CAJ | 학술논문

目的构建真核表达质粒pcGRA1,并观察其免疫小鼠的保护性. 方法经PCR从cDNA克隆中扩增出GRA1编码基因,定向克隆入pcDNA3的EcoRⅠ/XhoⅠ位点,从而获得pcGRA1.用限制性内切酶消化、PCR及序列测定对该重组质粒进行鉴定.将100 μg质粒于小鼠左后腿DNA肌注,于注射后2周和4周各加强1次;分别观察小鼠体内的特异性抗体滴度、GRA1基因在小鼠体内的分布、GRA1在肌细胞的表达以及免疫小鼠对弓形虫强毒株RH速殖子攻击后的保护性. 结果 DNA序列测定结果表明,将573 bp的GRA1编码阅读框定向克隆到pcDNA3 EcoRⅠ/XhoⅠ位点,成功构建了重组质粒pcGRA1;免疫小鼠血清中检测到特异性IgG;不同组织DNA为模板特异地扩增出GRA1编码基因;IFA检测pcGRA1免疫鼠肌细胞呈阳性反应;弓形虫RH株速殖子攻击感染pcGRA1免疫鼠,其存活时间长于对照组. 结论重组质粒pcGRA1免疫小鼠可诱导特异性免疫反应,对弓形虫强毒株的攻击感染具有一定的保护性.
목적구건진핵표체질립pcGRA1,병관찰기면역소서적보호성. 방법경PCR종cDNA극륭중확증출GRA1편마기인,정향극륭입pcDNA3적EcoRⅠ/XhoⅠ위점,종이획득pcGRA1.용한제성내절매소화、PCR급서렬측정대해중조질립진행감정.장100 μg질립우소서좌후퇴DNA기주,우주사후2주화4주각가강1차;분별관찰소서체내적특이성항체적도、GRA1기인재소서체내적분포、GRA1재기세포적표체이급면역소서대궁형충강독주RH속식자공격후적보호성. 결과 DNA서렬측정결과표명,장573 bp적GRA1편마열독광정향극륭도pcDNA3 EcoRⅠ/XhoⅠ위점,성공구건료중조질립pcGRA1;면역소서혈청중검측도특이성IgG;불동조직DNA위모판특이지확증출GRA1편마기인;IFA검측pcGRA1면역서기세포정양성반응;궁형충RH주속식자공격감염pcGRA1면역서,기존활시간장우대조조. 결론중조질립pcGRA1면역소서가유도특이성면역반응,대궁형충강독주적공격감염구유일정적보호성.
Objective To construct the eukaryotic expression recombinant plasmid, pcDNA3/GRA1 (pcGRA1), and evaluate the protective efficacy of pcGRA1-DNA vaccination in mice. Methods A gene GRA1 was amplified from a cDNA clone by using PCR technique and directly inserted into EcoRⅠ and XhoⅠ sites of pcDNA3 to generate plasmid pcGRA1. The eukaryotic expression recombinant plasmid pcGRA1 was identified by restriction endonuclease digestion, PCR and sequencing analysis. pcGRA1 was injected into the left leg muscle of mice for a dosage of 100 μg, and two booster vaccinations were given with the same dosage after 2 and 4 weeks, respectively. Control groups were injected with empty plasmid pcDNA3 and normal saline. The distribution of GRA1 gene in vaccinated mice, and expression of GRA1 in its muscle were examined. The survival time of vaccinated mice when challenged with virulent Toxoplasma gondii RH strain was observed. Results The recombinant plasmid pcGRA1 was constructed. The special IgG in sera from pcGRA1-plasmid immunized mice was detected with ELISA. A 573 bp GRA1 gene product was amplified from blood, spleen, lung, liver, heart, and muscle respectively, which were collected from pcGRA1 immunized mice. The immunoflurescence microscopy analysis showed that a strong labeling of the muscle tissues from pcDNA3-injected mice was observed, suggesting the GRA1 protein expression in pcGRA1 immunized mice. The data from in vivo challenge experiments indicates that vaccination of pcGRA1 DNA decreases acute virulence of T. gondii in mice, since the survival time of pcGRA1-injected mice was longer than that of two control ones. Conclusion Nude plasmid pcGRA1 DNA vaccination could induce protective immunity against virulent T. gondii RH strain challenge in mice.