肠道病毒71型类病毒颗粒在昆虫细胞Sf9中的表达

发布时间：2018-06-08 17:40

本文选题：肠道病毒71型 + P1基因　；参考：《北京协和医学院》2011年硕士论文

【摘要】：肠道病毒71型(Enterovirus 71, EV71)是手足口病(Hand, Foot, and Mouth Disease, HFMD)最重要的病原体。对于手足口病目前尚无有效治疗的药物,疫苗将成为预防EV71感染最有效的手段之一。用疫苗预防传染性疾病包括传统技术制备的减毒活疫苗和灭活疫苗,以及现代生物技术研制的新型疫苗。类病毒颗粒(Virus Like Particle, VLP)疫苗是新型疫苗中基因工程亚单位疫苗免疫原性最理想的一种形式。类病毒颗粒是由病毒外壳蛋白质所组成的不含遗传物质的壳状结构。由于其内部没有核酸,自身不具备复制性,所以作为疫苗安全性高,同时类病毒颗粒具有相对较完整的病毒结构,故其免疫原性强,这些优点使类病毒颗粒疫苗成为基因工程疫苗研究的热点。重组杆状病毒是一个很有前景的外源基因表达系统,能高效表达抗原性、免疫原性和生物活性较好的蛋白质,并能形成VLP。研制VLP除了有效的表达系统以外,还需要选择合适的抗原基因。EV71主要的抗原基因P1在未加工时免疫原性有限,当被蛋白酶3CD切割成病毒衣壳蛋白暴露在病毒表面后,免疫原性得到提高。基于以上原理及设想,本研究构建了含有EV71的P1基因和3CD基因的嵌合型重组质粒pFastBac Dual-P1-3CD,然后转化DH10感受态细胞,经同源重组得到重组杆状病毒质粒Bac-P1-3CD.将Bac-P1-3CD转染Sf9昆虫细胞以获得重组杆状病毒,收获感染重组杆状病毒的Sf9细胞,用SDS-PAGE, Western blot和电镜对基因表达产物进行检测和分析,评价了P1和3CD基因在昆虫细胞中表达,以及3CD蛋白酶对P1蛋白的切割作用,以及切割后病毒蛋白成分的抗原性。实验结果表明,经同源重组获得了在Sf9细胞中包装的重组杆状病毒；表达产物用SDS-PAGE检测到39kDa、32kDa、26kDa三条特异蛋白条带,与EV71 VP1、VPO、VP3蛋白大小相近,提示重组杆状病毒表达了P1蛋白和3CD蛋白酶,并且3CD蛋白酶切割了P1蛋白；Western blot检测到能与抗EV71血清反应的特异性条带,与EV71 VP1大小相近,证明表达产物为EV71特异性抗原；电镜下观察到形态学典型的EV71类病毒颗粒。这项研究证实了EV71 P1和3CD基因可以以嵌合形式通过重组杆状病毒在昆虫细胞中共表达,3CD蛋白酶可以切割P1蛋白得到VP1抗原成分,并能形成类病毒颗粒。这些研究结果不仅对研制EV71类病毒颗粒疫苗具有重要意义,也对研究小RNA病毒结构蛋白与非结构蛋白的相互作用提供了理论依据与技术手段。
[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.
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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