【作者】 侯强；

【导师】 仇华吉；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2006， 硕士

【摘要】 猪瘟病毒(classical swine fever virus;CSFV)E2囊膜糖蛋白是猪瘟病毒的主要结构蛋白之一,是猪瘟病毒中和抗体的主要靶标,是抗猪瘟病毒感染的主要保护性抗原;因此,猪瘟病毒E2蛋白是开发猪瘟新型疫苗、诊断试剂及研究猪瘟病毒致病机理的重要蛋白分子。 利用TRIzol试剂提取猪瘟病毒兔化弱毒疫苗(C株)的基因组RNA,经反转录获取其cDNA,再以cDNA为模板,扩增编码E2蛋白主要抗原区的DNA片段(378bp),再将扩增产物正确插入到原核表达载体pPROEX-HTb中,得到重组质粒pPROEX-tE2,然后以其转化大肠杆菌DH5α,经IPTG诱导,高效表达出了带有组氨酸标签的截短的重组蛋白(tE2),占细菌总蛋白的35%。经Western blotting分析和间接ELISA检测,该重组蛋白能被猪瘟病毒抗血清所识别。 用IPTG大量诱导重组质粒pPROEX-tE2转化菌后,通过亲和层析柱纯化重组蛋白tE2,以纯化的tE2蛋白为免疫原免疫BALB/c小鼠,然后取抗体转阳免疫小鼠的脾淋巴细胞,以PEG与SP2/0骨髓瘤细胞融合,经过3～5轮的筛选和纯化,最终获得一株能稳定分泌抗CSFV E2蛋白单克隆抗体的杂交瘤细胞株。经鉴定,该株单克隆抗体能够与CSFV C株和石门株特异性结合,且识别CSFVE2蛋白上的一个线性表位;该单克隆抗体重链为IgG1亚型,轻链为κ型。 总之,本研究利用原核表达系统高效表达了CSFV E2蛋白的主要抗原区,然后以该重组蛋白作为免疫原制备了一株能够稳定分泌抗E2蛋白单克隆抗体的杂交瘤细胞,为以后建立猪瘟诊断方法和研究CSFV E2蛋白的结构与功能提供了材料。更多还原

【Abstract】 The envelope glycoprotein E2 of classical swine fever virus (CSFV) is responsible for the elicitation of neutralizing antibodies. It is often used in developing new type vaccines, clinical diagnostic reagents and studying immunopathological mechanisms of the virus.A tuncated E2 gene encoding the major antigenic regions of E2 protein was amplified by RT-PCR from the genomic RNA of CSFV C-strain and cloned into pPROEX-HTb expression vector, resulting in pPROEX-tE2. The truncated E2 protein (tE2) was expressed with high-level in pPROEX-tE2-transformed Escherichi coli cells after induction by IPTG, as demonstrated by SDS-PAGE analysis. The recombinant protein could be recognized by CSFV antisera in either Western blotting or ELISA.The tE2 protein purified on a Ni-chelating HisTrap affinity column was used to immunize BALB/c mice, of which spleen cells were fused with SP2/0 cells with PEG3250 after 3 immunizations. A hybridoma cell line stably secreting monoclonal antibody (McAb) directed against the tE2 protein was selected by ELISA. The McAb was proved to be CSFV-specific and recognize a linear epitope on the E2 protein of CSFV C-strain or Shimen strain. The McAb was identified to be IgG1 subtype and kappa light chain.In summary, the truncated E2 protein of CSFV was expressed in E. coli and a hybridoma cell line secreting McAb directed against CSFV E2 protein was developed, which can be used to develop diagnostic assays and study the structure-function of CSFV E2 protein.更多还原