关键词: 刚地弓形虫, 基因重组, 表面抗原, 微线体蛋白, 棒状体蛋白, 鸡尾酒疫苗, 免疫原性

Abstract:

Objective  To construct a cocktail DNA vaccine that expresses multiple genes of Toxoplasma gondii and investigate its immunogenicity in mice. Methods Genes for surface antigens（SAG）, microneme（MIC）, and rhoptry protein（ROP） were amplified from genomic DNA of T. gondii and then cloned separately into eukaryotic fluorescent protein expression vectors pShuttle-CMV-MCS-EF1α-AmCyan, pLVX-IRES-Zsgreen and pLVX-IRES-rfp, to construct expression plasmids pShuttle-SAG1, pLVX-Zsgreen-MIC3 and pLVX-rfp-ROP2. 293F cells were transfected with a combination of the three plasmids using the polyethylenimine（PEI） method. Forty-eight hours later, the expression of the three genes was observed under a fluorescence microscope. In addition, 30 C57BL/6 mice were randomized to receive intramuscular injection of saline（50 μl, group A）, pShuttle+pLVX?鄄Zsgreen+pLVX-rfp empty plasmids（2 μg/μL, 17 μL of each, group B） and  pShuttle-SAG1+pLVX-Zsgreen-MIC3+ pLVX-rfp-ROP2 recombinant plasmid（2 μg/μL, 17 μL of each, group C）. After 28 days, anti-T. gondii antibody in mouse serum was detected by ELISA, to evaluate the immunogenicity of the vaccine. Results  The SAG1, MIC3 and ROP2 genes were amplified from the genomic DNA, with product sizes of 1, 1.1 and 1.7 kb. The eukaryotic expression plasmids pShuttle-SAG1, pLVX-Zsgreen-MIC3 and pLVX-rfp-ROP2 were constructed, and the corresponding fluorescences（blue, green and red） were observed after transfection. On day 28 after mouse vaccination, ELISA showed that the mean A450 values for serum IgG in groups A, B and C were（0.620±0.029）,（0.741±0.040） and（1.561±0.131）, respectively, with the group C value being significantly higher than the others（P<0.01）. Conclusion The cocktail DNA vaccine comprising T. gondii SAGl, MIC3 and ROP2 shows promising immunogenicity in mice, and the fluorescent protein expression vectors are reliable tools for expression of the target genes.

Key words: Toxoplasma gondii, Gene recombinant, Surface antigen, Microneme protein, Rhoptry protein, Cocktail DNA vaccine, Immunogenicity