AsiaⅠ型FMDV全衣壳蛋白基因在昆虫细胞中的表达与免疫活性分析  

Expression of the Whole Capsid Protein Gene P12A of Asia Ⅰ Foot-and-mouth Disease Virus in Insect Cells and Analysis of Its Immune Activity

作　　者：曹轶梅[1] 孙甲川[1] 卢曾军[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：《畜牧兽医学报》2009年第1期83-88,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家重大基础研究发展规划“973项目”(2005CB523201)

摘　　要：将AsiaⅠ型FMDV全衣壳蛋白P12A基因插入到带有蜂毒溶血肽信号序列的杆状病毒转移载体pMel-Bac中,构建重组转移载体pMel-P12A。然后将含有目的基因的转移载体与线性化的杆状病毒DNA共转染Sf9细胞,获得重组杆状病毒。以感染复数(MOI)10的重组病毒感染Sf9细胞,72 h后收获细胞,经Western blotting、间接免疫荧光检测,结果表明P12A基因在昆虫细胞中获得表达,相对分子质量约82 ku,与预测蛋白大小一致,且能被FMDV阳性血清所识别。免疫荧光检测表明表达蛋白主要集中在细胞膜部分。夹心ELISA检测结果表明一部分表达蛋白分泌至培养基中,而有一部分蛋白仍在细胞中未能分泌。表达蛋白加入等量佐剂乳化后免疫豚鼠,间接ELISA检测结果表明豚鼠免疫后15 d即产生了特异性FMDV抗体。本研究为空衣壳的体外组装及基因工程亚单位疫苗的研究进行了有益的探索。Structure protein gene P12A of FMDV from serotype Asia Ⅰ was inserted into the baculovirus transfer vector pMelBac-B with a melittin secretion signal sequence to gain recombinant plasmid of pMel-P12A. The recombinant transfer plasmids and linearized baculovirus DNA were co-transfected into sf9 insect cell to produce recombinant baculoviruses. After amplification of recombinant baculoviruses on cell passages, the recombinant viruses were seeded on sf9 cell with 10 MOI （multiplicity of infection）, and cells were harvested 72 hours post infection. The expressed products of P12A gene in insect cells were detected by Western blotting, Immunofluorescence and sandwich ELISA assay. The results demonstrated that the P12A gene was successfully expressed in insect cells. The products were 82 kDa protein and could be recognized by anti-FMDV serum. Some expressed proteins were secreted into cell culture medium, and some were still in the cell lysate, as shown in sandwich ELISA. Furthermore, the expressed proteins were injected to guinea pigs intramuscularly and anti-FMDV antibodies were detected by indirect ELISA at 15 days post-infection. The result showed that the baculovirus-expressed P12A protein was able to induce anti-FMDV antibodies in the second week after vaccination. This study laid a good foundation for the research of the assembly of FMDV empty capsids in vitro and the development of subunit vaccine.

关 键 词：FMDV 衣壳蛋白基因P12A 杆状病毒 SF9细胞 表达 

分 类 号：S852.659.6[农业科学—基础兽医学]