豬流行性腹瀉病毒N基因重組真核質粒構建及其表達
發(fā)布時間：2018-10-20 17:16
【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PEDV感染早期診斷的作用,根據(jù)PEDV CV777株N基因全序列設計一對特異性引物以擴增N基因,定向插入到pPM-CHis真核表達載體中構建重組表達質粒pPM-C-His-N,將重組質粒肌肉注射昆明系小鼠,以檢測其在小鼠體內(nèi)的表達水平;將pPM-C-His-N轉染至Vero細胞,分別在基因水平和蛋白水平對N基因的表達進行檢測,并采用間接免疫熒光試驗(indirect immunofluorescence assay,IFA)檢測N蛋白在細胞中的表達分布情況。結果表明,重組質粒在基因水平和蛋白水平均成功表達,免疫小鼠血清中抗體的效價為1∶51 200,Western-blot結果顯示,免疫血清能與重組N蛋白發(fā)生特異性反應,說明實驗成功構建了重組真核表達質粒pPM-C-His-N,且在Vero細胞內(nèi)檢測到了綠色熒光,為PEDV診斷方法的建立及致病機制的研究奠定基礎。
[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)大學動物科技學院;
【基金】：安徽省自然科學基金(1708085MC83) 現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術體系建設專項資金資助項目(CARS-36-生豬) 安徽省生豬產(chǎn)業(yè)技術體系資金資助項目
【分類號】：S852.651
[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)大學動物科技學院;
【基金】：安徽省自然科學基金(1708085MC83) 現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術體系建設專項資金資助項目(CARS-36-生豬) 安徽省生豬產(chǎn)業(yè)技術體系資金資助項目
【分類號】：S852.651
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