猪圆环病毒2型3E5单克隆抗体的制备与鉴定
毕业论文关键词：单克隆抗体3E5；抗体效价；鉴定；猪圆环2型病毒
Preparation and Identification of Monoclonal Antibody 3E5 against Porcine Circovirus Type 2
Abstract： In this study, the hybridoma cells cells secreting the monoclonal antibody 3E5 against porcine circovirus type 2（PCV2）were cultured in vitro. After collecting the supernatant of cell culture, the cells were used to immune the Balb/c mice by intraperitoneal injection. After more than 7 days, the ascites were collected. Finally, the antibody was identified with different immune methods and the titer of the supernatant and the ascites were measured. The experiment results proved that the titer of the McAbs in supernatant of the cell culture was 1: 3200, and the antibody titer in ascites was 1: 1600000. The indirect immunofluorescence assay (IFA) showed that that the ascites antibodies could react with PCV2-infected PK-15 cells. Western-blotting analysis showed that the monoclonal antibody was able to react with the purified PCV2-Cap protein (about 30kDa) specifically. This study laid the foundation for the specific differential diagnosis of this disease.
Key words: Monoclonal antibody; Antibody titer; Identification; Porcine circovirus type 2
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1材料与方法2
1.1细胞、毒株、实验动物及试剂 2
1.2杂交瘤细胞复苏2
1.3杂交瘤细胞传代2
1.4杂交瘤细胞冻存3
1.5单克隆抗体腹水的制备3
1.6单克隆抗体鉴定3
1.6.1效价的测定3
1.6.2 Western blot4
1.6.3间接免疫荧光（IFA）5
2 结果5
2.1效价的测定5
2.2 单克隆抗体特异性鉴定 6
2.2.1 Western blot6
2.2.2间接免疫荧光（IFA）6
3 讨论 7
致谢8
参考文献8
猪圆环病毒2型3E5单克隆抗体的制备与鉴定
猪圆环病毒（Porcine circovirus, PCV）属于圆环病毒属，它是单股环状，一种无囊膜的DNA病毒[1]。猪圆环病毒对外界的抵抗力相当强，在强酸和高温下也能存活一段时间，并且在氯仿作用下也不失活，从而为预防控制猪圆环病增加了一定的难度。从血清型方面分类，它可以被分为PCV1和PCV2两个亚型[2]，猪圆环病毒1型（PCV1）首次被发现于PK-15细胞系[3]，且被认为几乎无致病性。而猪圆环病毒2型(PCV2)却能够引起各个猪群大规模的疾病爆发，感染猪可以从鼻涕、粪便等排泄物中释放该病毒，再经过口腔、呼吸道等途径，感染不同年龄段的猪，是引起世界上养猪业巨大经济损失的主要猪病毒之一。
PCV2的核衣壳二十面体对称，其基因组有三种，全长分别为：1766bp、1767bp或1768bp，与PCV1相比，PCV2在基因组较为复杂，它的不同部位存在着一些小片段的缺失和插入[11]。其中其Cap蛋白是病毒的主要结构蛋白，大小约为30kDa[4]。 PCV2的主要免疫原也是改蛋白，并与宿主免疫有关[5]。经证实，PCV1与PCV2之间不存在抗原交叉反应[6]。
PCV2在临床上会引发多个疾病，如：断奶仔猪多系统衰竭综合征（PMWS），皮炎与肾病综合征（PNDS）[17]等。尤其是对于一些新生小猪来说，易感染率较高：极易发生高热、消瘦、咳嗽、腹泻、繁殖障碍和运动障碍等临床症状。这些症状的疾病被统称为猪圆环病毒病(Porcine circovirus, PCVD)或猪圆环病毒相关疾病（Porcine circovirus associated disease, PCVAD）[7] 。临床上证明，PCV2还容易与其他病原交叉混合感染，使患畜免疫力低下，增加其他疾病的易感性。其他调查表明，PCV2不仅在仔猪中易感，在商品猪和育肥猪中同样广泛存在。
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