本文選題：日本腦炎病毒 + NS1�。� 參考：《南京農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：日本腦炎病毒(Japanese encephalitis virus,JEV)是一種單股正鏈RNA病毒,可以引起公豬睪丸炎和母豬流產(chǎn),是一種人畜共患病毒,蚊子叮咬傳播。JEV編碼的蛋白有核心蛋白C,膜蛋白PrM/M和囊膜蛋白E以及(NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5)七個(gè)非結(jié)構(gòu)蛋白。JEV野毒株NS2A基因在翻譯過程中會(huì)因?yàn)楹颂求w-1移碼產(chǎn)生衍生物,與NS1蛋白C端融合,產(chǎn)生NS1'蛋白。JEV弱毒疫苗株SA14-14-2由于NS2A基因的66位核苷酸由G突變?yōu)锳,破壞了形成移碼功能的結(jié)構(gòu)基袖,因而失去了產(chǎn)生NS1'的能力。JEV NS1(NS1,)蛋白可以六聚體的形式分泌到感染動(dòng)物的血清中,因此,NS 1蛋白是JEV野毒感染的特征指標(biāo)。本研究用兔源日本腦炎病毒NS1蛋白多抗純化后作為捕獲抗體,用HRP標(biāo)記的JEV NS1'單克隆抗體作為檢測抗體構(gòu)建檢測JEV NS1'蛋白的雙抗體礎(chǔ)心ELISA。應(yīng)用大腸桿菌表達(dá)的NS1'蛋白作為陽性標(biāo)準(zhǔn)品,繪制蛋白濃度與OD450nm的關(guān)系曲線,初步確定其檢測的靈敏度。應(yīng)用該方法檢測JEV野毒與疫苗毒感染細(xì)胞上清與實(shí)驗(yàn)小鼠血清中NS1'蛋白,驗(yàn)證其檢測天然NS1'的能力。利用上述建立的方法對2014年實(shí)驗(yàn)室收集到的臨床豬血清進(jìn)行初步的血清學(xué)流行病學(xué)調(diào)查,測定豬群中JEV野毒的感染情況,為豬流行性乙型腦炎的防控提供參考,具體內(nèi)容如下1、日本腦炎病毒NS1和NS1'蛋白抗體的制備與鑒定原核表達(dá)JEV NS1蛋白,純化后三次免疫大白兔,制備兔源JEV NS1蛋白多克隆抗血清,Protein A純化。ELISA測定NS1兔源抗體效價(jià),western blot、IFA驗(yàn)證該純化多抗可以特異識別JEV NS1蛋白。復(fù)蘇本實(shí)驗(yàn)室制備保存的NS1'蛋白單克隆抗體雜交瘤細(xì)胞并制備腹水,得到的腹水經(jīng)過ProteinG純化單抗,western blot結(jié)果表明該單抗能與JEV的NS1'蛋白(約52kda)發(fā)生特異性反應(yīng),IFA結(jié)果表明該單抗能夠特異性的識別JEV NJ2008株感染的BHK細(xì)胞而與疫苗毒SA14-14-2感染的BHK細(xì)胞無反應(yīng)。用辣根過氧化物酶(HRP)標(biāo)記JEV NS1'單克隆抗體,通過間接ELISA測定兔源多抗與HRP標(biāo)記的鼠源單抗效價(jià)均在1:100000以上,這兩種抗體的制備為后續(xù)的雙抗體夾心ELISA方法的建立提供了良好的物質(zhì)基礎(chǔ)。2、檢測JEV NS1'蛋白雙抗體夾心ELISA的建立應(yīng)用純化的兔源JEV NS 1蛋白多抗作為捕獲抗體,HRP標(biāo)記的NS1'單克隆抗體作為檢測抗體,建立了檢測JEVNS1'蛋白的雙抗體夾心ELISA。其優(yōu)化條件為:兔源多抗包被濃度為10μg/ml,包被條件為4℃ 12h;用含2%BSA的磷酸緩沖液封閉,37℃作用2h;待檢豬血清樣品1:50稀釋加樣,37℃作用1.5h,HRP(辣根過氧化物酶)標(biāo)記NS1'蛋白單克隆抗體1:500稀釋,37℃作用1h;TMB顯色時(shí)間為37℃10min。判定標(biāo)準(zhǔn)為:樣品的OD450nm≥0.268時(shí)為陽性,樣品OD450nm≤0.238為陰性,介于0.198-0.218為疑似,利用體外原核表達(dá)的NS1'蛋白,利用上述建立的雙抗體夾心ELISA方法的檢測下限為0.29ng/ml,當(dāng)抗原的含量為25.12ng/ml時(shí)曲線趨于平緩,說明結(jié)合的抗原量達(dá)到飽和,同時(shí),當(dāng)?shù)鞍卓乖繛?.29ng/ml～4.64ng/ml時(shí),線性關(guān)系基本處于直線,有較好的相關(guān)性,利用該方法檢測JEV感染的BHK細(xì)胞裂解液與細(xì)胞上清,發(fā)現(xiàn)在感染后的48h到96h之間,NS1'蛋白的濃度開始上升,在96h達(dá)到峰值,且通過測定發(fā)現(xiàn)NS1'蛋白在分泌到細(xì)胞外的NS1'量與細(xì)胞內(nèi)的NS1'量基本一致,小鼠動(dòng)物實(shí)驗(yàn)表明分泌到小鼠血清中的NS1'蛋白與第7d左右有所上升,在第10d左右達(dá)到峰值,在14d左右開始下降,在21d仍舊能檢測到NS1'蛋白。3、2014年我國不同生態(tài)地區(qū)豬乙型腦炎野毒感染情況流行病學(xué)調(diào)查應(yīng)用上述建立的ELIS A對2014年本實(shí)驗(yàn)室收集的臨床豬血清進(jìn)行流行病學(xué)調(diào)查,樣品主要來源于1～9月份廣東、海南、江西、江蘇、山東、陜西、甘肅等7個(gè)豬群的產(chǎn)房仔豬、保育仔豬、育肥豬與母豬等臨床豬血清,經(jīng)過調(diào)查發(fā)現(xiàn)在氣候炎熱的季節(jié)(7～9月)中,樣品來源豬群JEV野毒感染率在40%左右,明顯高于3月份(23.4%)、5月份(33.86%)收集到的血清,在氣候寒冷的1月份野毒感染率為0%,同時(shí)針對蚊蟲較多的敏感季節(jié)(7～9月份)可以看出乙腦的野毒感染呈現(xiàn)明顯的地域分布特征,江西(53.53%)、廣東(39.69%)、江蘇(37.54%)等地區(qū)乙腦的野毒感染率明顯高于甘肅(18.13%)、海南(17.27%)等地區(qū),且乙腦野毒感染在豬群中主要集中在產(chǎn)房仔豬(48.80%)與母豬上(47.62%),而保育仔豬(26.87%)與育肥豬(24.46%)相對較少,從上述的流行病學(xué)調(diào)查中可以看出乙腦的野毒感染呈現(xiàn)明顯的季節(jié)性、區(qū)域性、豬群類別性,這對后期JEV的防控及流行病學(xué)的開展提供了參考。
[Abstract]:Japanese encephalitis virus (Japanese encephalitis virus, JEV) is a single strand of positive chain RNA virus, which can cause boar orchitis and sow abortion. It is a zoonotic virus. The protein of.JEV encoded by mosquito bites is the core protein C, membrane protein PrM/M and membrane protein E and seven non structural proteins. In the process of translation, the NS2A gene of the.JEV wild strain produces derivatives due to the ribosome -1 transcoding, and is fused with the C terminal of the NS1 protein to produce the NS1'protein.JEV weakly toxic vaccine strain SA14-14-2 because the 66 bit nucleotide of the NS2A gene is changed from G to A. It is secreted into the sera of infected animals in the form of six polymers. Therefore, NS 1 protein is a characteristic index of JEV virus infection. This study used rabbit source of Japanese encephalitis virus NS1 protein as a capture antibody and HRP labeled JEV NS1'monoclonal antibody as a detection antibody to construct a double antibody base ELISA. application for detecting JEV NS1' protein. NS1'protein expressed in Escherichia coli was used as a positive standard, and the relationship between protein concentration and OD450nm was plotted and the sensitivity of the detection was preliminarily determined. The method was used to detect the NS1' protein in the cell supernatant of JEV and vaccine infected cells and the serum of experimental mice, and to verify its ability to detect natural NS1'. The method established above was used in 2014. A preliminary serological epidemiological survey was carried out in the clinical pig serum collected by the laboratory to determine the infection of JEV wild virus in the pigs and to provide reference for the prevention and control of Japanese encephalitis B. The specific contents are as follows: 1, the preparation and identification of the NS1 and NS1'protein antibodies of Japanese encephalitis virus and the identification of the prokaryotic expression JEV NS1 protein, and the purification of the purified protein after the purification of the Japanese encephalitis virus. Rabbit, prepared rabbit source JEV NS1 protein polyclonal antiserum, Protein A purified.ELISA assay of NS1 rabbit antibody titer, Western blot, IFA confirmed that the purified polyclonal antibody can specifically identify JEV NS1 protein. The results of stern blot showed that the monoclonal antibody could react specifically with the NS1'protein (about 52kda) of JEV. The results of IFA showed that the monoclonal antibody could specifically identify BHK cells infected by JEV NJ2008 strain and no response to the BHK cells infected by the vaccine virus SA14-14-2. The titer of rat source mAb of rabbit source polyclonal and HRP markers is above 1:100000. The preparation of these two antibodies provides a good material basis for the establishment of the subsequent double antibody sandwich ELISA method, and the establishment of the JEV NS1'protein double antibody sandwich ELISA and the purified rabbit source JEV NS 1 egg white polyclonal antibody as the capture antibody and NS1' monomer for HRP labeling. The clonal antibody was used as a detection antibody to establish a double antibody sandwich ELISA. for detection of JEVNS1'protein. The optimum condition was that the concentration of the rabbit source polyclonal package was 10 u g/ml and the envelope was 4 centigrade 12h; it was sealed with 2%BSA phosphoric acid buffer and was 2H at 37 C; the samples were diluted at 1:50, at 37, 1.5h, and HRP (horseradish peroxidase) labeled N S1'protein monoclonal antibody 1:500 diluted, 37 C action 1H; TMB color time is 37 C 10min. determination standard: sample OD450nm > 0.268 positive, sample OD450nm < 0.238 as negative, 0.198-0.218 as suspected, using in vitro prokaryotic expression of the NS1' protein, using the above established dual antibody sandwich ELISA method detection limit of 0.29ng is 0.29ng /ml, when the content of antigen is 25.12ng/ml, the curve tends to be slow, indicating that the amount of antigen binding is saturated. At the same time, when the protein antigen content is 0.29ng/ml to 4.64ng/ml, the linear relationship is basically in a straight line and has a good correlation. This method is used to detect the BHK cell lysate and cell supernatant of JEV infection, and the 48h to 96 after infection is found. Between H, the concentration of NS1'protein began to rise and peak at 96h, and the NS1' quantity of NS1'protein secreted to the cell was found to be the same as that of NS1' in the cell. The mouse experiment showed that the NS1'protein secreted into the serum of mice increased, at the peak at 10d, and began to decline around 14d. The epidemiological investigation of NS1'protein.32014 was still detected by 21d in different ecological regions of China, and the epidemiological investigation of wild virus infection in swine encephalitis in different ecological regions in China was used to investigate the epidemiological investigation of the clinical pig serum collected by this laboratory in 2014. The samples were mainly derived from Guangdong, Hainan, Jiangxi, Jiangsu, Shandong, Shaanxi, Gansu in 2014. In the delivery room, piglets, piglets, fattening pigs and sows in the 7 pigs, it was found that in the hot season (7~9 months), the rate of JEV wild virus infection was about 40%, obviously higher than that in March (23.4%), and in May (33.86%), the rate of wild virus infection was 0% in the cold climate of January. In the sensitive season of mosquitoes (7~9 months), it can be seen that the wild venom infection of the encephalitis B has obvious regional distribution characteristics. The rate of wild venom infection in Jiangxi (53.53%), Guangdong (39.69%), Jiangsu (37.54%) and other regions is obviously higher than that of Gansu (18.13%), Hainan (17.27%) and other regions, and the wild venom infection of the brain is mainly concentrated in the delivery room. Pigs (48.80%) and sows (47.62%), and the conserved piglets (26.87%) and fattening pigs (24.46%) are relatively less. From the above epidemiological investigation, it can be seen that the wild venom infection in the brain of ethyl acetate shows a distinct seasonal, regional, type of pig group, which provides a reference for the prevention and control of the late JEV and the development of epidemiology.
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S852.4

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8 李焱;Vero細(xì)胞膜上與日本腦炎病毒結(jié)合分子的初步篩選[D];第四軍醫(yī)大學(xué);2010年

9 黃藝珠;日本腦炎病毒E蛋白多表位疫苗的設(shè)計(jì)及其在小鼠模型上的免疫效力評價(jià)[D];南京農(nóng)業(yè)大學(xué);2009年

10 唐青海;日本腦炎病毒W(wǎng)He株基因組特征及其疫苗的研究[D];西北農(nóng)林科技大學(xué);2007年

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