【摘要】：試驗(yàn)旨在通過真核表達(dá)系統(tǒng)表達(dá)豬圓環(huán)病毒2型(porcine circovirus type 2,PCV2)CAP蛋白。以PCV2TZ0601株為模板,將PCV2CAP全基因及CAP去除信號肽的基因編碼序列克隆至pOET3載體上,酶切與測序鑒定正確后,將重組質(zhì)粒pOET3-CAP及pOET3-CAP-X轉(zhuǎn)染sf9昆蟲細(xì)胞。采用flashBAC桿狀病毒表達(dá)系統(tǒng)表達(dá)PCV2CAP及去除信號肽的CAP蛋白,通過間接免疫熒光法、SDS-PAGE和Western blotting鑒定目的蛋白的表達(dá)。結(jié)果表明,真核表達(dá)質(zhì)粒pOET3-CAP及pOET3-CAP-X構(gòu)建成功,目的基因在sf9昆蟲細(xì)胞中高效表達(dá),得到的蛋白經(jīng)SDS-PAGE和Western blotting鑒定,在25~35ku處有蛋白條帶,表達(dá)的蛋白質(zhì)可被PCV2陽性血清識別。試驗(yàn)結(jié)果為進(jìn)一步制備PCV2亞單位疫苗及診斷抗原試劑盒的研發(fā)奠定了基礎(chǔ)。
[Abstract]:The aim of this study was to express porcine circovirus type 2 (PCV2) CAP protein by eukaryotic expression system. Using PCV2TZ0601 strain as template, the whole PCV2CAP gene and the gene coding sequence of CAP removing signal peptide were cloned into pOET3 vector. The recombinant plasmids pOET3-CAP and pOET3-CAP-X were transfected into sf9 insect cells after confirmed by restriction enzyme digestion and sequencing. The flashBAC baculovirus expression system was used to express PCV2CAP and CAP protein which removed the signal peptide. The expression of the target protein was identified by indirect immunofluorescence staining and SDS-PAGE and Western blotting. The results showed that the eukaryotic expression plasmids pOET3-CAP and pOET3-CAP-X were successfully constructed. The target gene was highly expressed in sf9 insect cells. The obtained protein was identified by SDS-PAGE and Western blotting, and the expressed protein could be recognized by PCV2 positive serum. The results laid a foundation for the further preparation of PCV2 subunit vaccine and diagnostic antigen kit.
【作者單位】： 天津瑞普生物技術(shù)股份有限公司瑞普生物研究院;中國農(nóng)業(yè)大學(xué)動(dòng)物醫(yī)學(xué)院;
【基金】：瑞普生物研究院動(dòng)物疫病流行病學(xué)調(diào)查專項(xiàng)基金(RPBVI20130010)
【分類號】：S852.651
，

本文編號：2159576