SjESG-2A锤头状核酶表达载体构建及其抗日本血吸虫虫卵发育研究  

Construction and identification of the eukaryotic expression vector containing genes of Hammerhead Ribozyme targeting SjESG-2A and study on its effection of anti-Schistosoma japonicum ovulation development in infected BALB/c mice

作　　者：胡永轩[1] 周月兰[1] 肖建华[1] 杨秋林[1] 黄家芳[1] 

机构地区：[1]南华大学病原生物研究所,衡阳421001

出　　处：《中国人兽共患病学报》2008年第4期343-346,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金项目(No.30771879/C030112)资助湖南省自然科学基金项目(No.05JJ30043)资助

摘　　要：目的设计并合成日本血吸虫中国大陆株卵壳蛋白基因2A(eggshell protein gene 2A,ESG-2A)的特异性锤头状核酶并构建真核表达载体。探讨其在BALB/c小鼠体内的抗日本血吸虫虫卵发育作用。方法运用计算机软件人工设计并合成核酶基因(RZ),将核酶基因克隆到真核表达载体pcDNA3.1(+)中,采用双酶切、琼脂糖凝胶电泳及DNA序列测定方法鉴定阳性克隆。以日本血吸虫尾蚴感染BALB/c小鼠,建立动物模型。大量抽提阳性重组子,分别在小鼠感染时、感染后2w、感染后4w,将其经后腿胫前肌免疫BALB/c小鼠,共3次。小鼠感染6w后计数成虫负荷及肝组织虫卵数。ELISA法检测小鼠脾淋巴细胞培养上清IFN-γ和IL-4的水平。结果经酶切电泳及DNA测序证实合成的核酶基因序列正确并已被准确克隆入pcDNA3.1(+)的XbaI和EcoR I酶切位点之间,命名为pcRz-ESG-2A。在核酶组,IFN-γ的水平呈上升趋势,IL-4的水平呈先上升后下降;在空载体组和生理盐水组,IFN-γ的水平呈上升趋势,IL-4的水平也呈上升趋势。核酶组免疫BALB/c小鼠能诱生减虫率25.64%和减卵率44.21%。结论成功合成SjESG-2A特异性的锤头状核酶基因并构建了该基因的真核表达载体。核酶在已感染日本血吸虫尾蚴的BALB/c小鼠体内具有抗日本血吸虫虫卵发育的作用。To construct and identify the eukaryotic expression vector with genes of hammerhead ribozyme targeting Schistosorna japonicum eggshell protein gene 2A （SjESG-2A） mRNA and to investigate its effection on antbSchistosorna japonicum ovulation development in infected BALB/c Mice. According to design by computer , two specific restriction sites Xba I and EcoR I were added to the both ends of the ribozyme gene, then the modified ribozyme gene was synthesized and cloned into the eukaryotic expression vector pcDNA3.1 （ ＋ ） . The positive recombinants were screened by tolerance of ampicillin , and plasmids were extracted from the positive recombinants and digested by Xba I and EcoR I , and then were analyzed by agarose gel electrophoresis and DNA sequencing. Sixty female BALB/c mice were devided into three groups. Each mice was challenged with 40 cercariae of S. japonicum Chinese strain. Each mouse of control group was injected with 100 g of pcDNA3. 1（＋） or NS by intramuscle. Experiment group were injected with 100 g of pcRz-ESG-2A. Each mouse was immunized at infected, 2 weeks and 4 weeks. Then the mice were killed and perfused 45 days after challenge. The numbers of recovered worms and hepatic eggs were counted. The production of IFN-γ and IL-4 in mice was assayed by ELISA. The recombinants containing the ri-bozyme gene were successfully selected by restriction endonuelease digestion and agarose gel eleetrophoresis , and were proved by DNA automatic sequencing. In ribozyme group, IFN-γ was rising among the program, IL-4 was rising first, then back up. In the control groups, IFN-γ and IL-4 was rising. The worm and egg reduction rates of ribozyme group were 36. 32% and 44.21% respectively in comparison with the control group. The eukaryotic expression vectors with genes of hammerhead ribozyme targeting SjESG-2A mRNA（pcRz-ESG- 2A） were constructed successfully , and it could induced partial protection against S. japonicum ovulation development in infected BALB/c mice.

关 键 词：日本血吸虫(中国大陆株) 卵壳蛋白基因2A 锤头状核酶 虫卵发育 

分 类 号：R383[医药卫生—医学寄生虫学]