基于ORF2基因表达产物的猫杯状病毒间接ELISA方法的建立及其初步应用

发布时间：2018-05-19 23:26

本文选题：猫杯状病毒 + 原核表达　；参考：《吉林农业大学》2015年硕士论文

【摘要】：猫杯状病毒(Feline calicivirus,FCV),为杯状病毒科杯状病毒属中成员,是一种单股正链RNA病毒。病毒无囊膜,直径35-39nm。FCV能够引起猫和猫科动物口腔及上呼吸道疾病。该病是一种多发型、流行性、具有高度传染性的传染病,临床上称为猫传染性-鼻结膜炎。患病的动物表现为发烧、鼻炎、口炎、眼分泌物增多,单独的FCV感染常不引起致命性疾病,和其他病原混合感染时,可表现跛行、肺炎、胃肠炎、肾炎等,严重时导致动物的死亡。该病不仅危害家养的猫和圈养的猫科动物,还严重威胁野生猫科动物。疫苗接种是对该病的最好预防措施,但并不能完全清除病原,使患病动物成隐形带毒者,持续向外界环境中排毒,成为传染源。此外,由于FCV的易变性,也降低了疫苗的免疫保护作用。由于FCV极其容易变异及该病临床症状的多样性,给该病毒和该病的检测和诊断带来了一定的困难。病毒的分离培养、电镜观察、RT-PCR、荧光定量PCR、中和实验、琼脂扩散实验都能检测该病毒。这些方法需要熟练的实验室操作技能。血清学方法既可以评价疫苗的免疫保护效果,也有助于疾病的诊断,但是,目前国内尚无商品化血清学检测试剂盒。所以,开展血清学诊断方法的研究对疾病的预防、控制十分必要。FCV具有3个开放阅读框(ORF),其中,ORF2编码能够诱导机体产生有效抗体的衣壳蛋白,包含多个抗原识别位点。本研究根据本实验室保存的1株豹源猫杯状病毒基因序列设计一对引物,扩增了ORF2中1 734 bp的核苷酸序列,将其克隆至表达载体pET-28a上,成功构建了pET-28a-FCV1 734重组质粒。将该质粒转化至大肠杆菌表达菌株中,诱导表达产物经SDS-PAGE和Western blot检测,证明所表达的蛋白具有良好的反应原性。纯化提取该蛋白,作为猫杯状病毒间接ELISA方法的包被抗原。利用方阵法,确定酶标二抗的的最佳工作浓度为1:5 000,抗原最佳包被浓度为2.1250μg/mL,一抗最佳稀释度为1:200。优化最佳反应条件,确定了阴、阳性血清的临界值:当OD490值大于0.2570判定为阳性,小于0.2390判定为阴性,介于0.2570-0.2390之间的判定为疑似。利用本研究建立的猫杯状病毒间接ELISA方法对采自吉林省49份、上海188份和广西47份猫血清进行检测,结果表明,来源于吉林省猫血清阳性率69.39%(34/49),上海地区猫血清阳性率91.49%(172/188),广西省猫血清阳性率89.71%(38/47),血总阳性率85.92%(244/288)。
[Abstract]:Feline calicivirus, a member of the genus calix virus, is a single-stranded positive strand RNA virus. The virus has no capsule and diameter 35-39nm.FCV can cause oral and upper respiratory diseases in cats and cats. The disease is a multi-hairstyle, epidemic, highly infectious disease, clinically known as feline infectious-rhinoconjunctivitis. The sick animals show fever, rhinitis, stomatitis, increased secretion of the eye, FCV infection alone often does not cause fatal diseases, and other pathogens mixed infection, can show claudication, pneumonia, gastroenteritis, nephritis, etc. In serious cases, animals die. The disease not only endangers domestic cats and captive cats, but also threatens wild cats. Vaccination is the best preventive measure to the disease, but it can not completely eliminate the pathogen, make the sick animal become invisible virus carrier, continuously detoxify the environment and become the source of infection. In addition, due to the variability of FCV, the immune protection of the vaccine is also reduced. It is difficult to detect and diagnose FCV because of its easy variation and variety of clinical symptoms. The virus was isolated and cultured, RT-PCR, fluorescence quantitative PCR, neutralization test and Agar diffusion assay were all able to detect the virus. These methods require skilled laboratory skills. Serological methods can not only evaluate the immune protection of vaccines, but also help the diagnosis of diseases. However, there is no commercial serological test kit in China. Therefore, it is necessary to develop serological diagnostic methods for disease prevention and control. FCV has three open reading frames, in which ORF2 encodes capsid protein which can induce the body to produce effective antibodies and contains many antigen recognition sites. In this study, a pair of primers were designed according to the gene sequence of a leopard source cat calix virus. The nucleotide sequence of 1 734 BP in ORF2 was amplified and cloned into the expression vector pET-28a. The recombinant plasmid pET-28a-FCV1 734 was successfully constructed. The plasmid was transformed into Escherichia coli expression strain, and the induced expression product was detected by SDS-PAGE and Western blot. The result showed that the expressed protein had good reactivity. The protein was purified and extracted and used as the coating antigen of the indirect ELISA method. By using square matrix method, the optimum working concentration of enzyme labeled second antibody was determined to be 1:5 000, the best coating concentration of antigen was 2.1250 渭 g / mL, and the best dilution of the first antibody was 1: 200. The optimal reaction conditions were optimized and the critical value of negative and positive serum was determined. When the OD490 value was greater than 0.2570, it was determined as positive, less than 0.2390 as negative, and between 0.2570-0.2390 as suspected. The indirect ELISA method was used to detect the serum samples of 49 cats from Jilin province, 188 from Shanghai and 47 from Guangxi. The positive rate of cat serum in Jilin Province was 69.39%. The positive rate of cat serum in Shanghai area was 91.49%. The positive rate of cat serum in Guangxi Province was 89.71%. The total positive rate of blood was 85.92% / 288%.
【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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