【摘要】：目的 構(gòu)建漢坦病毒(hantavirus,HV)莒南擴(kuò)增株JUN05株核衣殼蛋白(nucleocapsid protein,NP)的熒光蛋白表達(dá)載體;將HV NP基因與EGFP基因在BHK21細(xì)胞中融合表達(dá),熒光顯微鏡下觀察融合蛋白的表達(dá)強(qiáng)度,以及融合蛋白在細(xì)胞中定位和分布,免疫組化觀察融合蛋白的免疫反應(yīng)性,從而為新型HV診斷試劑研制與開(kāi)發(fā),以及NP結(jié)構(gòu)與功能的研究奠定基礎(chǔ)。 方法 根據(jù)HV莒南擴(kuò)增株JUN05株NP基因的序列和表達(dá)載體pEGFP-N1的多克隆位點(diǎn)序列,設(shè)計(jì)引物,通過(guò)PCR的方法擴(kuò)增NP全基因。PCR產(chǎn)物回收后以核酸內(nèi)切酶BglⅡ、PstⅠ雙酶切,克隆至同樣雙酶切的pEGFP-N1載體上,并保持NP基因與EGFP基因的讀碼框一致,CaCl_2法轉(zhuǎn)化并篩選陽(yáng)性克隆。通過(guò)PCR方法和雙酶切方法鑒定,并通過(guò)DNA測(cè)序技術(shù)最終鑒定表達(dá)載體的構(gòu)建情況。將重組質(zhì)粒以脂質(zhì)體法轉(zhuǎn)染BHK21細(xì)胞,轉(zhuǎn)染24h后固定細(xì)胞,熒光顯微鏡檢和免疫組化技術(shù)觀察融合蛋白的表達(dá)并拍照記錄。 結(jié)果 1.構(gòu)建了含有HV NP基因的熒光蛋白表達(dá)載體,酶切鑒定、PCR鑒定、及測(cè)序結(jié)果表明載體構(gòu)建成功。 2.測(cè)序結(jié)果表明,NP基因成功連入EGFP基因上游,二者讀碼框一致無(wú)移位,與NP基因在克隆載體中的序列比對(duì),無(wú)PCR擴(kuò)增導(dǎo)致的堿基突變,并且NP基因末端的終止密碼子TAA被去除。并添加了kozak序列,為高效表達(dá)提供了基因水平的改造。 3.熒光顯微鏡下觀察,NP-EGFP融合蛋白在BHK21細(xì)胞中得到高效表達(dá),細(xì)胞漿內(nèi)充滿了致密的綠色熒光,熒光呈核周分布,達(dá)到了以綠色熒光蛋白作
[Abstract]:Objective to construct the fluorescent protein expression vector of nucleocapsid protein (nucleocapsid protein,NP) of Hantaan virus (hantavirus,HV) JUN05 strain. The fusion protein of HV NP gene and EGFP gene were expressed in BHK21 cells. The expression intensity of fusion protein, localization and distribution of fusion protein were observed under fluorescence microscope, and the immunoreactivity of fusion protein was observed by immunohistochemistry. It lays a foundation for the research and development of a new HV diagnostic reagent and the study of the structure and function of NP. Methods according to the sequence of NP gene of JUN05 strain amplified by HV and the polyclonal site sequence of expression vector pEGFP-N1, primers were designed and the whole NP gene was amplified by PCR method. The products were digested by Bgl 鈪，

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