狂犬病病毒M蛋白单克隆抗体的制备
毕业论文关键词：狂犬病病毒cvs11-M蛋白；单抗；杂交瘤细胞融合；基因工程技术
Preparation of monoclonal antibody against RABV M protein
Abstract：Rabies is a zoonotic disease caused by rabies virus infection that pose a serious threat to the health of humans and animals. The morbidity of the disease is close to 100%, which is a serious public safety problem. Nearly 59,000 people worldwide die of rabies each year, especially in China, India and other developing countries. M protein as a structural protein of rabies virus plays an important role in the morphological determinants, transcriptional replication and budding of viruses. At the same time, it can induce the apoptosis of infected cells and affect the immune response of the host.In-depth study of M protein in the pathogenesis of RABV infection and its mechanism, specific high reactivity of antibodies is essential. Therefore, in this study, the M protein of cvs11 strain was selected to prepare monoclonal antibodies which used genetic engineering technology and hybridoma cell fusion. The study was conducted to provide a basis for the detection of rabies virus and the study of rabies virus infection.
Key words: Rabies Virus cvs11-M protein;Monoclonal Antibody;Hybridoma Cell Fusion;Genetic engineering technology
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1 狂犬病病毒cvs11毒株M蛋白克隆与纯化2
1.1 实验材料2
1.1.1 试剂，载体，菌种2
1.1.2 溶液的配制3
1.1.3 仪器设备4
1.2 实验方法4
1.2.1 cvs11-M的克隆4
1.2.2 蛋白表达6
1.2.3 纯化蛋白6
2 狂犬病病毒M蛋白单克隆抗体的制备6
2.1 实验材料6
2.1.1 实验动物与细胞，试剂6
2.1.2 主要器材6
2.1.3 试剂配制6
2.2 实验方法7
2.2.1 BALB/c 小鼠的免疫程序7
2.2.2 M蛋白血清效价的测定7
2.2.3 杂交瘤细胞株的建立7
3 实验结果8
3.1 cvs11-M克隆至PET28a载体8
3.2 重组蛋白的诱导表达及纯化9
3.3 三免后小鼠血清中抗体效价的测定9
3.4 杂交瘤细胞株的建立10
3.5 单克隆抗体的鉴定11
4 讨论11致谢13
参考文献13
狂犬病病毒M蛋白单克隆抗体的制备
引言
狂犬病作为一种人畜共患病，发病后死亡率接近100%[1]。该病毒属于弹状病毒科狂犬病病毒属，病毒粒子呈子弹状，主要存在于患者动物的延脑，大脑皮质，小脑，脊髓等中枢神经系统中，唾液腺和唾液也有大量病毒，并随唾液排出体外。RABV主要感染患病动物的中枢神经系统，侵犯神经元，可以使所有温血动物受感染，宿主为犬，野生动物，蝙蝠等，具有嗜神经性[2]。本病经患病动物咬伤而感染，少数情况下可由病犬，病猫舔舐健康动物的伤口而感染。潜伏期较长，患者发病后的临床症状分为狂暴型和麻痹型，前期会表现出一种特殊的神情，精神沉郁，后期会致意识的模糊不清、恐水、高度兴奋、狂暴撕咬、日渐消瘦、最后大量流涎，不久后四肢麻痹，卧地不起，见水惶恐，并因中枢神经衰竭而昏迷或死亡[3]，整个病程约6-8天，病理组织上，以非化脓性脑炎和神经细胞胞浆内出现内基小体为主要特征[4]。
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