本文選題：中山病　切入點(diǎn)：VP7蛋白　出處：《黑龍江八一農(nóng)墾大學(xué)》2017年碩士論文

【摘要】：中山病是由中山病病毒(Chuzan virus,CHUV)引起的一種蟲(chóng)媒性傳染病,感染牛羊等家畜,臨床癥狀以流產(chǎn)、早產(chǎn)、死產(chǎn)、產(chǎn)生先天性異常犢牛為主要特征。中山病來(lái)源于日本,國(guó)內(nèi)外相關(guān)研究較少。為了解中山病在我國(guó)的流行狀況以控制本病在我國(guó)的發(fā)生和發(fā)展,本研究開(kāi)展了我國(guó)中山病流行病學(xué)調(diào)查及間接ELISA抗體檢測(cè)方法的研究。對(duì)我國(guó)黑龍江、內(nèi)蒙古、新疆、山東、江蘇、湖北、廣西、云南和海南9個(gè)省區(qū)的543份牛羊血清,用經(jīng)典的病毒微量中和試驗(yàn)方法進(jìn)行中山病的流行病學(xué)調(diào)查。結(jié)果表明:海南68份牛羊血清樣品中陽(yáng)性樣品39份,陽(yáng)性率高達(dá)57.35%;黑龍江的80份樣品檢測(cè)結(jié)果均為陰性;其他各省的血清陽(yáng)性率介于6%-48.65%之間,氣候溫暖地區(qū)感染率較高;對(duì)新疆、湖北、廣西、云南4個(gè)省區(qū)牛和羊的易感性進(jìn)行比較,除新疆(羊陽(yáng)性率比牛高)外,其他地區(qū)牛的陽(yáng)性率均比羊高,牛血清陽(yáng)性率在55.56%-58.6%,羊在32.73%-33.33%之間;對(duì)新疆、湖北、廣西3個(gè)省區(qū)不同季節(jié)中山病的感染率進(jìn)行比較,秋季(9-11月)感染率(16.67%-43.33%)最高,明顯高于春季和夏季。為建立一種簡(jiǎn)便快速的檢測(cè)中山病抗體的間接ELISA方法,本研究將攜帶群特異性抗原決定簇的CHUV VP7蛋白作為檢測(cè)抗原,參考CHUV廣西分離株GX871毒株的S7基因序列設(shè)計(jì)引物,經(jīng)RT-PCR擴(kuò)增得到VP7基因,克隆至p ET-28a(+)表達(dá)載體中,經(jīng)IPTG誘導(dǎo)表達(dá)、SDS-PAGE和Western blot鑒定,獲得重組VP7蛋白。用純化的VP7蛋白作為包被抗原,用方陣滴定法確定包被抗原的最佳使用濃度,經(jīng)過(guò)對(duì)ELISA術(shù)式進(jìn)行優(yōu)化和完善,初步建立了檢測(cè)中山病抗體的間接ELISA方法。其臨界值分別為0.384-0.347,即OD450nm大于0.384為陽(yáng)性,小于0.347為陰性,二者之間為可疑;對(duì)阿卡斑病毒(Akabane virus,AKAV)、牛輪狀病毒(Rotaviruses,RV)、藍(lán)舌病病毒(Bluetongue virus,BTV)、口蹄疫病毒(Foot and mouth virus,FMDV)、牛病毒性腹瀉-粘膜病(Bovine viral diarrhoea virus,BVDV)、牛傳染性鼻氣管炎(Infectious bovine rhinotracheitis virus,IBRV)、牛副流感3型(Parainfluenza virus type3,PIV3)陽(yáng)性血清檢測(cè)結(jié)果均為陰性;批內(nèi)和批間重復(fù)試驗(yàn)的變異系數(shù)分別小于7.1%和13.2%;與中和試驗(yàn)符合率為83.05%。用所建立的間接ELISA檢測(cè)方法對(duì)我國(guó)黑龍江、新疆、廣西和云南四省區(qū)121份血清樣品進(jìn)行了檢測(cè),其陽(yáng)性率分別為0%、13.33%、39.39%和64.28%,與微量中和試驗(yàn)檢測(cè)結(jié)果一致。綜上所述,中山病在我國(guó)被調(diào)查的8個(gè)省區(qū)均有不同程度的流行,氣候溫暖地區(qū)感染率較高,秋季陽(yáng)性率較高,牛比羊更易感,符合蟲(chóng)媒病的流行規(guī)律。本研究建立的以重組VP7蛋白為抗原的間接ELISA方法,具有良好的特異性和敏感性,且成本較低,可用于中山病血清的高通量檢測(cè)。
[Abstract]:Zhongshan disease is caused by Zhongshan disease virus (Chuzan virus, CHUV) caused by an insect borne infectious diseases, infection of cattle and other livestock, clinical symptoms of abortion, premature birth, stillbirth and congenital anomalies were characterized. Zhongshan disease originated from Japan, less relevant research at home and abroad. In order to understand the disease epidemic situation in Zhongshan our country to control the disease in the occurrence and development of our country, this paper carried out research on detection method of investigation of Zhongshan disease epidemiology in China and the indirect ELISA antibody in China. Heilongjiang, Inner Mongolia, Xinjiang, Shandong, Jiangsu, Hubei, Guangxi, Yunnan and Hainan 9 provinces 543 cattle and sheep serum, were epidemiological investigation of Zhongshan disease virus neutralization test using classical methods. The results show that 68 samples of Hainan cattle and sheep serum samples positive in 39 samples, the positive rate is as high as 57.35% in Heilongjiang; 80 samples test results were negative for each other; Between the serum positive rate between 6%-48.65%, a higher rate of warm climate in the region of Xinjiang, Hubei, infection; Guangxi, Yunnan 4 provinces and the susceptibility of cattle and sheep were compared, in addition to Xinjiang (the positive rate of sheep than cattle, cattle in other high) positive rates than sheep, bovine serum positive rate in 55.56%-58.6% sheep, 32.73%-33.33%; in Xinjiang, Hubei, Guangxi 3 provinces were compared in different seasons in Zhongshan disease infection rate in autumn (9-11 months) (16.67%-43.33%) the highest infection rate was significantly higher than that in spring and summer. For the indirect ELISA method to establish a simple and rapid detection of Zhongshan disease antibody, the research group will carry the specific epitope of CHUV VP7 protein as antigen, primers S7 gene sequences of reference CHUV Guangxi isolate GX871 strain, VP7 gene was amplified by RT-PCR, cloned into P (+) ET-28a expression vector, expression of SDS induced by IPTG. -PAGE and Western blot identification, obtain the recombinant VP7 protein. The purified VP7 protein as antigen to determine the optimal concentration of coating antigen by matrix titration method, through the optimization and improvement of the ELISA operation, the initial establishment of the indirect ELISA method for detection of Zhongshan disease antibody. Its critical value was 0.384-0.347, i.e. OD450nm was more than 0.384 less than 0.347 positive, negative, between the two as suspicious of Akabane virus (Akabane; virus, AKAV), bovine rotavirus (Rotaviruses, RV), bluetongue virus (Bluetongue virus, BTV of foot-and-mouth disease virus (Foot), and mouth virus, FMDV), bovine viral diarrhea (Bovine viral diarrhoea virus, BVDV), infectious bovine rhinotracheitis (Infectious bovine rhinotracheitis virus, IBRV), bovine parainfluenza virus type 3 (Parainfluenza virus, type3, PIV3) detection results of positive serum were negative; intra and inter repeated test The coefficient of variation of the test were less than 7.1% and 13.2%; and with indirect ELISA neutralization test rate of 83.05%. by using the proposed detection method on China's Heilongjiang, Xinjiang, Guangxi and Yunnan four provinces of 121 serum samples were detected, the positive rates were 0%, 13.33%, 39.39% and 64.28%, and the micro neutralization test results consistent. To sum up, Zhongshan disease was investigated in China's 8 provinces have different levels of popularity, a higher rate of infection is higher in autumn warm climates, the positive rate of cattle sheep are more susceptible, meet the epidemic regularity of insect borne diseases. This study established the recombinant VP7 protein as antigen by indirect ELISA method, with good specificity and sensitivity, and low cost, can be used for high-throughput serum Zhongshan disease detection.

【學(xué)位授予單位】：黑龍江八一農(nóng)墾大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S855.3

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本文編號(hào)：1722061