【作者】 李敏；

【导师】 黄勇；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2011， 硕士

【摘要】 鸡传染性支气管炎(Avian Infectious Bronchitis, IB)是一种由鸡传染性支气管炎病毒(Avian Infectious Bronchitis Virus, IBV)引起的急性、高度接触传染性呼吸道疾病。它主要引起鸡呼吸道或肾脏感染,蛋鸡的产蛋数量和产蛋品质下降等。IBV为冠状病毒(Coronavirus)γ亚群的代表成员,由于其基因组易发生突变和重组,加之不同血清型之间缺乏交叉保护作用,虽然疫苗一直在广泛的使用,但IB仍不断地爆发和流行,给养禽业带来了严重的经济损失。单克隆抗体针对病毒特定的抗原表位结构,具有较强的特异性,对该病的诊断及基因工程疫苗的设计具有重要意义。N蛋白是诱导机体免疫应答和细胞免疫应答的重要免疫原蛋白。本研究以IBV N蛋白作为研究对象,制备了针对N蛋白的单克隆抗体,对IBV的诊断、预防和控制具有一定的意义。实验首先从鸡胚培养的IBV ZY3毒株尿囊液中提取基因组RNA,然后利用软件对其N蛋白抗原表位进行预测分析,设计一对特异性引物对免疫原性好、亲水性强、表位可能性大的保守片段进行了扩增,片段大小为818 bp；将其与表达载体pET-32a(+)连接并转入大肠杆菌BL21中诱导表达,SDS-PAGE检测表达的重组N蛋白大小为50kDa;用Ni-NTA Superflow纯化重组N蛋白后,采用抗鸡IBV血清与之进行Western blot反应以检测其反应原性,结果显示重组蛋白反应原性良好。采用纯化的重组N蛋白加以弗氏佐剂对BALB/c小鼠进行免疫,通过间接ELISA方法检测,发现免疫后的小鼠均能产生高效价的抗体,可用于细胞融合。将SP2/0细胞与免疫小鼠脾细胞进行融合,融合后2周左右用间接ELISA方法对其进行抗体效价检测,将筛选到的阳性细胞孔进行3-4次亚克隆化,最终得了2株能稳定分泌抗重组N蛋白抗体的杂交瘤细胞,分别命名为2833和4E10。经测定,2株单抗亚类分别为IgGl和IgM,轻链均为K链；通过腹水诱导法大量制备的腹水和细胞培养上清抗体效价分别大于106和1：640；这两株单克隆抗体具有良好的特异性,均能与IBV N蛋白发生特异性的反应；经检测,2株杂交瘤细胞均具有良好的传代稳定性。这两株抗IBV N蛋白单克隆抗体研制的成功,对进一步研究IBV N蛋白及建立鸡传染性支气管炎的诊断方法有重要意义。更多还原

【Abstract】 Avian infectious bronchitis (IB) is an acute and highly contagious disease in chickens which caused by infectious bronchitis virus (IBV). It can lead to a decline in the weight gain and feed conversion of chickens, decrease egg production and quality, and also induce respiratory or kidney infections chicks. IBV is a Coronaviruses (the family Coronaviridae, genera Coronavirus), representative member of subgroupγ. Because of the high mutation frequency and recombination of IBV and little cross-protection between serologically distinct viruses, although the vaccine has been used, IB is still ongoing outbreak and epidemic. It caused serious economic losses in poultry industry. Monoclonal antibody (McAbs) can aim directly at specified epitope structure, and have a strong specificity, it show great significance on diagnosis of the disease and the design of genetic engineering vaccine. N Protein is a amajor immunogenic protein which induce the immunoresponse, especially the cellular immunological response. IBV N protein is regard as a target protein in our study, we prepared a monoclonal antibody against the target protein, and it has a certain significance on the diagnosis, prevention and control of IBV.In this study, IBV genomic RNA was extracted from chicken embryo allantoic fluid. Using software to predict and analysis the epitope of N protein, A pair of specific primers was designed to amplify the fragments which showed good immunogenic, hydrophilicity and epitope possibility, amplified fragment was 818 bp. It ligated into the vector pET-32a(+) and then transformed into E. coli BL21. After induction of expression, the SDS-PAGE results showed that a protein about 50 kDa. Purified on a column packed with Ni-NTA His-Bind superflow, the recombinant protein then reaction with chicken anti-IBV serum by western blot to test their antigenicity, the results showed good antigenicity of recombinant proteins.BALB/c mice were immunized with recombinant N protein mixed with an equal volume of Freund’s adjuvant. Detected by indirect ELISA, it found that after immunization, mice produced high titers of antibodies, and can be used for cell fusion. SP2/0 cells and immune mouse spleen cells were fused, and indirect ELISA method was used for detection of antibody titers 2 weeks after fusion. Two McAbs designated as 2B33 and 4E10 were prepared. The subclass of them were IgG1 and IgM respectively and their light strand wereκ. The antibody titers of ascites antibody preparaed by ascites induction method and cell culture supernatant were greater than 106 and 1:640 respectively. The two monoclonal antibodies had good specificity, and could specifically recognize the N protein. The indirect ELISA result indicated that they both have good stability passage.This successful development of the two McAbs against IBV N protein is important to further research on IBV N protein and the diagnosis of avian infectious bronchitis更多还原