本文選題：腸道病毒71型 + P1基因�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2011年碩士論文

【摘要】：腸道病毒71型(Enterovirus 71, EV71)是手足口病(Hand, Foot, and Mouth Disease, HFMD)最重要的病原體。對(duì)于手足口病目前尚無(wú)有效治療的藥物,疫苗將成為預(yù)防EV71感染最有效的手段之一。用疫苗預(yù)防傳染性疾病包括傳統(tǒng)技術(shù)制備的減毒活疫苗和滅活疫苗,以及現(xiàn)代生物技術(shù)研制的新型疫苗。類(lèi)病毒顆粒(Virus Like Particle, VLP)疫苗是新型疫苗中基因工程亞單位疫苗免疫原性最理想的一種形式。類(lèi)病毒顆粒是由病毒外殼蛋白質(zhì)所組成的不含遺傳物質(zhì)的殼狀結(jié)構(gòu)。由于其內(nèi)部沒(méi)有核酸,自身不具備復(fù)制性,所以作為疫苗安全性高,同時(shí)類(lèi)病毒顆粒具有相對(duì)較完整的病毒結(jié)構(gòu),故其免疫原性強(qiáng),這些優(yōu)點(diǎn)使類(lèi)病毒顆粒疫苗成為基因工程疫苗研究的熱點(diǎn)。 重組桿狀病毒是一個(gè)很有前景的外源基因表達(dá)系統(tǒng),能高效表達(dá)抗原性、免疫原性和生物活性較好的蛋白質(zhì),并能形成VLP。研制VLP除了有效的表達(dá)系統(tǒng)以外,還需要選擇合適的抗原基因。EV71主要的抗原基因P1在未加工時(shí)免疫原性有限,當(dāng)被蛋白酶3CD切割成病毒衣殼蛋白暴露在病毒表面后,免疫原性得到提高。 基于以上原理及設(shè)想,本研究構(gòu)建了含有EV71的P1基因和3CD基因的嵌合型重組質(zhì)粒pFastBac Dual-P1-3CD,然后轉(zhuǎn)化DH10感受態(tài)細(xì)胞,經(jīng)同源重組得到重組桿狀病毒質(zhì)粒Bac-P1-3CD.將Bac-P1-3CD轉(zhuǎn)染Sf9昆蟲(chóng)細(xì)胞以獲得重組桿狀病毒,收獲感染重組桿狀病毒的Sf9細(xì)胞,用SDS-PAGE, Western blot和電鏡對(duì)基因表達(dá)產(chǎn)物進(jìn)行檢測(cè)和分析,評(píng)價(jià)了P1和3CD基因在昆蟲(chóng)細(xì)胞中表達(dá),以及3CD蛋白酶對(duì)P1蛋白的切割作用,以及切割后病毒蛋白成分的抗原性。 實(shí)驗(yàn)結(jié)果表明,經(jīng)同源重組獲得了在Sf9細(xì)胞中包裝的重組桿狀病毒；表達(dá)產(chǎn)物用SDS-PAGE檢測(cè)到39kDa、32kDa、26kDa三條特異蛋白條帶,與EV71 VP1、VPO、VP3蛋白大小相近,提示重組桿狀病毒表達(dá)了P1蛋白和3CD蛋白酶,并且3CD蛋白酶切割了P1蛋白；Western blot檢測(cè)到能與抗EV71血清反應(yīng)的特異性條帶,與EV71 VP1大小相近,證明表達(dá)產(chǎn)物為EV71特異性抗原；電鏡下觀察到形態(tài)學(xué)典型的EV71類(lèi)病毒顆粒。這項(xiàng)研究證實(shí)了EV71 P1和3CD基因可以以嵌合形式通過(guò)重組桿狀病毒在昆蟲(chóng)細(xì)胞中共表達(dá),3CD蛋白酶可以切割P1蛋白得到VP1抗原成分,并能形成類(lèi)病毒顆粒。這些研究結(jié)果不僅對(duì)研制EV71類(lèi)病毒顆粒疫苗具有重要意義,也對(duì)研究小RNA病毒結(jié)構(gòu)蛋白與非結(jié)構(gòu)蛋白的相互作用提供了理論依據(jù)與技術(shù)手段。
[Abstract]:Enterovirus 71 (EV71) is the most important pathogen of hand, foot and mouth disease (HFMD). The vaccine will be one of the most effective methods to prevent EV 71 infection for hand, foot and mouth disease. Vaccines are used to prevent infectious diseases, including live attenuated vaccines and inactivated vaccines prepared by traditional technology, and new vaccines developed by modern biotechnology. Virus like Particle (VLP) vaccine is the most ideal form of immunogenicity of genetic engineering subunit vaccine. Viroid particles are shell-like structures consisting of viral shell proteins that do not contain genetic material. Because it has no nucleic acid inside, it has no replicability, so it is safe as a vaccine and has relatively complete virus structure, so it has strong immunogenicity. The recombinant baculovirus is a promising foreign gene expression system, which can efficiently express antigenicity, immunogenicity and bioactivity of proteins. And can form VLP. In addition to the effective expression system, the development of VLP requires the selection of suitable antigen gene. EV71 main antigen gene P1 has limited immunogenicity when it is not processed, and when it is cut into virus capsid protein by protease 3CD, it is exposed to virus surface. The immunogenicity was improved. Based on the above principles and assumptions, the chimeric recombinant plasmid pFastBac Dual-P1-3CD1 containing EV71 and 3CD gene was constructed, then transformed into DH10 receptive cells, and the recombinant baculovirus plasmid Bac-P1-3CDwas obtained by homologous recombination. Bac-P1-3CD was transfected into Sf9 insect cells to obtain recombinant baculovirus. Sf9 cells infected with recombinant baculovirus were harvested. Expression of P1 and 3CD genes in insect cells was evaluated by SDS-PAGE, Western blot and electron microscope. The cleavage of P1 protein by 3CD protease and the antigenicity of protein components of the virus after cleavage. The results showed that recombinant baculovirus packaged in Sf9 cells was obtained by homologous recombination. Three specific protein bands were detected by SDS-PAGE, which were similar to the size of EV71 VP1VPOVP3 protein, suggesting that the recombinant baculovirus expressed P1 protein and 3CD protease. And 3CD protease cleavage P1 protein blot to detect the specific band which can react with EV71 serum, which is close to the size of EV71 VP1, which proves that the expressed product is EV71 specific antigen, and the typical EV71 virus particles were observed under electron microscope. This study confirmed that EV71P1 and 3CD genes can be expressed in chimeric form by recombinant baculovirus in insect cells to express p3CD protease can cut P1 protein to obtain VP1 antigen components, and can form virus-like particles. These results are not only of great significance to the development of EV71 virus vaccine, but also provide theoretical basis and technical means for the study of the interaction between structural protein and non-structural protein of small RNA virus.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R373

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