【摘要】：為探明豬流行性腹瀉病毒(porcine epidemic diarrhea virus,PEDV)N基因編碼的蛋白對(duì)PEDV感染早期診斷的作用,根據(jù)PEDV CV777株N基因全序列設(shè)計(jì)一對(duì)特異性引物以擴(kuò)增N基因,定向插入到pPM-CHis真核表達(dá)載體中構(gòu)建重組表達(dá)質(zhì)粒pPM-C-His-N,將重組質(zhì)粒肌肉注射昆明系小鼠,以檢測(cè)其在小鼠體內(nèi)的表達(dá)水平;將pPM-C-His-N轉(zhuǎn)染至Vero細(xì)胞,分別在基因水平和蛋白水平對(duì)N基因的表達(dá)進(jìn)行檢測(cè),并采用間接免疫熒光試驗(yàn)(indirect immunofluorescence assay,IFA)檢測(cè)N蛋白在細(xì)胞中的表達(dá)分布情況。結(jié)果表明,重組質(zhì)粒在基因水平和蛋白水平均成功表達(dá),免疫小鼠血清中抗體的效價(jià)為1∶51 200,Western-blot結(jié)果顯示,免疫血清能與重組N蛋白發(fā)生特異性反應(yīng),說明實(shí)驗(yàn)成功構(gòu)建了重組真核表達(dá)質(zhì)粒pPM-C-His-N,且在Vero細(xì)胞內(nèi)檢測(cè)到了綠色熒光,為PEDV診斷方法的建立及致病機(jī)制的研究奠定基礎(chǔ)。
[Abstract]:The recombinant expression plasmid pPM-C-His-N, was inserted into the eukaryotic expression vector of pPM-CHis. The recombinant plasmid was injected intramuscularly into Kunming mice to detect its expression level in mice. PPM-C-His-N was transfected into Vero cells. The expression of N gene was detected at gene level and protein level, and the distribution of N protein in cells was detected by indirect immunofluorescence assay (indirect immunofluorescence assay,IFA). The results showed that the recombinant plasmid was successfully expressed at the gene level and protein level. The titer of antibody in the serum of immunized mice was 1:51 200m Western-blot. The results showed that the immunized serum could react specifically with the recombinant N protein. The results showed that the recombinant eukaryotic expression plasmid pPM-C-His-N, was successfully constructed and the green fluorescence was detected in Vero cells, which laid a foundation for the establishment of PEDV diagnostic method and the study of pathogenesis.
【作者單位】： 安徽農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院;
【基金】：安徽省自然科學(xué)基金(1708085MC83) 現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系建設(shè)專項(xiàng)資金資助項(xiàng)目(CARS-36-生豬) 安徽省生豬產(chǎn)業(yè)技術(shù)體系資金資助項(xiàng)目
【分類號(hào)】：S852.651

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