猪弓形虫重组卡介苗的构建及其免疫保护性  

Construction of recombinant BCG with Rhomboid-IL-2 and its immunoprotection on infection with Toxoplasma gondii in pigs

作　　者：于艳辉[1,2] 杨升 姜鑫 信彩岩 李显赫 王伟荣 李建华 张西臣 宫鹏涛 YU Yan-hul;YANG Sheng;JIANG Xin;XIN Cai-yan;LI Xian-he;WANG Wei-rong;LI Jian-hua;ZHANG Xi-chen;GONG Peng-tao(Clinical Lab,The Second Hospital of Jilin University,Changchun 130021,Jilin Province,China)

机构地区：[1]吉林大学第二医院检验科,吉林长春130021 [2]吉林大学动物医学学院,吉林长春130062

出　　处：《中国生物制品学杂志》2018年第7期695-700,707,共7页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划项目(20130206023NY)

摘　　要：目的构建弓形虫Rhomboid基因和猪IL-2基因的重组卡介苗(BCG),并分析其免疫保护效果。方法应用RT-PCR技术分别扩增弓形虫Rhomboid基因和猪IL-2基因,克隆至p MD18-T载体中,双酶切鉴定及测序正确后,将弓形虫Rhomboid基因和猪IL-2基因串联后分别连接至穿梭表达载体pMV261和整合表达载体pMV361中,电转化BCG,制备重组BCG pMV261-IL-2-Rho和pMV361-IL-2-Rho。分别经颈部肌肉注射免疫猪,共3次,每次间隔2周,免疫剂量均为108 CFU/只,采用ELISA法检测免疫前后猪体内抗体(IgG)滴度、Th1型及Th2型细胞因子的变化情况;第3次免疫后2周通过腹腔注射的方式接种弓形虫China I株速殖子1×108个/只进行攻虫试验,观察重组BCG对猪的免疫保护效果。结果成功构建了重组BCG p MV261-IL-2-Rho和p MV361-IL-2-Rho。重组BCG免疫猪后,随着免疫次数的增加,血清中抗体滴度也逐渐升高,且与PBS对照组相比,差异有统计学意义(P<0.01)。3次免疫后,pMV261-IL-2-Rho组血清Th1型细胞因子IL-2、IFNγ和IL-12的表达水平均明显提高,与PBS对照组相比,差异有统计学意义(P<0.05),而pMV361-IL-2-Rho组血清IL-2、IFNγ和IL-12的水平虽略有升高,但与PBS对照组相比,差异无统计学意义(P>0.05);各组在免疫过程中Th2型细胞因子IL-4水平均未发生明显变化。攻虫后各组猪出现精神沉郁,食欲下降,体温升高等症状,耐过发病期后的猪,食欲逐渐恢复,体温渐趋正常,各种症状减轻,且各组猪在攻虫后20 d均无死亡。结论构建的基因重组BCG pMV261-IL-2-Rho对猪弓形虫感染具有一定的免疫保护作用。Objective To construct a recombinant BCG（r BCG）with Toxoplasma gondii Rhomboid gene and porcine IL-2（Po IL-2）gene, and observe its immunoprotection. Methods Rhomboid gene of Toxoplasma gondii and Po IL-2 gene were amplified by RT-PCR and cloned into p MD18-T vector. After identification by restriction analysis and sequencing,Rhomboid and Po IL-2 genes were linked and subcloned into pMV261 and pMV361 vectors respectively. The constructed recombinant plasmids were transformed to BCG by electroporation to obtain r BCG pMV261-IL-2-Rho and pMV361-IL-2-Rho, with which pigs were immunized by intramuscular injection each at a dosage of 10^8 CFU for 3 times respectively,each at an interval of 2 weeks. The IgG antibody titers and Th1 or Th2 cytokines in pigs before and after immunization were determined by ELISA. The pigs were challenged with tachyzoites of Toxoplasma gondii China I strain at a dosage of 1 × 10^8 tachyzoites 2 weeks after the third immunization to observe the immunoprotection of r BCG. Results Recombinant BCG pMV261-IL-2-Rho and pMV361-IL-2-Rho were successfully constructed. The serum antibody titers in immunized pigs increased gradually with the increasing dose, which showed significant difference with that in PBS control group（P〈0. 01）. After the third immunization, the serum IL-2, IL-12 and IFNγ levels of pigs in pMV261-IL-2-Rho group increased significantly as compared with those in PBS control group（P〈0. 05）. However, the levels of the cytokines in pMV361-IL-2-Rho group increased slightly, which showed no significant difference with those in PBS control group（P〉0. 05）. The IL-4 levels in various groups showed no significant change during immunization. After challenge with tachyzoites of Toxoplasma gondii, the pigs in various groups showed the symptoms of droopy appearance, loss of appetite and raised body temperature,which were relieved after onset period, and no deaths of pigs were observed within 20 d after challenge. Conclusion The constructed rBCG pMV261-IL-2-Rho showed

关 键 词：弓形虫 Rhomboid基因 IL-2基因 重组卡介苗 免疫保护 

分 类 号：R382.5[医药卫生—医学寄生虫学] R392.33[医药卫生—基础医学]