具有免疫反应活性的猪源重组抗体的原核表达及鉴定

发布时间：2018-09-11 07:52

【摘要】：为制备与猪繁殖与呼吸障碍综合征病毒(PRRSV)具有反应活性的猪源化原核表达抗体(Pr Ab),本实验通过流式细胞技术,以PRRSV GP5蛋白多肽及抗猪Ig G抗体,筛选出能够与PRRSV抗原表位结合的阳性B淋巴细胞,利用RT-PCR从筛选得到的B细胞的mRNA中扩增出编码抗体重链可变区(VH)基因序列和轻链可变区(VL)基因序列,将扩增的可变区基因序列分别与本实验室前期克隆的猪源抗体轻链恒定区(CL)和重链恒定区(CH)基因序列连接后,以Linker序列将轻链和重链全长基因序列相连,构建全长的猪源抗体编码基因(H-linker-L)。将该基因克隆于原核表达载体p ET-28a中进行原核表达及纯化。Western blot鉴定结果显示,表达的重组蛋白能够被抗猪IgG抗体所识别,表明表达的重组蛋白为PrAb;经PRRSV包被的ELISA鉴定,PrAb与PRRSV弱毒株CH1R和强毒株HuN4呈阳性反应,表明所制备的PrAb为PRRSV特异性抗体。本实验为进一步制备诊断PRRS的猪源化重组抗体奠定了基础。
[Abstract]:In order to prepare porcine prokaryotic expression antibody (Pr Ab),) which reacts with porcine reproductive and respiratory syndrome virus (PRRSV), PRRSV GP5 protein polypeptide and anti-porcine Ig G antibody were used by flow cytometry. The positive B lymphocytes which could bind to PRRSV epitope were screened. The (VH) gene sequence of heavy chain variable region and the (VL) gene sequence of light chain variable region were amplified by RT-PCR from the mRNA of B cell selected. The amplified variable region gene sequence was linked to the light chain constant region (CL) and the heavy chain constant region (CH) gene of porcine antibody cloned in our laboratory, respectively. The light chain and heavy chain full-length gene sequences were linked by Linker sequence. The full-length porcine antibody encoding gene (H-linker-L) was constructed. The recombinant protein was cloned into prokaryotic expression vector p ET-28a for prokaryotic expression and purified. Western blot analysis showed that the expressed recombinant protein could be recognized by anti porcine IgG antibody. The results showed that the expressed recombinant protein was identified by ELISA coated with PrAb; and identified as a positive reaction with PRRSV attenuated CH1R and virulent HuN4, indicating that the prepared PrAb was PRRSV specific antibody. This study laid a foundation for the further preparation of porcine recombinant antibodies for diagnosis of PRRS.
【作者单位】：中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/猪传染病研究室;
【基金】：哈尔滨市杰出青年基金项目(2014RFYYJ003)
【分类号】：S852.65

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