通过耐酸性改造在昆虫细胞中组装FMDV空衣壳结构  

Synthesis of Foot-and-Mouth Disease Virus Empty Capsids in Insect Cells Through Acid-Resistant Modification

作　　者：曹轶梅[1] 卢曾军[1] 孙甲川[1] 孙普[1] 郭建宏[1] 刘在新[1] 

机构地区：[1]中国农业科学院兰州兽医研究所/家畜疫病病原生物学国家重点实验室/农业部畜禽病毒学重点实验室,兰州730046

出　　处：《中国农业科学》2009年第3期1069-1077,共9页Scientia Agricultura Sinica

基　　金：国家重大基础研究发展规划项目(2005CB523201)

摘　　要：【目的】口蹄疫病毒(FMDV)对酸很敏感,当pH值低于7时,二十面体对称的衣壳结构就会裂解成12S的五聚体。而昆虫细胞培养基的正常pH值在6.3左右,很难应用杆状病毒表达系统在昆虫细胞中组装天然的FMDV空衣壳结构。本研究旨在通过耐酸性改造,应用杆状病毒表达载体在昆虫细胞中组装产生AsiaⅠ型FMDV空衣壳结构。【方法】应用定点突变技术改变VP3上的H140和H143为亮氨酸,以提高空衣壳对酸的耐受性。将改造和未改造的P12A基因和3C基因插入带双启动子的杆状病毒转移载体pFastBacTM Dual中,通过在大肠杆菌内转座重组,获得重组杆粒,转染Sf9细胞,获得两株表达FMDV全衣壳蛋白的重组杆状病毒BacmP12A3C和BacP12A3C。重组杆状病毒经增殖后感染High FiveTM细胞,进行目的蛋白的表达。【结果】通过Western blotting检测表明目的基因均获得表达,且衣壳蛋白被3C蛋白酶成功地加工裂解。双抗体夹心ELISA和免疫荧光检测结果表明表达蛋白主要集中在细胞膜上,且具有很好的抗原性。通过电镜观察到P12A基因改造的重组杆状病毒在昆虫细胞内产生了直径为25～30nm的空衣壳结构,而P12A基因未改造的重组杆状病毒观察到很多直径小得多的结构。【结论】本研究首次用电子显微镜在昆虫细胞中观察到FMDV完整的空衣壳结构,为基因工程亚单位疫苗和新型诊断试剂的研发奠定了基础。[ Objective ] Foot-and-mouth disease virus （FMDV）, a non-enveloped picornavirus, is sensitive to acidic conditions. At pH below 7 the icosahedral virus capsid dissociates into 12 pentamers. It is difficult to synthesize FMDV capsids in insect cells for the influence of low pH of insect cell medium. This study aimed at assembling empty capsid-like particles of Asia I FMDV in insect cells by site-specific mutation of related amino acid residues to improve pH-stability of empty capsids. [ Method ] Two amino acid residues were site-specifically mutated to get modified P12A gene named mP12A （H140L and H143L in VP3）. Both P12A and mP12A were then inserted into a transfer vector pFastBacTM Dual under the control of PH promoter, respectively. The 3C gene was placed under the control of P10 promoter. Two recombinant baculoviruses were generated by transfecting recombinant bacmids into St9 cells. High FiveTM cells were infected with two recombinant baculoviruses after multiplication in Sf9 cells. [Result] The target proteins were successfully expressed in insect cells by Western blotting, and that expressed capsid proteins could be processed by expressed viral 3C proteinase. The capsid proteins were mainly expressed near the inner membrane of insect ceils as revealed by immunofluorescent assay, and that small amounts were released into culture medium as showed by sandwich ELISA. Furthermore, empty capsid-like particles were observed in lystate of High FiveTM cells infected with recombinant baculoviruses with mutated P12A under electron microscope. Whereas, many small-sized particles were observed in lystate of High FiveTM cells infected with recombinant baculovirus without mutation in P12A. The observed empty capsid-like particles were about 25-30 nm in diameter. [ Conclusion ] This is the first report on the observations of empty capsid-like particles of FMDV in insect cells by microscopy. The potential utility of these recombinant, non-infectious, FMDV empty capsids for diagnostic and vaccine purposes is apparen

关 键 词：FMDV 耐酸性改造 昆虫细胞 组装 空衣壳结构 

分 类 号：S852.65[农业科学—基础兽医学]