嗜肺军团菌flaA基因DNA疫苗的构建及其免疫保护性研究

目的构建flaA基因的DNA疫苗,观察其对嗜肺军团菌感染的免疫保护作用。方法用限制性核酸内切酶从重组质粒pET32a-flaA上切下flaA基因,亚克隆到真核表达载体pcDNA3.1(+),重组子经限制性酶切分析、PCR鉴定正确后,命名为pcDNA3.1-flaA。将18只BALB/c雌性小鼠随机分为3组,pcDNA3.1-flaA实验组肌肉接种pcDNA3.1-flaA质粒100μg,2w后加强免疫一次,2个对照组分别肌肉注射等体积的生理盐水和100μg空质粒,加强免疫后3w小鼠鼻腔滴注嗜肺军团菌Lp1菌液进行攻击感染,感染4w后,检查小鼠肺组织带菌量,观察肺组织病理形态学改变。结果成功构建了真核表达重组质粒pcDNA3.1-flaA,攻击感染实验表明,pcDNA3.1-flaA免疫组肺组织带菌量显著少于生理盐水对照组和空质粒对照组(P<0.05),且肺组织病理变化症状较pcDNA3.1对照组和生理盐水对照组的肺轻微。结论由flaA基因构建的嗜肺军团菌DNA疫苗能诱导小鼠产生保护性免疫。

Construction and immunoprotection of DNA vaccine against Legionella pneumophila carrying flaA gene

To construct DNA vaccine containing flaA gene and observe its immunoprotection effect against Legionella pneumophila infection,the flaA gene was obtained from reombinant plasmid pET32a-flaA by restriction endonuclease digestion and was subcloned into eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid, named pcDNA3.1-flaA, was identified by restriction analysis, PCR and DNA sequencing analysis. Eighteen female BALB/c mice of 6-8 weeks old were grouped randomly into three groups, the pcDNA3.1-flaA group were immunized intramuscularly with recombinant plasmid pcDNA3.1-flaA, the blank plasmid group and normal saline group were injected with blank plasmid pcDNA3.1(+) and normal saline respectively. Immunization was boosted twice with the same dosages at two weeks interval. Three weeks after last immunization, all mice were challenged with Legionella pneumophila serotype 1. 4 weeks after challenging late, the bacterial colony counts and the pathological changes in lungs were determined and observed to evaluate the protective immunity of DNA vaccine. The results showed that the recombinant plasmid pcDNA3.1-flaA was constructed, in which the bacterial colony of Legionella pneumophila in the pcDNA3.1-flaA group were significantly lower than that of the normal saline group and the pcDNA3.1(+) group(P<0.05), the pathological changes of lungs in the pcDNA3.1-flaA group showed little blank pcNDA3.1(+) group and normal saline group. It is concluded that the recombinant plasmid pcDNA3.1-flaA was constructed successfully, and protective immunity against Legionella pneumophila can be induced by the DNA vaccine containing flaA gene, Changed in comparison with the,thas providing a good experimental basis for further development of DNA vaccine against Legionella pneumophila.