猪细小病毒单克隆抗体的研制及其抗原表位的筛选

发布时间：2018-07-18 18:49

【摘要】： 猪细小病毒(Porcine parvovirus,PPV)属于细小病毒科细小病毒属的DNA病毒,是引起母猪繁殖障碍性疾病的主要病原之一。PPV病自出现至今,在世界各主要养猪国家和地区广泛存在,给养猪业带来了很大负面影响,一直是猪病研究的热点之一。迄今为止,对PPV抗原结构还不是完全清楚。本研究制备了15株抗PPV的单克隆抗体(McAbs),应用噬菌体展示技术对效价高、特性好的4株McAbs筛选了对应的模拟表位,以期为诊断试剂开发和表位疫苗研制奠定基础。将PPV分离株BQ传代培养纯化后的病毒免疫BALB/c小鼠,取脾细胞与SP2/0细胞在聚乙二醇作用下融合,用间接ELISA法进行筛选,以有限稀释法稀释扩大培养,3轮筛选以后得到15株分泌抗PPV的McAbs细胞株,分别对这些细胞株进行了如下生物学特性鉴定:染色体计数发现其核内染色体数均在80~110条之间,符合杂交瘤细胞染色体数规律;特异性实验表明该15株McAbs细胞株,仅特异性地与PPV发生反应,而不与其他引起母猪繁殖障碍性疾病的病原:猪圆环病毒2型(PCV2)、猪伪狂犬病毒(PRV)、猪生殖和呼吸综合征病毒(PRRSV)等发生交叉反应;将杂交瘤细胞按一定量(5×105～5×106个/ml)注射液体石蜡免疫一周后的BALB/c小鼠,制得的腹水抗体效价介于1:2 560～1:20 480之间;单抗亚型鉴定表明所有McAbs均为IgG1亚型,而所有McAbs的轻链均为κ型;Western blot结果显示15株McAbs中2株针对VP1发生反应,12株针对VP2、VP3发生反应,还有1株单抗没有印记。以15株McAbs中特异性强、效价高且有代表性的识别VP1的C4和识别VP2的E2、B4、F6的4株McAbs分别为配基,从噬菌体随机12肽库中筛选抗体识别的模拟肽。经过3轮的淘筛和间接ELISA试验,获得了分别与4株McAbs反应的阳性噬菌体克隆。经测序F6阳性克隆的氨基酸序列高度一致,均为LMGKRRIQRPRI,与VP2有一定同源性;E2和B4阳性克隆与VP2同源性则很低;C4得到了以“-RKR-”为核心序列的VP1抗原模拟表位。动物实验表明阳性噬菌体免疫BALB/c小鼠可以产生一定的抗体效价。这些模拟表位的获得为进一步分析PPV的结构与功能的研究奠定基础,也可以开发以表位为基础的诊断试剂和多表位基因工程疫苗。
[Abstract]:Porcine parvovirus (PPV) is a DNA virus belonging to parvovirus genus of parvovirus family. It is one of the main pathogens causing reproductive disorders in sows. It has brought great negative effect to pig industry and has been one of the hot spots in pig disease research. Up to now, the structure of PPV antigen is not completely clear. In this study, 15 McAbs anti-PPV monoclonal antibodies (McAbs) were prepared. Phage display technique was used to screen the corresponding mimic epitopes of 4 McAbs strains with high titer and good properties, so as to lay a foundation for the development of diagnostic reagents and epitope vaccines. BALB / c mice were immunized with purified PPV virus isolated by passage. Spleen cells and SP2 / 0 cells were fused with polyethylene glycol (PEG) and screened by indirect Elisa. A total of 15 McAbs cell lines secreting anti-PPV were obtained after three rounds of screening by dilution and dilatation with a limited dilution method. The biological characteristics of these cell lines were identified as follows: the chromosome count showed that the number of chromosomes in the nucleus was between 80 and 110. In accordance with the rule of chromosome number of hybridoma cells, the specific experiment showed that the 15 McAbs cells reacted specifically with PPV. But not cross reaction with other diseases causing reproductive disorders in sows: porcine circovirus type 2 (PCV2), porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSv). The ascites antibody titers of BALB / c mice immunized with a certain amount (5 脳 10 ~ 5 ~ 5 脳 10 ~ 6 / ml) of liquid paraffin a week after immunization with hybridoma cells were found to be within the range of 1:2 560: 120 480, and all McAbs were identified as IgG1 subtype. The light chain of all McAbs was 魏 -type blot. The results showed that 2 of the 15 McAbs reacted to VP1, 12 of them reacted to VP2VP3, and 1 McAbs had no imprinting. Four McAbs strains with strong specificity, high titer and representative recognition of VP1 and VP2, respectively, were used as ligand. The mimic peptides were screened from phage random 12 peptide library. After three rounds of screening and indirect Elisa tests, positive phage clones which reacted with 4 McAbs strains were obtained. The amino acid sequences of the F6 positive clones were highly consistent, all of them were LMGKRRIQRPRI, and the E2 and B4 positive clones had some homology with VP2, but they had very low C 4 sequence homology with VP2, and the mimic epitopes of VP1 antigen with "-RKR-" as the core sequence were obtained. Animal experiments showed that positive phage immunized BALB / c mice could produce a certain antibody titer. The acquisition of these mimic epitopes lays a foundation for further analysis of the structure and function of PPV, and can also be used to develop epitope based diagnostic reagents and multiepitope genetic engineering vaccines.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

【相似文献】