【摘要】 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。更多还原

【Abstract】 Objective To construct the recombinant encoding rhoptry protein ROP2 and major surface protein 1(P30) of Toxoplasma gondii and to evaluate initially the immunological effect of ROP2-P30 by DNA vaccine.Methods The ROP2-P30 fused gene of T.gondii was amplified by using PCR from the constructed pET28b/ROP2-P30,and then the recombinant eukaryotic expression plasmid pcDNA3.1/ROP2-P30 was constructed.After being identified by PCR and restriction enzyme cleavage,pcDNA3.1/ROP2-P30 was transfected into COS-7 cells and Western blot analysis was employed to examine the recombinant ROP2-P30 expression in the COS-7 cells.Subsequently,groups of female BALB/c mice were immunized intramuscularly in their hinds with 100 μg of pcDNA3.1/ROP2-P30 mixing with 10 U of hyaluronidase.Taking the soluble T.gondii extract for coating antigen,ELISA was used for determination of antibody IgG in serum from immunized mice.Two weeks after the last vaccination,the experimental mice were challenged with 100 tachyzoites of the RH strain of T.gondii,and then the survival of these animals was recorded.Results Analysis of PCR and restriction enzyme cleavage confirmed that the recombinant eukaryotic expression plasmid pcDNA3.1/ROP2-P30 was precisely constructed.Western blot indicated that the products which were transiently expressed in COS-7 cells could be recognized by serum from immunized rabbits with rROP2-P30.A specific antibody IgG in serum from mice immunized with pcDNA3.1/ROP2-P30,detected by soluble T.gondii extract was significantly generated while no corresponding antibody production was observed in the control mice.The survival time of mice immunized with pcDNA3.1/ROP2-P30 was longer than that of control mice against tachyzoites infection.Conclusion pcDNA3.1/ROP2-P30 was constructed successfully.The specific humoral immune response was induced directly by DNA vaccine of pcDNA3.1/ROP2-P30,and exhibited potential protection against T.gondii challenge infection.So the recombinant fusion ROP2-P30 expressed by pcDNA3.1/ROP2-P30 exhibited immunogenicity,and could have the potential to be developed as a candidate antigen for vaccine.更多还原