菊粉对铜绿假单胞菌flgE基因DNA疫苗的免疫增强作用

The immune enhancement of inulin on flgE gene DNA vaccine of Pseudomonas aeruginosa

为研究菊粉对铜绿假单胞菌flgE基因DNA疫苗的免疫增强作用,本研究将铜绿假单胞菌的flgE基因克隆于真核表达载体pcDNA3.1(+)中制备DNA疫苗,分别以该DNA疫苗(100μg/只)和菊粉佐剂+DNA疫苗免疫BALB/c小鼠(质粒100μg/只,终浓度20%菊粉),同时设置菊粉灌胃对照组(600 mg/kg)以及灭活疫苗(100μL/只)、鞭毛蛋白(100μL/只)、pcDNA3.1(+)空载体(100μg/只)和PBS对照组(100μL/只)。每两周免疫一次,共免疫3次。利用间接ELISA检测免疫后血清特异性抗体水平,MTT法检测免疫小鼠脾淋巴细胞增殖水平,双抗夹心ELISA检测IFN-γ分泌情况,计算强毒攻击后各组小鼠的存活数及保护率。结果显示,菊粉佐剂组和菊粉灌胃组小鼠产生的抗体水平与DNA疫苗组相比无明显差异(p>0.05),且显著低于灭活疫苗组和鞭毛蛋白组(p<0.05);菊粉佐剂组对小鼠脾淋巴细胞的刺激值(SI值)和IFN-γ水平与鞭毛蛋白组相当,明显高于菊粉灌胃组和DNA疫苗组(p<0.05);菊粉佐剂组和菊粉灌胃组对小鼠的保护率均高于DNA疫苗组,但低于灭活疫苗组,表明以菊粉预先灌胃和以菊粉为佐剂均可在一定程度上提高flgE基因DNA疫苗对小鼠的保护效率,但仍不及传统的灭活疫苗。本实验为铜绿假单胞菌DNA疫苗的研究及菊粉在DNA疫苗中的应用奠定基础。

To study the immune enhancement of inulin on flg E gene DNA vaccine against Pseudomonas aeruginosain mice,flg E gene was cloned into the eukaryotic expression vector pcDNA3.1(+) for preparing the DNA vaccine. BALB/c mice were vaccinated with DNA vaccine(100 μg/mice) and inulin-adjuvant DNA vaccine(100 μg/mice, final inulin concentration is 20%) at2 weeks intervals, and 3 times in all. Inulin gavage group(600 mg/kg), inactivated vaccine group(100 μL/mice), flagellin vaccine group(100 μL/mice), pcDNA3.1(+) empty vector group(100 pg/mice) and PBS group(100 μL/mice) were set as controls. The serum antibodies, spleniceen lymphocyte proliferation and IFN-γ secretion were detected by indirect ELISA, MTT and Double-antibodyies-sandwich-ELISA, respectively. The mice were challenged with Pseudomonas aeruginosa on week 2 post the third immunization, the survival number and protection rate were countedderived. The results showed that there was no difference in the antibody levels among the inulin-adjuvant group, inulin gavage group and DNA vaccine groups(p>0.05), and the levels of antibodies in these three groups were significantly lower than the inactive vaccine and flagellin vaccine groups(p<0.05). The SI value and levels of IFN-γ induced with flagellin protein of Pseudomonas aeruginosain inulin-adjuvant group were equally to theflagellin vaccine group and significantly higher than the inulin gavage group and DNA vaccine groups(p<0.05). The protection rate of inulin-adjuvant group and inulin gavage group were higher the DNA vaccine group and lower than the inactive vaccine group. The results indicated inulin could enhance the protective effectof flgE DNA vaccine of Pseudomonas aeruginosato some extent, but still inferior to the inactivated vaccine. The goal of this study was to lay the foundation for the development of DNA vaccine against the Pseudomonas aeruginosa and for the application of inulin in DNA vaccine.