【摘要】：目的:構(gòu)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(jié)構(gòu)域融合基因的重組質(zhì)粒,在大腸桿菌中表達(dá)諾如病毒P結(jié)構(gòu)域與EV71中和抗原表位的嵌合蛋白。方法:根據(jù)已報(bào)道的3個(gè)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(dá)使用的偏好性優(yōu)化和設(shè)計(jì)各線性中和抗原表位的核苷酸序列,將這些表位以單個(gè)或不同的組合克隆至含諾如病毒P結(jié)構(gòu)域和GST標(biāo)簽的質(zhì)粒中,經(jīng)測序確認(rèn)后,分別轉(zhuǎn)化到E.coli BL21(DE3)感受態(tài)細(xì)胞中,通過IPTG誘導(dǎo)融合蛋白表達(dá)。用GST融合蛋白純化磁珠對融合蛋白進(jìn)行純化,最后通過免疫印跡法確認(rèn)融合蛋白的表達(dá)及嵌合蛋白的抗原性。結(jié)果:測序結(jié)果表明,成功地構(gòu)建了含EV71病毒3個(gè)單表位和4個(gè)串聯(lián)中和抗原表位的諾如病毒P結(jié)構(gòu)域重組質(zhì)粒,而且這7個(gè)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(dá)。免疫印跡分析表達(dá)蛋白的抗原性結(jié)果表明,表達(dá)的嵌合蛋白都能與抗諾如病毒P結(jié)構(gòu)域抗血清反應(yīng)。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(yīng)。結(jié)論:成功地在大腸桿菌中表達(dá)了諾如病毒P結(jié)構(gòu)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(jià)疫苗及檢測方法的研發(fā)奠定了基礎(chǔ)。
[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(xué)科學(xué)院&北京協(xié)和醫(yī)學(xué)院醫(yī)學(xué)生物學(xué)研究所感染和免疫實(shí)驗(yàn)室;
【基金】：國家自然科學(xué)基金(81571549) 云南省重點(diǎn)新產(chǎn)品開發(fā)專項(xiàng)項(xiàng)目(2016BC004) 中國醫(yī)學(xué)科學(xué)院醫(yī)學(xué)與健康科技創(chuàng)新工程協(xié)同創(chuàng)新團(tuán)隊(duì)項(xiàng)目(2016-12M-3-026)資助項(xiàng)目
【分類號】：R373

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