【摘要】 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。更多还原

【Abstract】 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.更多还原