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猪脑心肌炎病毒非结构蛋白2C的原核表达与间接ELISA方法的建立

标题: 猪脑心肌炎病毒非结构蛋白2C的原核表达与间接ELISA方法的建立
英文标题: Prokaryotic Expression of Nonstructural Protein 2C of Porcine EMCV and Establishment of Indirect ELISA
作者: 刘兰亚
出版时间: 2009-01-01
所在大学: 河北农业大学
关键词: EMCV,2C基因,克隆,原核表达,ELISA,血清学调查
英文关键词: EMCV,2C gene,Clone,Prokaryotic expression,ELISA,Serosurvey
论文级别: 硕士
学位: 学位论文
导师: 宋勤叶
专业: 预防兽医学
提交时间: 2009
摘要: 猪脑心肌炎是由脑心肌炎病毒(encephalomyocarditis viurs,EMCV)引起猪的以脑炎、心肌炎或心肌周围炎为主要特征的急性传染病.本研究针对EMCV非结构蛋白2C基因设计合成了一对特异性引物,通过RT-PCR获得了长为1062 bp的2C基因序列,构建了重组原核表达质粒pGEX-6P-2C,并在大肠杆菌BL21细胞中成功表达了62 ku预期大小的重组2C蛋白,蛋白表达量约为33.9%.重组2C蛋白于室温保存72 h,-20℃和-80℃分别保存12月,冻融9次未见降解.Western blot分析表明,重组2C蛋白可以被猪源或兔源抗EMCV阳性血清识别. 以纯化的重组2C蛋白作为包被抗原建立了检测EMCV抗体的间接ELISA方法,确定了方法的最佳工作条件和阴阳性临界值,检测了方法的特异性、敏感性、重复性,并用该方法分析了EMCV感染动物2C抗体的产生动态.试验结果表明,间接ELISA的阴阳性临界值为0.27,以1 μg/mL的重组2C蛋白于37℃包被1 h后4℃过夜,用含5%小牛血清的PBST于37℃封闭1 h,待检血清作1?20倍、酶标二抗作1?400倍稀释后分别于37℃反应1 h和45 min后,加底物后于室温避光显色15 min为最佳工作浓度和工作条件.重组2C抗原与猪抗CSFV、PCV2、PPV、PRV和PRRSV等5种阳性血清不发生交叉反应,EMCV可以阻断重组抗原与脑心肌炎阳性血清的特异性反应.该ELISA方法与血清中和试验的符合率为95.7%(22/23),相对敏感性为90.9%(10/11);同一时间批内重复性试验的变异系数<10%,不同时间批间重复性试验的变异系数<15%.应用该方法可以在EMCV感染仔猪后4 d,检测到相应抗体,感染后7 d抗体水平最高,随后逐渐下降,直至感染后21 d仍然可以检测到较低水平的2C抗体. 应用建立的间接ELISA方法检测了2006~2008年间河北省不同地区31个规模化猪场的1306份血清样本.结果显示,EMCV抗体阳性率平均为13.48%,其中经产母猪的抗体阳性率最高(27.68%),其次为后备母猪和哺乳仔猪(分别为16.67%和2.86%),育成猪EMCV抗体阳性率最低(1.98%). 上述研究结果表明,重组EMCV 2C蛋白具有良好的稳定性和反应原性;以其作为包被抗原,建立的间接ELISA方法可以用于猪脑心肌炎的早期诊断和血清流行病学调查;河北省猪群中存在EMCV感染现象,尤以经产母猪的感染率最高.
英文摘要: Pocrine encephalomyocarditis is an acute infectious disease of swine caused by encephalomyocarditis virus(EMCV)and characterized by encephalitis, myocarditis or inflammation around cardiac muscles. In this study, first, a pair of specific primer to EMCV nonstructural protein 2C gene was designed and synthesized and 2C gene sequence of 1062 bp was obtained by RT-PCR. Moreover, recombinant expression plasmid pGEX-6P-2C was constructed and predicted recombinant nonstructural protein 2C of 62 ku was expressed in E.coli BL21 cells at the rate of about 33.9%. No visible degradation was found when recombinant protein 2C was kept in room temperature for 72 h, -20℃and -80℃for12 months, or when it underwent freeze- thawing for 9 times. And Western blotting showed that recombinant protein 2C could be recognized by positive pig or rabbit anti-serum to EMCV.Second, an indirect ELISA was developed to detect antibody against EMCV with purified recombinant protein 2C as coating antigen, reaction condition and working concentrations optimized, cut-off value confirmed, specificity, sensitivity and repetition of assay detected, and serum antibody against protein 2C dynamically analysed. Results indicated that the cutoff value of ELISA was 0.27; optimal reaction condition and working concentration went as follows: incubating the coating 100μL of recombinant antigen (1μg/mL) at 37℃for 1 h and at 4℃for overnight; blocking the uncoated part at 37℃for 1 h with PBST containing fetal bovine serum of 5%; diluting serum sample or the HRP conjugated to secodary antibody (goat anti-pig IgG-HRP) with blocking buffers at 1:20 or 1:400 and incubating it at 37℃for 1 h or 45 min; reacting with HRP protected from light at room temperature for 15 min after adding substrate. Moreover,recombinant 2C protein had no cross-reaction with positive sera of other 4 swine diseases (CSF, PCV2, PPV , PR and PRRS infection); EMCV could block the specific combination of recombinant antigen with positive serum of pocrine encephalomyocarditis. The concordance between this ELISA and neutralization test was 95.7%(22/23), and the relative sensitivity was 90.9%(10/11). The coefficient of variant (CV) in repetition test at the same time was under 10%, and that at different times under 15%. Antibody against EMCV was detected in sera of piglets on 4 d post infected (dpi). The antibody reached to the highest on 7 dpi, and then gradually declined but lower antibody to 2C could still be detected until 21dpi.Third, 1306 serum samples collected from different areas in Hebei Province were detected by the indirect ELISA. The results showed that an average positive rate of antibody to EMCV was 13.48%, in which the highest antibody positive rate occurred to postpartum sows (27.68%), the secondary to gilts and suckling pigs (16.67%and 2.86%) and the lowest to growing pigs (1.98%).In summary, the recombinant EMCV 2C protein was of high stability and reactinogenicity.Using it as antigen, indirect ELISA could be applied to early diagnosis of pocrine encephalomyocarditis and seroepidemiological survey. At swine farms of Hebei Province, EMCV infection had occured with a high infection rate in postpartum sow.