嗜肺军团菌flaA基因DNA疫苗的构建及其免疫保护性研究  

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

作　　者：张雷[1,2] 张莉[1] 陈建平[3] 王涛[1] 杨志伟[3] 李金福[3] 

机构地区：[1]大理学院基础医学院病原生物学综合实验室 [2]四川大学华西医学中心基础医学与法医学院,成都610041 [3]四川大学华西医学中心基础医学与法医学院

出　　处：《中国人兽共患病学报》2007年第4期362-365,共4页Chinese Journal of Zoonoses

基　　金：国家自然科学基金资助(No.30300302)

摘　　要：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。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 peDNA3.1-flaA, the blank plasmid group and normal saline group were injected with blank plasmid pcDNA3.1（＋） and norreal saline respeetively. 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 eounts and the pathological changes in lungs were determined and observed to evaluate the protective immunity of DNA vaeeine. 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 peDNA3.1（＋） group（P〈0.05）, the pathological changes of lungs in the pcDNA3. 1-flaA group showed little blank peN- DA3.1（＋） group and normal saline group. It is concluded that the recombinant plasmid pcDNA3.1-flaA was constructed sueeessfully, and protective immunity against Legionella pneumophila can be induced by the DNA vaccine containing flaA gene, Changed in eomparison with the,thas providing a good experimental basis for further development of DNA vaccine against Legionella pneumophila.

关 键 词：嗜肺军团菌 flaA基因 DNA疫苗 保护性免疫 

分 类 号：R378[医药卫生—病原生物学]