嵌合O型口蹄疫病毒多表位基因猪细小病毒VLPs载体疫苗的构建

Construction of recombinant porcine parvovirus-like particles formed by hybrid VP2 protein carrying multi-epitope VP1genes of foot-and-mouth disease virus type O

综合运用生物信息学和分子生物学技术,模拟PPV VP2蛋白表面不同Loop区插入O型FMDV VP1蛋白上多表位基因(VP1:21～40、141～160和200～213残基)空间构象,并在VP2 N端引入通用型辅助性T淋巴细胞表位(PADRE),人工合成嵌合基因并克隆到杆状病毒转移载体pFastBac HTA中,转化E.coli DH10Bac感受态细胞,经三抗筛选,获得重组杆状病毒表达质粒rBac-FMDV VP1∶PPV VP2,用脂质体法转染Sf9细胞。对rBac-FMDVVP1∶PPV VP2感染的Sf9细胞,用间接免疫荧光试验(IFA)检测,具有特异性荧光;用Western-blotting分析,在64 000处出现一条特异蛋白条带;电镜观察,重组VP2蛋白能够自主组装成病毒样颗粒。红细胞凝集试验证实,表达的嵌合蛋白PPV∶VP2-FMDV∶VP1具有与全病毒类似的血凝活性。

This experiment aimed to construct insertion mutants in the surface of porcine parvovirus （PPV） capsids, and to investigate the possibility of inserting exogenous antigen gene into the loops using structural and antigenic data. Muhi-epitope VP1 of foot-and-mouth virus （FMDV） type O （three amino acid residues 21-40, 141-160 and 200-213 ）linked in tandem with short spacer sequence GGGS （Gly- Gly-Gly-Ser） was used as a model. Pan DR T helper epitopes （PADRE） was inserted into the VP2 N terminus of PPV. The hybrid nucleotides fragment was synthesized and cloned into contribution vector pFastBac HTA to get recombinant plasmid pFastBac-FMDV VP1 ： PPV VP2, which was then transformed into competent Escherichia coli DH10Bac cells growing on LB plate containing three kinds of antibiotics. After selection, the recombinant expression bacmid rBac-FMDV VP1 ： PPV VP2 was obtained and then was transfected into insect cell Sf9 with lipofectamine reagent. The infected Sf9 cell generated specific fluorescent light by the indirect immunofluorescence assay （IFA）. A protein band of at approximate molecular weight of 64 000 was detected by Western blotting technique, and the self-assembly virus-like particles （VLP） was observed under electron microscopy. Moreover, the expressed protein PPV ： VP2-FMDV ： VP1 was similar to PPV in haemagglutinating activity.