荧光蛋白报告载体观察刚地弓形虫SAGl、MIC3、ROP2鸡尾酒DNA疫苗的表达及其免疫原性的研究  

Expression of Cocktail DNA Vaccine Comprising Toxoplasma gondii SAGl, MIC3 and ROP2 Using Fluorescent Protein-Reporting Vectors and Evaluation of Its Immunogenicity

作　　者：王燕娟[1] 曹建平[1] 孙雅雯[1] 徐馀信[1] 沈玉娟[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所,卫生部寄生虫病原与媒介生物学重点实验室,世界卫生组织热带病合作中心,上海200025

出　　处：《中国寄生虫学与寄生虫病杂志》2015年第5期368-371,376,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：上海市卫生局青年科研项目(No.20124Y038);国家自然科学基金(No.81202301)~~

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(microneme,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-EF1α-Am Cyan,pLVX-IRES-Zsgreen及pLVX-IRES-rfp中,构建pShuttle-SAG1,pLVX-Zsgreen-MIC3和pLVX-rfp-ROP2表达质粒。通过聚乙烯亚胺法用混合质粒转染293F细胞48 h,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(50μl)、pShuttle+pLVX-Zsgreen+pLVX-rfp混合空质粒(2μg/μl,各17μl)、pShuttle-SAG1+pLVX-Zsgreen-MIC3+pLVX-rfp-ROP2混合重组质粒(2μg/μl,各17μl)。免疫28 d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1.7 kb的SAG1、MIC3和ROP2序列。成功构建了pShuttle-SAG1、pLVX-Zsgreen-MIC3和pLVX-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28 d,ELISA测得A、B、C组IgG抗体的吸光度(A450值)分别为(0.620±0.029)、(0.741±0.040)、(1.561±0.131),C组显著高于A、B组(P<0.01)。结论刚地弓形虫SAG1、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。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.

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 

分 类 号：R382.5[医药卫生—医学寄生虫学]