【摘要】：腦心肌炎病毒(Encephalomyocarditis virus,EMCV)是一種自然疫源性人畜共患病�？梢愿腥径喾N動(dòng)物和人,引起以腦炎、心肌炎或心肌周?chē)诪橹鞯囊活?lèi)高度傳染病。豬是EMCV的易感宿主,引起哺乳仔豬的高死亡率和妊娠母豬的繁殖障礙。給養(yǎng)豬業(yè)帶來(lái)了極大的經(jīng)濟(jì)損失。多數(shù)豬場(chǎng)雖然未用EMCV的疫苗進(jìn)行免疫,但血清學(xué)調(diào)查表明,我國(guó)規(guī)�；i場(chǎng)的豬的血清陽(yáng)性率達(dá)50%以上。表明豬群中EMCV的野毒感染較為普遍,因此需要對(duì)EMCV的流行進(jìn)行檢測(cè)和監(jiān)控。為了對(duì)EMCV的流行病學(xué)進(jìn)行調(diào)查和分析,我們參考GenBank中的序列,人工合成一段含有VP1抗原的DNA序列,轉(zhuǎn)化到BL21感受態(tài)細(xì)胞中,誘導(dǎo)表達(dá)蛋白并純化。將純化的VP1蛋白包被ELISA板,建立檢測(cè)豬腦心肌炎病毒抗體的間接ELISA的診斷方法,并對(duì)ELISA檢測(cè)條件進(jìn)行優(yōu)化。最佳包被緩沖液為PH9.6的碳酸鹽緩沖液;最佳包被蛋白的濃度為用1mg/ml;4℃包被過(guò)夜;封閉條件是5%milk作為封閉液,37℃,封閉1h;待檢血清的最佳稀釋倍數(shù)為1:100倍稀釋,待檢血清的孵育條件為37℃,0.5h;HRP標(biāo)記二抗抗體的最佳稀釋倍數(shù)為1:1000倍稀釋,反應(yīng)條件為37℃,孵育1.5h;TMB顯色時(shí)間為室溫下20min,在OD450進(jìn)行讀數(shù),并對(duì)數(shù)據(jù)進(jìn)行分析。通過(guò)對(duì)2106份豬血清進(jìn)行檢測(cè),用TG-ROC軟件對(duì)檢測(cè)的數(shù)據(jù)進(jìn)行分析統(tǒng)計(jì),確定本方法的判定標(biāo)準(zhǔn)是:OD值大于0.5的為陽(yáng)性;OD值小于0.4的為陰性;界于兩者之間的為可疑。應(yīng)用該方法,檢測(cè)了山東省區(qū)域內(nèi)豬的血清2106份,OD值大于0.5的數(shù)值有984份,陽(yáng)性率為46.72%;OD值小于0.4的數(shù)值有985份,陰性率為46.77%;界于兩者之間的數(shù)值有137份,為可疑值,可疑率為6.51%。結(jié)果證明山東省內(nèi)的豬群,大部分存在EMCV抗體,EMCV感染非常普遍。本實(shí)驗(yàn)為山東省內(nèi)的EMCV的感染的流行病學(xué)監(jiān)測(cè)及診斷提供了有效手段,并可以大概了解山東省內(nèi)豬群的EMCV的感染狀況。
[Abstract]:Cerebral myocarditis virus (Encephalomyocarditis virus,EMCV) is a natural zoonotic disease. Can infect a variety of animals and people, causing encephalitis, myocarditis, or perimyocardial inflammation is a high-level infectious disease. Pig is a susceptible host of EMCV, which causes high mortality of suckling piglets and reproductive disturbance of pregnant sows. It has brought great economic losses to the pig industry. Although most pig farms were not immunized with EMCV vaccine, serological investigation showed that the seropositive rate of pigs in large-scale pig farms in China was more than 50%. The results showed that the wild virus infection of EMCV was common in pigs, so it was necessary to detect and monitor the prevalence of EMCV. In order to investigate and analyze the epidemiology of EMCV, we synthesized a sequence of DNA containing VP1 antigen and transformed it into BL21 competent cells, induced the expression of protein and purified it. The purified VP1 protein was coated with Elisa board to establish an indirect ELISA diagnostic method for detection of antibodies against porcine encephalomyocarditis virus. The detection conditions of ELISA were optimized. The optimal coating buffer was carbonate buffer of PH9.6, the optimal concentration of coated protein was 1 mg / ml 路ml ~ (- 1) for the night at 4 鈩，

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