發(fā)布時間：2018-08-06 15:38

【摘要】：目的表達(dá)并純化人甲型流感病毒H1N1亞型NS1蛋白。方法用RT-PCR法從人甲型流感病毒株A/PR/8/34(H1N1)中擴增NS1基因,克隆入原核表達(dá)載體pTXB1,構(gòu)建重組原核表達(dá)質(zhì)粒pTXB1/NS1,經(jīng)酶切鑒定后,轉(zhuǎn)化大腸桿菌BL21(DE3),IPTG誘導(dǎo)表達(dá),SDS-PAGE分析表達(dá)形式和表達(dá)量。經(jīng)幾丁質(zhì)柱親和層析純化表達(dá)蛋白,串聯(lián)飛行時間質(zhì)譜儀檢測其相對分子質(zhì)量。結(jié)果所構(gòu)建的重組表達(dá)質(zhì)粒pTXB1/NS1序列完整,插入的基因片段全長690bp。以1.0mmol/LIPTG37℃誘導(dǎo)4h,重組蛋白表達(dá)量最高,占菌體總蛋白的50%以上。破菌上清及沉淀中均有目的蛋白表達(dá)。純化的NS1蛋白純度達(dá)95%以上,相對分子質(zhì)量約為26000。結(jié)論已成功表達(dá)并純化了人甲型流感病毒H1N1亞型NS1蛋白,為其進(jìn)一步的研究奠定了基礎(chǔ)。
[Abstract]:Objective to express and purify human influenza A virus H1N1 subtype NS1 protein. Methods the NS1 gene was amplified by RT-PCR from human influenza A virus strain A/PR/8/34 (H1N1) and cloned into the prokaryotic expression vector pTXB1. The recombinant prokaryotic expression plasmid pTXB1 / NS1 was constructed. The recombinant plasmid pTXB1 / NS1 was identified by restriction endonuclease digestion, and then transformed into E. coli BL21 (DE3) BL21 (DE3) to induce expression by SDS-PAGE. The expressed protein was purified by chitosan column affinity chromatography and its molecular weight was determined by tandem time of flight mass spectrometer. Results the pTXB1/NS1 sequence of the recombinant expression plasmid was complete and the inserted gene fragment was 690 BP. When induced at 1.0mmol/LIPTG37 鈩，

本文編號：2168179