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目的 构建弓形虫单价基因疫苗 pcDNA3 1 p3 0及复合基因疫苗 pcDNA3 1 p3 0 ROP2 ,并比较两种疫苗对小鼠的免疫保护性。 方法 用聚合酶链反应 (PCR)从弓形虫RH株基因组DNA中分别扩增编码弓形虫主要表面抗原 p3 0和弓形虫棒状体蛋白 2 (ROP2 )的基因片段 ,经T A克隆 ,将p3 0单价基因及 p3 0 ROP2复合基因片段分别插入真核细胞表达载体pcDNA3 1,构建重组真核表达质粒 pcDNA3 1 p3 0及pcDNA3 1 p3 0 ROP2。分别免疫BALB/c小鼠 ,设磷酸缓冲盐溶液 (PBS)组、pcDNA3 1空质粒组为对照 ;酶联免疫吸附测定 (ELISA)检测血清特异性IgG抗体 ;弓形虫速殖子腹腔攻击感染观察小鼠生存时间。 结果 获得 pcDNA3 1 p3 0、pcDNA3 1 p3 0 ROP2重组表达质粒 ,用 pcDNA3 1 p3 0 ROP2免疫的小鼠 ,其IgG抗体吸光度 (A490 =2 0 5 1± 0 3 3 7)高于用 pcDNA3 1 p3 0的吸光度 (A490 =1 892± 0 3 69) (P <0 0 5 )。攻击感染弓形虫后小鼠生存时间 ,用 pcDNA3 1 p3 0 ROP2免疫的小鼠 ,较用 pcDNA3 1 p3 0的明显延长 (P <0 0 1)。 结论 弓形虫不同生活阶段的抗原复合基因疫苗较单价基因疫苗具有更好的免疫保护性。
Objective To construct the Toxoplasma gondii monovalent gene vaccine pcDNA3 1 p3 0 and the composite gene vaccine pcDNA3 1 p3 0 ROP2 and compare the immunoprotective effects of the two vaccines on mice. Methods Gene fragments of Toxoplasma major surface antigen p3 0 and toxoplasma gondii protein 2 (ROP2) were amplified by polymerase chain reaction (PCR) from genomic DNA of Toxoplasma gondii RH strains respectively. After TA cloning, p30 monovalent Gene and p3 0 ROP2 gene fragment were inserted into eukaryotic expression vector pcDNA3 1 to construct recombinant eukaryotic expression plasmid pcDNA3 1 p3 0 and pcDNA3 1 p3 0 ROP2. BALB / c mice were immunized respectively with phosphate buffer saline (PBS) group and pcDNA3 1 empty plasmid group as control; serum specific IgG antibody was detected by enzyme-linked immunosorbent assay (ELISA); Toxoplasma gondii Mouse survival time. Results The pcDNA3 1 p3 0 and pcDNA3 1 p3 0 ROP2 recombinant plasmids were obtained. The IgG antibody absorbance (A490 = 2051 ± 0 3 3 7) in mice immunized with pcDNA3 1 p3 0 ROP2 was higher than that in pcDNA3 1 p3 0 absorbance (A490 = 1892 ± 0 3 69) (P <0 05). The survival time of mice after challenge with Toxoplasma gondii was significantly longer than that of pcDNA3 1 p3 0 mice immunized with pcDNA3 1 p3 0 ROP2 (P <0.01). Conclusion Toxoplasma gondii antigen composite gene vaccine at different stages of life than monovalent gene vaccine has better immune protection.