【摘要】：目的:構建腸道病毒71型(Enterovirus71,EV71)的線性中和抗原表位與諾如病毒P結構域融合基因的重組質粒,在大腸桿菌中表達諾如病毒P結構域與EV71中和抗原表位的嵌合蛋白。方法:根據(jù)已報道的3個EV71線性中和抗原表位的氨基酸序列,按大腸桿菌密碼子表達使用的偏好性優(yōu)化和設計各線性中和抗原表位的核苷酸序列,將這些表位以單個或不同的組合克隆至含諾如病毒P結構域和GST標簽的質粒中,經(jīng)測序確認后,分別轉化到E.coli BL21(DE3)感受態(tài)細胞中,通過IPTG誘導融合蛋白表達。用GST融合蛋白純化磁珠對融合蛋白進行純化,最后通過免疫印跡法確認融合蛋白的表達及嵌合蛋白的抗原性。結果:測序結果表明,成功地構建了含EV71病毒3個單表位和4個串聯(lián)中和抗原表位的諾如病毒P結構域重組質粒,而且這7個含線性中和抗原表位的嵌合蛋白在大腸桿菌中都以可溶形式得到了表達。免疫印跡分析表達蛋白的抗原性結果表明,表達的嵌合蛋白都能與抗諾如病毒P結構域抗血清反應。除了含單表位的SP55和SP28嵌合蛋白外,其它的嵌合蛋白均能與抗EV71病毒的抗血清反應。結論:成功地在大腸桿菌中表達了諾如病毒P結構域和EV71病毒中和抗原表位的嵌合蛋白,且具有抗原性,這為諾如病毒和EV71病毒的二價疫苗及檢測方法的研發(fā)奠定了基礎。
[Abstract]:Aim: to construct the recombinant plasmid of the fusion gene of the linear neutralizing antigen epitope of enterovirus 71 (Enterovirus71,EV71) and the P domain of norovirus, and to express the chimeric protein of the P domain of norovirus and EV71 neutralizing antigen epitope in Escherichia coli. Methods: according to the reported amino acid sequences of three EV71 linear neutralizing antigen epitopes, the nucleotide sequences of each linear neutralizing antigen epitope were optimized and designed according to the preference of E. coli codon expression. These epitopes were cloned into plasmids containing norovirus P domain and GST tag in a single or different combination. After sequencing, these epitopes were transformed into E.coli BL21 (DE3) competent cells, and the fusion protein expression was induced by IPTG. The fusion protein was purified by GST fusion protein and the expression of fusion protein and the antigenicity of chimeric protein were confirmed by Western blotting. Results: sequencing results showed that the recombinant plasmids containing 3 single epitopes and 4 tandem neutralizing antigen epitopes of EV71 virus were successfully constructed. Moreover, the seven chimeric proteins containing linear neutralizing antigen epitopes were all expressed in soluble form in Escherichia coli. The results of immunoblotting analysis showed that the expressed chimeric proteins could react with anti-norovirus P domain antiserum. Except SP55 and SP28 chimeric proteins containing monoepitopes, the other chimeric proteins can react with antiserum against EV71 virus. Conclusion: the chimeric proteins of P domain and neutralizing epitope of EV71 virus were successfully expressed in Escherichia coli, which laid a foundation for the development of bivalent vaccine and detection method for Norovirus and EV71 virus.
【作者單位】： 中國醫(yī)學科學院&北京協(xié)和醫(yī)學院醫(yī)學生物學研究所感染和免疫實驗室;
【基金】：國家自然科學基金(81571549) 云南省重點新產(chǎn)品開發(fā)專項項目(2016BC004) 中國醫(yī)學科學院醫(yī)學與健康科技創(chuàng)新工程協(xié)同創(chuàng)新團隊項目(2016-12M-3-026)資助項目
【分類號】：R373

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