检测日本脑炎病毒NS1’蛋白双抗体夹心ELISA的建立及其应用

发布时间：2018-06-25 03:47

本文选题：日本脑炎病毒 + NS1　；参考：《南京农业大学》2015年硕士论文

【摘要】：日本脑炎病毒(Japanese encephalitis virus,JEV)是一种单股正链RNA病毒,可以引起公猪睾丸炎和母猪流产,是一种人畜共患病毒,蚊子叮咬传播。JEV编码的蛋白有核心蛋白C,膜蛋白PrM/M和囊膜蛋白E以及(NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5)七个非结构蛋白。JEV野毒株NS2A基因在翻译过程中会因为核糖体-1移码产生衍生物,与NS1蛋白C端融合,产生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突变为A,破坏了形成移码功能的结构基袖,因而失去了产生NS1'的能力。JEV NS1(NS1,)蛋白可以六聚体的形式分泌到感染动物的血清中,因此,NS 1蛋白是JEV野毒感染的特征指标。本研究用兔源日本脑炎病毒NS1蛋白多抗纯化后作为捕获抗体,用HRP标记的JEV NS1'单克隆抗体作为检测抗体构建检测JEV NS1'蛋白的双抗体础心ELISA。应用大肠杆菌表达的NS1'蛋白作为阳性标准品,绘制蛋白浓度与OD450nm的关系曲线,初步确定其检测的灵敏度。应用该方法检测JEV野毒与疫苗毒感染细胞上清与实验小鼠血清中NS1'蛋白,验证其检测天然NS1'的能力。利用上述建立的方法对2014年实验室收集到的临床猪血清进行初步的血清学流行病学调查,测定猪群中JEV野毒的感染情况,为猪流行性乙型脑炎的防控提供参考,具体内容如下1、日本脑炎病毒NS1和NS1'蛋白抗体的制备与鉴定原核表达JEV NS1蛋白,纯化后三次免疫大白兔,制备兔源JEV NS1蛋白多克隆抗血清,Protein A纯化。ELISA测定NS1兔源抗体效价,western blot、IFA验证该纯化多抗可以特异识别JEV NS1蛋白。复苏本实验室制备保存的NS1'蛋白单克隆抗体杂交瘤细胞并制备腹水,得到的腹水经过ProteinG纯化单抗,western blot结果表明该单抗能与JEV的NS1'蛋白(约52kda)发生特异性反应,IFA结果表明该单抗能够特异性的识别JEV NJ2008株感染的BHK细胞而与疫苗毒SA14-14-2感染的BHK细胞无反应。用辣根过氧化物酶(HRP)标记JEV NS1'单克隆抗体,通过间接ELISA测定兔源多抗与HRP标记的鼠源单抗效价均在1:100000以上,这两种抗体的制备为后续的双抗体夹心ELISA方法的建立提供了良好的物质基础。2、检测JEV NS1'蛋白双抗体夹心ELISA的建立应用纯化的兔源JEV NS 1蛋白多抗作为捕获抗体,HRP标记的NS1'单克隆抗体作为检测抗体,建立了检测JEVNS1'蛋白的双抗体夹心ELISA。其优化条件为:兔源多抗包被浓度为10μg/ml,包被条件为4℃ 12h;用含2%BSA的磷酸缓冲液封闭,37℃作用2h;待检猪血清样品1:50稀释加样,37℃作用1.5h,HRP(辣根过氧化物酶)标记NS1'蛋白单克隆抗体1:500稀释,37℃作用1h;TMB显色时间为37℃10min。判定标准为:样品的OD450nm≥0.268时为阳性,样品OD450nm≤0.238为阴性,介于0.198-0.218为疑似,利用体外原核表达的NS1'蛋白,利用上述建立的双抗体夹心ELISA方法的检测下限为0.29ng/ml,当抗原的含量为25.12ng/ml时曲线趋于平缓,说明结合的抗原量达到饱和,同时,当蛋白抗原含量为0.29ng/ml～4.64ng/ml时,线性关系基本处于直线,有较好的相关性,利用该方法检测JEV感染的BHK细胞裂解液与细胞上清,发现在感染后的48h到96h之间,NS1'蛋白的浓度开始上升,在96h达到峰值,且通过测定发现NS1'蛋白在分泌到细胞外的NS1'量与细胞内的NS1'量基本一致,小鼠动物实验表明分泌到小鼠血清中的NS1'蛋白与第7d左右有所上升,在第10d左右达到峰值,在14d左右开始下降,在21d仍旧能检测到NS1'蛋白。3、2014年我国不同生态地区猪乙型脑炎野毒感染情况流行病学调查应用上述建立的ELIS A对2014年本实验室收集的临床猪血清进行流行病学调查,样品主要来源于1～9月份广东、海南、江西、江苏、山东、陕西、甘肃等7个猪群的产房仔猪、保育仔猪、育肥猪与母猪等临床猪血清,经过调查发现在气候炎热的季节(7～9月)中,样品来源猪群JEV野毒感染率在40%左右,明显高于3月份(23.4%)、5月份(33.86%)收集到的血清,在气候寒冷的1月份野毒感染率为0%,同时针对蚊虫较多的敏感季节(7～9月份)可以看出乙脑的野毒感染呈现明显的地域分布特征,江西(53.53%)、广东(39.69%)、江苏(37.54%)等地区乙脑的野毒感染率明显高于甘肃(18.13%)、海南(17.27%)等地区,且乙脑野毒感染在猪群中主要集中在产房仔猪(48.80%)与母猪上(47.62%),而保育仔猪(26.87%)与育肥猪(24.46%)相对较少,从上述的流行病学调查中可以看出乙脑的野毒感染呈现明显的季节性、区域性、猪群类别性,这对后期JEV的防控及流行病学的开展提供了参考。
[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.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4

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