本文選題：貓杯狀病毒 + 原核表達　； 參考：《吉林農業(yè)大學》2015年碩士論文

【摘要】：貓杯狀病毒(Feline calicivirus,FCV),為杯狀病毒科杯狀病毒屬中成員,是一種單股正鏈RNA病毒。病毒無囊膜,直徑35-39nm。FCV能夠引起貓和貓科動物口腔及上呼吸道疾病。該病是一種多發(fā)型、流行性、具有高度傳染性的傳染病,臨床上稱為貓傳染性-鼻結膜炎�；疾〉膭游锉憩F(xiàn)為發(fā)燒、鼻炎、口炎、眼分泌物增多,單獨的FCV感染常不引起致命性疾病,和其他病原混合感染時,可表現(xiàn)跛行、肺炎、胃腸炎、腎炎等,嚴重時導致動物的死亡。該病不僅危害家養(yǎng)的貓和圈養(yǎng)的貓科動物,還嚴重威脅野生貓科動物。疫苗接種是對該病的最好預防措施,但并不能完全清除病原,使患病動物成隱形帶毒者,持續(xù)向外界環(huán)境中排毒,成為傳染源。此外,由于FCV的易變性,也降低了疫苗的免疫保護作用。由于FCV極其容易變異及該病臨床癥狀的多樣性,給該病毒和該病的檢測和診斷帶來了一定的困難。病毒的分離培養(yǎng)、電鏡觀察、RT-PCR、熒光定量PCR、中和實驗、瓊脂擴散實驗都能檢測該病毒。這些方法需要熟練的實驗室操作技能。血清學方法既可以評價疫苗的免疫保護效果,也有助于疾病的診斷,但是,目前國內尚無商品化血清學檢測試劑盒。所以,開展血清學診斷方法的研究對疾病的預防、控制十分必要。FCV具有3個開放閱讀框(ORF),其中,ORF2編碼能夠誘導機體產(chǎn)生有效抗體的衣殼蛋白,包含多個抗原識別位點。本研究根據(jù)本實驗室保存的1株豹源貓杯狀病毒基因序列設計一對引物,擴增了ORF2中1 734 bp的核苷酸序列,將其克隆至表達載體pET-28a上,成功構建了pET-28a-FCV1 734重組質粒。將該質粒轉化至大腸桿菌表達菌株中,誘導表達產(chǎn)物經(jīng)SDS-PAGE和Western blot檢測,證明所表達的蛋白具有良好的反應原性。純化提取該蛋白,作為貓杯狀病毒間接ELISA方法的包被抗原。利用方陣法,確定酶標二抗的的最佳工作濃度為1:5 000,抗原最佳包被濃度為2.1250μg/mL,一抗最佳稀釋度為1:200。優(yōu)化最佳反應條件,確定了陰、陽性血清的臨界值:當OD490值大于0.2570判定為陽性,小于0.2390判定為陰性,介于0.2570-0.2390之間的判定為疑似。利用本研究建立的貓杯狀病毒間接ELISA方法對采自吉林省49份、上海188份和廣西47份貓血清進行檢測,結果表明,來源于吉林省貓血清陽性率69.39%(34/49),上海地區(qū)貓血清陽性率91.49%(172/188),廣西省貓血清陽性率89.71%(38/47),血總陽性率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%.
【學位授予單位】：吉林農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65

【參考文獻】

相關期刊論文 前1條

1 王祥生,夏咸柱,范泉水,張建賓,劉全;虎暴發(fā)錐蟲病一起[J];中國獸醫(yī)科技;1999年07期

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本文編號：1912175