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目的构建弓形虫棒状体蛋白2(ROP2)和膜表面蛋白1(P30)融合的重组真核表达质粒,观察融合抗原ROP2-P30以DNA免疫方式在体内的免疫学效应。方法以重组质粒pET28b/ROP2-P30为模板,利用分子克隆技术构建重组真核表达质粒pcDNA3.1/ROP2-P30,经PCR和酶切鉴定正确后,体外转染COS-7细胞,Westernblot检测ROP2-P30表达。重组质粒pcDNA3.1/ROP2-P30以每鼠100μg混合透明质酸酶10U的剂量肌肉注射免疫BALB/c雌性小鼠,以弓形虫虫体裂解抗原作包被抗原,ELISA法测定免疫小鼠血清IgG抗体,免疫结束2周后,以约100个弓形虫速殖子攻击感染小鼠,观察小鼠生存状况。结果PCR和酶切鉴定表明重组质粒pcDNA3.1/ROP2-P30构建正确;West-ernblot显示该重组质粒在COS-7细胞中瞬时表达的产物可被重组ROP2-P30免疫兔血清识别;ELISA检测重组质粒免疫小鼠血清特异性IgG抗体水平升高;重组质粒免疫小鼠感染弓形虫后的存活时间较对照组有所延长。结论重组真核表达质粒pcDNA3.1/ROP2-P30构建成功,用该重组质粒DNA直接免疫小鼠,能够诱导产生特异的体液免疫反应,具有一定的免疫保护性;该重组质粒表达的融合抗原分子ROP2-P30具有免疫原性,可作为疫苗候选抗原深入研究。
Objective To construct a recombinant eukaryotic expression plasmid fused with ROP2 and membrane surface protein 1 (P30), and to observe the immunological effect of the fusion antigen ROP2-P30 in vivo by DNA immunization. Methods Recombinant eukaryotic expression plasmid pcDNA3.1 / ROP2-P30 was constructed by using recombinant plasmid pET28b / ROP2-P30 as a template. The recombinant plasmid pcDNA3.1 / ROP2-P30 was identified by PCR and restriction enzyme digestion. -P30 expression. Recombinant plasmids pcDNA3.1 / ROP2-P30 were intramuscularly immunized BALB / c female mice with a dose of 10U per 100μg of mixed hyaluronidase to coat the antigen with Toxoplasma gondii lysed antigen, and the serum was detected by ELISA IgG antibody. Two weeks after the end of immunization, mice were challenged with about 100 Toxoplasma gondii tachyzoites to observe the survival of mice. Results PCR and restriction enzyme digestion showed that the recombinant plasmid pcDNA3.1 / ROP2-P30 was constructed correctly. West-ernblot showed that the transient expression product of the recombinant plasmid in COS-7 cells could be recognized by recombinant ROP2-P30 rabbit serum. Serum-specific IgG antibodies raised in plasmid-immunized mice; the survival time of mice immunized with recombinant plasmids was longer than that of the control group. Conclusion The recombinant eukaryotic expression plasmid pcDNA3.1 / ROP2-P30 was constructed successfully. The direct immunization of mice with the recombinant plasmid DNA can induce specific humoral immune response and have certain immunoprotective properties. The fusion antigen molecule expressed by the recombinant plasmid ROP2-P30 is immunogenic and can be further studied as a vaccine candidate antigen.