中文摘要：

目的构建能同时表达多个基因的刚地弓形虫DNA鸡尾酒疫苗并初步观察其免疫原性．方法从基因组DNA扩增刚地弓形虫表面抗原（surface antigen，SAG）、微线体蛋白（mictoneine．MIC）和棒状体蛋白（rhoptry protein，ROP）基因片段，克隆到真核荧光表达载体pShuttle．CMV—MCS—EFlct—AmCvan，pLVX．IRES．Zs．green及pLVX—IRES．却中，构建pShuttle—SAGl，pLVX．Zsgreen—MIC3和pLVX．却．ROP2表达质粒。通过聚乙烯亚胺法用混合质粒转染293F细胞48h，荧光显微镜下观察3个基因的表达情况。将30只C57BL／6雄性小鼠随机分为A、B、C组，分别肌内注射生理盐水（50μ1）、pShuttle＋pLVX—Zsgreen＋pLVX—rrp混合空质粒（2μg/μl，各17μl）、pShuttle—SAGl＋pLVX．Zsgreen—MIC3＋pLVX．rfp．ROP2混合重组质粒（2μg／μl，各17μl）。免疫28d后，ELISA检测血清抗刚地弓形虫IgG抗体水平，初步评价该疫苗的免疫原性。结果从刚地弓形虫基因组成功扩增出1、1．1及1．7kb的SAGl、MIC3和ROP2序列。成功构建了pShuttle—SAGl、pLVX—Zsgreen—MIC3和DLVX．rfp．ROP2真核荧光表达质粒，转染293F细胞后观察到相应的蓝、绿、红报告荧光。免疫小鼠后28d，ELISA测得A、B、C组IgG抗体的吸光度（AⅧ值）分别为（0．620±0．029）、（0．741±0．040）、（1．561±O．131），C组显著高于A、B组（P〈0．01）。结论刚地弓形虫SAGI、MIC3和ROP2基因混合重组质粒组成的DNA鸡尾酒疫苗能诱导小鼠产生良好的免疫原性．且多个荧光蛋白真核表达载体能够较好地指示目的基因的表达。

英文摘要：

Objective To construct a cocktail DNA vaccine that expresses multiple genes of Toxoplasma gortdii 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-EFlα-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 Ixl, group A）, pShuttle＋pLVX-Zsgreen＋pLVX-rfp empty plasmids（2 μg/μL, 17 μL of each, group B） and pShuttle-SAGl＋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 A45o 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 SAG1, MIC3 and ROP2 shows promising immunogenicity in mice, and the fluorescent protein expression vectors are reliable tools for expression of the target genes.