弓形虫GRA1基因核酸疫苗的实验研究  

Immunoprotection by a nucleic acid vaccine from the GRA1 gene of Toxoplasma gondii

作　　者：刘慧颖[1,2] 孙玲[1] 李淑红[1] 宫鹏涛[3] 李建华[3] 张西臣[3] 

机构地区：[1]吉林大学白求恩医学院病原生物学教研室,吉林长春130021 [2]长春市绿园区疾病预防控制中心,吉林长春130062 [3]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国病原生物学杂志》2012年第3期181-185,234,共6页Journal of Pathogen Biology

基　　金：国家科技支撑计划课题(No.2008BAD96B11-3);吉林省发改委资助项目(No.200603)

摘　　要：目的构建弓形虫致密颗粒蛋白GRA1基因的真核重组表达质粒,研究其在Hela细胞中的表达情况及其对动物弓形虫感染的免疫保护作用。方法以弓形虫总RNA为模板,利用设计合成的一对引物,通过RT-PCR方法体外扩增GRA1基因cDNA片段,构建pVAX1-GRA1重组真核表达质粒,并将其转染Hela细胞,验证其在真核细胞中的表达;用pVAX1-GRA1真核表达质粒注射免疫小鼠,通过检测血清特异IgG水平及血CD4+、CD8+细胞百分率并观察弓形虫感染小鼠的存活时间,评价其免疫保护力。结果 PCR扩增出573bp的GRA1开放阅读框,成功构建重组表达质粒pVAX1-GRA1。用间接免疫荧光方法在重组质粒转染后的Hela细胞中检测到特异蛋白,Western blot证实该蛋白具有反应原性,SDS-PAGE检测该蛋白分子质量单位为24ku。用构建的核酸疫苗免疫小鼠,随着免疫次数的增加血清特异性IgG抗体滴度逐渐增加,CD4+与CD8+T淋巴细胞百分率与对照组相比差异有统计学意义(P<0.01)。弓形虫RH速殖子攻击感染疫苗免疫组小鼠存活时间为(165±23.1)d,PBS对照组、pVAX1对照组分别为(144±16.3)d和(148±16.3)d,差异有统计学意义(P<0.05)。结论构建的真核表达质粒pVAX1-GRA1有一定的免疫保护性,为弓形虫基因疫苗的进一步研究奠定了基础。Objective To construct a recombinant eukaryotic expression plasmid containing the dense granule protein 1（GRA1） gene of Toxoplasma gondii and to express this gene in Hela cells and study its immunological protection.Methods T.gondii total RNA was used as a template to design a pair of primers to amplify cDNA fragments of GRA1.The recombinant eukaryotic expression plasmid pVAX1-GRA1 was constructed and transfected into Hela cells to test its expression in cells.Its protective effect was evaluated based on the serum levels of IgG and percentage of CD4 and CD8 cells.The survival time of vaccinated mice challenged with T.gondii was observed.Results PCR amplification produced an open reading frame of GRA1 of about 573 bp and the recombinant expression plasmid pVAX1-GRA1 was constructed.Specific protein in Hela cells transfected with pVAX1-GRA1 was detected.The protein was found to have a certain degree of immunogenicity.Results indicated that the DNA vaccine provided protection,increasing the antibody titer and significantly increasing（P0.01） the percentage of CD4^＋ and CD8^＋ lymphocytes compared to the control group.The results of SDS-PAGE revealed that the molecular weight of the recombinant protein was 24 ku.Mice immunized with the vaccine had an increased survival time（165±23.1）d compared to control mice given PBS.The pVAX1 control group died within（144±16.3）d and（148±16.3）d of the challenge,and the difference in survival time was statistically significant（P〈0.05）.Conclusion A nucleic acid vaccine containing pVAX1-GRA1 can provide partial immunoprotection and is a promising candidate for use in future studies of vaccination against T.gondii.

关 键 词：刚地弓形虫 GRA1基因 核酸疫苗 

分 类 号：R382.5[医药卫生—医学寄生虫学]