本文主要研究内容
作者高佩佩（2019）在《猪瘟病毒E0、E2基因的原核表达及间接ELISA方法建立》一文中研究指出：猪瘟(classical swine fever,CSF)是由猪瘟病毒(classical swine fever virus,CSFV)引起的一种急性、烈性、高度接触性传染病。CSFV自19世纪在美国被首次报道以来,对世界养猪业的健康发展造成了严重的影响。猪瘟作为世界卫生组织(OIE)规定的A类疫病,世界上仍主要通过免疫弱毒疫苗的方法来对其进行防控。传统猪瘟弱毒疫苗免疫效力好,给猪群提供了较好的保护。但使用这种类型的疫苗后无法区分感染抗体和疫苗抗体。E2亚单位疫苗是最早投入使用也是当前惟一投入使用的猪瘟标记疫苗。免疫E2亚单位疫苗后不仅能够对抗CSFV野毒株,而且很容易通过检测抗E0蛋白的抗体区分免疫猪和感染猪。在CSFV编码的结构蛋白中,E0和E2糖蛋白均可诱导机体产生中和抗体,同时也是建立CSFV血清学检测方法的主要抗原。因此,在免疫E2亚单位疫苗的基础上,可以利用CSFV的E0和E2基因构建的检测方法,用来分析疫苗免疫效果以及初步区分疫苗抗体和感染抗体,这为进一步开发猪瘟检测试剂盒提供了参考。本实验进行了一系列的研究:(1)根据GeneBank中登录的猪瘟病毒E0和E2基因序列,并参考pET32a载体和蛋白的特点进行特异性引物的设计与合成,并且在引物的上下游5,端分别插入EcoRI和XhoI两种限制性内切酶的酶切位点。E0和E2基因经RT-PCR扩增,得到基因片段分别为699bp和558bp的条带,经克隆测序鉴定后的质粒命名为pMD19T-E0,pMD19T-E2。将pET32a线性载体与克隆质粒中切下的E0和E2基因连接构建重组表达质粒pET32a-E0,pET32a-E2,随后将重组质粒转入宿主菌BL21(DE3)中并进行诱导表达,经SDS-PAGE检测有与预期大小相合的蛋白大量表达,且蛋白主要存在于包涵体中。利用镍柱亲和层析法对pET32a-E0、pET32a-E2蛋白进行纯化,Westernblotting显示pET32a-E0、pET32a-E2蛋白的反应原性较好。(2)利用纯化的pET32a-E0重组蛋白作为抗原,构建了间接ELISA方法。对反应条件的优化结果显示:pET32a-E0重组蛋白的最适抗原包被浓度为0.6μg/mL,包被时间为37℃2 h,4℃过夜;最佳封闭条件为PBST缓冲液稀释的胎牛血清(5%)于37℃恒温反应1 h;血清和酶标抗体的最适稀释度分别为1∶100和1∶20000,两者均于37℃反应1 h;TMB底物显色液于室温暗处反应10 min。基于pET32a-E0蛋白建立的方法重复性试验(批内和批间)显示变异系数小于10%,且与PRRSV、PCV、PRV、PEDV、TGEV阳性血清间不存在抗原交叉反应,具有较高的重复性与特异性。(3)利用纯化的pET32a-E2重组蛋白作为包被抗原进行了间接ELISA方法的建立,对反应条件的优化和选择结果显示pET32a-E2重组蛋白的适宜反应浓度为1.6μg/mL于37℃恒温反应2 h后置于4℃过夜;采用PBST缓冲液溶解的脱脂奶粉(5%)进行封闭效果最好,37℃恒温反应1 h;血清和酶标二抗的最适稀释倍数分别为1∶80和1∶20000,两者均于37℃恒温反应1 h;TMB底物显色液于暗处室温反应15min最佳。所建立的方法重复性试验(批内和批间)显示变异系数小于10%,且与PRRSV、PCV、PRV、PEDV、TGEV阳性血清间不存在抗原交叉反应,具有较高的重复性与特异性。该方法为CSFV抗体的检测提供了一种相对实用的ELISA诊断方法。
Abstract
zhu wen (classical swine fever,CSF)shi you zhu wen bing du (classical swine fever virus,CSFV)yin qi de yi chong ji xing 、lie xing 、gao du jie chu xing chuan ran bing 。CSFVzi 19shi ji zai mei guo bei shou ci bao dao yi lai ,dui shi jie yang zhu ye de jian kang fa zhan zao cheng le yan chong de ying xiang 。zhu wen zuo wei shi jie wei sheng zu zhi (OIE)gui ding de Alei yi bing ,shi jie shang reng zhu yao tong guo mian yi ruo du yi miao de fang fa lai dui ji jin hang fang kong 。chuan tong zhu wen ruo du yi miao mian yi xiao li hao ,gei zhu qun di gong le jiao hao de bao hu 。dan shi yong zhe chong lei xing de yi miao hou mo fa ou fen gan ran kang ti he yi miao kang ti 。E2ya chan wei yi miao shi zui zao tou ru shi yong ye shi dang qian wei yi tou ru shi yong de zhu wen biao ji yi miao 。mian yi E2ya chan wei yi miao hou bu jin neng gou dui kang CSFVye du zhu ,er ju hen rong yi tong guo jian ce kang E0dan bai de kang ti ou fen mian yi zhu he gan ran zhu 。zai CSFVbian ma de jie gou dan bai zhong ,E0he E2tang dan bai jun ke you dao ji ti chan sheng zhong he kang ti ,tong shi ye shi jian li CSFVxie qing xue jian ce fang fa de zhu yao kang yuan 。yin ci ,zai mian yi E2ya chan wei yi miao de ji chu shang ,ke yi li yong CSFVde E0he E2ji yin gou jian de jian ce fang fa ,yong lai fen xi yi miao mian yi xiao guo yi ji chu bu ou fen yi miao kang ti he gan ran kang ti ,zhe wei jin yi bu kai fa zhu wen jian ce shi ji he di gong le can kao 。ben shi yan jin hang le yi ji lie de yan jiu :(1)gen ju GeneBankzhong deng lu de zhu wen bing du E0he E2ji yin xu lie ,bing can kao pET32azai ti he dan bai de te dian jin hang te yi xing yin wu de she ji yu ge cheng ,bing ju zai yin wu de shang xia you 5,duan fen bie cha ru EcoRIhe XhoIliang chong xian zhi xing nei qie mei de mei qie wei dian 。E0he E2ji yin jing RT-PCRkuo zeng ,de dao ji yin pian duan fen bie wei 699bphe 558bpde tiao dai ,jing ke long ce xu jian ding hou de zhi li ming ming wei pMD19T-E0,pMD19T-E2。jiang pET32axian xing zai ti yu ke long zhi li zhong qie xia de E0he E2ji yin lian jie gou jian chong zu biao da zhi li pET32a-E0,pET32a-E2,sui hou jiang chong zu zhi li zhuai ru su zhu jun BL21(DE3)zhong bing jin hang you dao biao da ,jing SDS-PAGEjian ce you yu yu ji da xiao xiang ge de dan bai da liang biao da ,ju dan bai zhu yao cun zai yu bao han ti zhong 。li yong nie zhu qin he ceng xi fa dui pET32a-E0、pET32a-E2dan bai jin hang chun hua ,Westernblottingxian shi pET32a-E0、pET32a-E2dan bai de fan ying yuan xing jiao hao 。(2)li yong chun hua de pET32a-E0chong zu dan bai zuo wei kang yuan ,gou jian le jian jie ELISAfang fa 。dui fan ying tiao jian de you hua jie guo xian shi :pET32a-E0chong zu dan bai de zui kuo kang yuan bao bei nong du wei 0.6μg/mL,bao bei shi jian wei 37℃2 h,4℃guo ye ;zui jia feng bi tiao jian wei PBSThuan chong ye xi shi de tai niu xie qing (5%)yu 37℃heng wen fan ying 1 h;xie qing he mei biao kang ti de zui kuo xi shi du fen bie wei 1∶100he 1∶20000,liang zhe jun yu 37℃fan ying 1 h;TMBde wu xian se ye yu shi wen an chu fan ying 10 min。ji yu pET32a-E0dan bai jian li de fang fa chong fu xing shi yan (pi nei he pi jian )xian shi bian yi ji shu xiao yu 10%,ju yu PRRSV、PCV、PRV、PEDV、TGEVyang xing xie qing jian bu cun zai kang yuan jiao cha fan ying ,ju you jiao gao de chong fu xing yu te yi xing 。(3)li yong chun hua de pET32a-E2chong zu dan bai zuo wei bao bei kang yuan jin hang le jian jie ELISAfang fa de jian li ,dui fan ying tiao jian de you hua he shua ze jie guo xian shi pET32a-E2chong zu dan bai de kuo yi fan ying nong du wei 1.6μg/mLyu 37℃heng wen fan ying 2 hhou zhi yu 4℃guo ye ;cai yong PBSThuan chong ye rong jie de tuo zhi nai fen (5%)jin hang feng bi xiao guo zui hao ,37℃heng wen fan ying 1 h;xie qing he mei biao er kang de zui kuo xi shi bei shu fen bie wei 1∶80he 1∶20000,liang zhe jun yu 37℃heng wen fan ying 1 h;TMBde wu xian se ye yu an chu shi wen fan ying 15minzui jia 。suo jian li de fang fa chong fu xing shi yan (pi nei he pi jian )xian shi bian yi ji shu xiao yu 10%,ju yu PRRSV、PCV、PRV、PEDV、TGEVyang xing xie qing jian bu cun zai kang yuan jiao cha fan ying ,ju you jiao gao de chong fu xing yu te yi xing 。gai fang fa wei CSFVkang ti de jian ce di gong le yi chong xiang dui shi yong de ELISAzhen duan fang fa 。
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论文作者分别是来自河北农业大学的高佩佩，发表于刊物河北农业大学2019-09-19论文，是一篇关于猪瘟病毒论文,蛋白论文,蛋白论文,诊断方法论文,河北农业大学2019-09-19论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自河北农业大学2019-09-19论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：猪瘟病毒论文; 蛋白论文; 诊断方法论文; 河北农业大学2019-09-19论文;