弓形虫主要表面抗原p30单价及复合基因疫苗的构建

目的　构建弓形虫单价基因疫苗 pcDNA3.1-p30及复合基因疫苗 pcDNA3.1-p30-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 p30和弓形虫棒状体蛋白 2 (ROP2 )的基因片段 ,经T-A克隆 ,将p30单价基因及 p30-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-p30及pcDNA3.1-p30-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-p30、pcDNA3.1-p30-ROP2重组表达质粒 ,用 pcDNA3.1-p30-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-p30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-p30-ROP2免疫的小鼠 ,较用 pcDNA3.1-p30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。

Construction of Monovalent and Compound Nucleic Acid Vaccines Against Toxoplasma gondii with Gene Encoding p30

Objective To construct a monovalent gene vaccine pcDNA3 ^1-p30 and a compound gene vaccine pcDNA3 ^1-p30-ROP2 and assess the protective effect of the two vaccines against Toxoplasma gondii. Methods The sequences encoding p30 and ROP2 were amplified from the genomic DNA of T.gondii RH strain by polymerase chain reaction (PCR) and inserted into eukaryotic vector pcDNA3 ^1 to construct pcDNA3 ^1-p30 and pcDNA3 ^1-p30-ROP2. Mice were injected with the recombinant plasmid to observe the immunoprotectivity of the nucleic acid vaccine by using ELISA for detection of total IgG and observing the survival time after tachyzoites challenge. Results The recombinant plasmids pcDNA3 ^1-p30 and pcDNA3 ^1-p30-ROP2 were constructed. Mice in pcDNA3 ^1-p30-ROP2 group showed higher IgG(P<0 ^05) and survived longer than those in pcDNA3 ^1-p30 group(P<0 ^01)after challenged with T.gondii. Conclusion Compound vaccine of genes from different stages of T.gondii elicits stronger immunoprotectivity in mice than a single gene vaccine.