胞,经IPTG诱导表达,使用SDS-PAGE和Western blotting分析表达产物的特性。利用Ni柱纯化该重组蛋白,采用NC膜皮下包埋法免疫Balb/C小鼠,取免疫后的脾细胞与SP2/0细胞进行融合后筛选杂交瘤细胞株,检测杂交瘤细胞株的特性并制备单克隆抗体。SDS-PAGE和Western blotting结果表明,该重组蛋白表达正确,约为20ku,能被抗PRRSV的阳性血清特异性识别。超声后用Ni柱纯化,经SDS-PAGE分析可得单一的目的条带。NC膜皮下包埋法免疫效果良好,通过细胞融合、ELISA筛选获得3株能稳定分泌抗PRRSV N蛋白抗体的杂交瘤细胞株(H7、F7、C8),制备出高特异性的针对N蛋白的H7单抗。IFA与Western blot-ting结果显示制备的单抗可与病毒N蛋白产生特异性反应。亚型鉴定为IgG2b型,染色体分析证实杂交瘤细胞染色体数目正确。结果成功实现了N蛋白的原核表达,并获得高特异性的单克隆抗体,为PRRSV N蛋白抗原的检测奠定基础。

In this experiment,the nucleoprotein（N） gene of porcine reproductive and respiratory syndrome virus（PRRSV） amplified by RT-PCR was cloned into pet-30a vector and expressed in Escherichia coli BL21,then induced with IPTG.The expressed protein was identified by SDS-PAGE and confirmed by Western blotting.The recombinant N protein was purified by Ni.The BALB/C mice were immunized through NC filter immunization,the splenocytes of the immunized mice were fused with SP2/0 cells,the hybridoma cells were screened and confirmed to prepare monoclonal antibodies.The SDS-PAGE and Western blotting showed that the recombinant N protein was 20 ku in size and specifically reacted with anti-PRRSV positive serum.After purifying in Ni,the recombinant N protein was shown as one specific band in SDS-PAGE.The NC filter immunization was a good method.After cells fusion and screening,three hybridoma cells which produced McAbs steadily were screened by ELISA,named H7,F7,C8.the special anti-nucleocapsid protein monoclonal antibody-H7 was prepared.IFA and Western blotting assays showed that the monoclonal antibody reacted with PRRSV N protein specially.The McAb belong to IgG2b.The chromosomes analysis of the hybridoma cells confirmed the correct number of chromosomes.Finally,the procaryotic expression and purification of recombinant N protein and its highly specific monoclonal antibody were successfully achieved.This sudy laid the foundation for the analysis of N protein.