弓形虫gra14基因真核表达载体的构建及其免疫保护性研究  

CONSTRUCTION OF EUKARYOTIC EXPRESSION VECTOR OF TOXOPLASMA GONDII GRA14 GENE AND ITS IMMUNOPROTECTIVE EFFECT

作　　者：陈晓恒[1] 郑斌[1] 丁建祖[1] 楼涤[1] 童群波[1] 孔庆明[1] 陈睿[1] 陆绍红[1] 

机构地区：[1]浙江省医学科学院寄生虫病研究所,杭州310013

出　　处：《寄生虫与医学昆虫学报》2015年第4期205-211,共7页Acta Parasitologica et Medica Entomologica Sinica

基　　金：浙江省科技计划项目（2013C33195,2014C33230,2015C37108）;浙江省医学熏点学科群建设（XKQ-OIO-001,XKQ-009-003）;浙江省卫生高层次创新人才培养工程项目（C31001）

摘　　要：构建弓形虫致密颗粒蛋白14 （GRA14）的真核表达质粒,研究其对小鼠的免疫保护力.扩增弓形虫RH强毒株gra14基因并构建pVAX1-gra 14真核表达质粒,免疫BALB/c小鼠.60只小鼠随机分成4组,实验组小鼠肌肉注射100 μg pVAX1-gra14质粒,对照组分别注射PBS（100 μL/只）和pVAX1空质粒（100μg/只）,空白对照不作处理.共免疫3次,每次间隔14d.ELISA法检测免疫后IgG、亚型抗体和细胞因子水平.攻击实验后比较小鼠生存率,分析pVAX1-gra 14的免疫保护作用.结果显示成功扩增gra14基因并构建真核表达载体,Western blot结果显示目的基因成功表达并具有反应原性,pVAX1-gra14免疫后小鼠的IgG抗体水平显著升高,IFN-γ、IL-2、IL-4和IL-10的表达量分别为652.47±14.55、455.53±7.88、228.13±14.51和252.13±14.20 pg/mL,同时免疫后实验组小鼠的生存时间相比对照组显著延长,平均生存时间为14.1 ±1.3 d.以上结果表明,pVAX1-gra14能够诱导特异细胞免疫和体液免疫反应,产生一定的免疫保护作用.A recombinant eukaryotic expression plasmid encoding Toxoplasma gondii dense granule protein 14 （GRA14） was constructed and its protective effect was studied. Gral4 gene of Toxoplasma gondii RH strain was amplified and inserted into the pVAX1 vector. Sixty BALB/c mice were randomly divided into four groups, each mouse in the experimental group was injected intramuscularly with 100 μg recombinant pVAX1-gra14 plasmids biweekly for three times, and each of the control groups mice was injected intramuscularly with 100 μL PBS, 100 μg blank vectors or nothing, respectively. The induced IgG, subtype antibody and cytokine levels were detected by ELISA. The survival rate of mice after challenge was compared to evaluate the immune protective effect. The results showed that the gral4 gene was amplified and the eukaryotic expression vector was well constructed, western blot analysis showed that the target gene was successfully expressed and had reactogenicity, the IgG antibody levels of mice injected with pVAXl-gral4 were significantly increased, the expression of IFN-γ, IL-2, IL-4 and IL-10 were 652. 47 ± 14. 55, 455.53± 7.88, 228.13± 14. 51 and 252. 13 ± 14. 20 pg/mL, respectively, and the survival time of mice immunized with pVAX1-gral4 was significantly prolonged compared with control groups, the mean survival time was 14. 1 ±1.3 days. These results suggested that, pVAXl-gral4 was capable of inducing the specific cellular and humoral immune responses and inducing immune protection against Toxoplasma gondii infection.

关 键 词：刚地弓形虫 致密颗粒蛋白14 DNA疫苗 免疫 Gra14 

分 类 号：Q782[生物学—分子生物学]