编码弓形虫致密颗粒抗原-1(GRA1)质粒DNA免疫小鼠诱导抗弓形虫感染的保护性(英文)

INDUCTION OF PROTECTIVE IMMUNITY AGAINST TOXOPLASMA GONDII INFECTION IN MICE BY DNA IMMUNIZATION WITH A PLASMID ENCODING THE DENSE GRANULE ANTIGEN-1 (GRA1) GENE

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100μg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573bp的GRA1编码阅读框定向克隆到pcDNA3EcoRⅠ/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。

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 EcoR I and Xho I 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 100μg, 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 To：coplasrna gondii RH strain was observed. Results The recombinant plasmid pcGRA1 was constructed. The special IgG in sera from pcGRAl-plasmid immunized mice was detected with EI.ISA. 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 ob- served, 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.