铜绿假单胞菌flgE基因菊粉季铵盐纳米DNA的制备与免疫效果研究  

Preparation and Immune efficacy of flgE inulin quaternary ammonium salt nanoparticle DNA vaccine of Pseudomonas aeruginosa

作　　者：宫强[1] 李雅静[1] 翟文汉 牛明福[1] 秦翠丽[1] GONG Qiang;LI Ya-jing;ZHAI Wen-han;NIU Ming-fu;QIN Cui-li(Henan University of Science and Technology/Henan Engineering Research Center of Food Microbiology,Luoyang 471023,China)

机构地区：[1]河南科技大学河南省食品微生物工程技术研究中心,河南洛阳471023

出　　处：《中国预防兽医学报》2021年第9期985-990,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：河南省自然科学基金项目(162300410068)。

摘　　要：为提高铜绿假单胞菌flgE基因DNA疫苗的免疫效果,本实验对菊粉进行阳离子化后获得带正电荷的菊粉季铵盐,以其包裹裸DNA疫苗后制备成菊粉季铵盐纳米DNA疫苗,结果显示,flgE基因壳菊粉季铵盐纳米DNA疫苗在扫描电镜下呈现较为规则的圆球型,粒径100 nm~200 nm。以裸DNA疫苗和菊粉季铵盐纳米DNA疫苗免疫BALB/c小鼠,同时设置灭活疫苗、pcDNA3.1(+)空载体和PBS对照组。免疫1周时经断尾采血并分离血清,采用间接ELISA检测免疫后血清特异性抗体水平,结果显示小鼠免疫后菊粉季铵盐纳米DNA疫苗诱导的血清抗体水平虽低于灭活疫苗,但明显高于裸DNA疫苗(p<0.05)。同时每次免疫2周后,每组各随机取5只小鼠分离制备脾淋巴细胞悬液,采用MTT法检测免疫小鼠脾淋巴细胞增殖情况,双抗夹心ELISA测定脾淋巴细胞分泌的IFN-γ和IL-2的水平,结果显示,纳米DNA疫苗组小鼠的刺激指数(SI)及脾淋巴细胞分泌的IFN-γ和IL-2含量与灭活疫苗组相当,显著高于裸DNA疫苗组(p<0.05)。三免2周后以1.8×10^(10)cfu/只铜绿假单胞菌CAU0792菌液攻击各组小鼠,且计算存活数及保护率,结果显示灭活疫苗组、菊粉季铵盐纳米DNA疫苗组和裸DNA疫苗组的保护率分别为90%、75%和55%,表明菊粉季铵盐可增强裸DNA疫苗诱导的免疫应答水平和保护效果。本实验首次将菊粉进行季铵盐化后使其带有正电荷,有利于结合DNA分子形成纳米颗粒,从而增强了DNA疫苗的免疫原性,为铜绿假单胞菌DNA疫苗的研制及新型DNA疫苗佐剂的开发奠定了基础。To improve the immune efficacy of Pseudomonas aeruginosa flgE gene DNA vaccine,the inulin quaternary ammonium salt with positive charge was obtained by cationization of inulin.Then the nanoparticles DNA vaccines of flgE gene was constructed using inulin quaternary ammonium salt as vector and adjuvant.The results showed that inulin quaternary ammonium salt nanoparticle DNA vaccine presented regular spherical shape and the particle size was between 100 nm-200 nm under the scanning electronic microscope.BALB/c mice were vaccinated with the the naked DNA vaccine and the inulin quaternary ammonium salt nanoparticle DNA vaccine.Meanwhile,the inactivated vaccine group,pc DNA3.1(+)empty vector group and PBS control group were sets.After one week immunization,blood samples from tail were collected and the serum was separated.The serum specific antibody level was detected by indirect ELISA.The results showed that the antibody levels induced by nanoparticles DNA vaccine was lower than that induced by the inactivated vaccine,but significantly higher than that induced by the naked DNA vaccine group(p<0.05).At the same time,two weeks after each immunization,five mice were randomly selected from each group to prepare spleen lymphocyte suspension.Then,MTT assay was used to detect the proliferation of splenic lymphocytes in immunized mice,and the levels of IFN-γand IL-2 secreted by splenic lymphocytes were determined by doubleantibody sandwich ELISA.The results showed that the stimulation index(SI)values and the concentrations of IFN-γand IL-2 secreted by spleen lymphocytes in the nanoparticles DNA vaccine group were similar to those in the inactivated vaccine group,and significantly higher than those in the naked DNA vaccine group(p<0.05).After two weeks of third vaccination,the mouse in each group were challenged with the virulent P.aeruginosa strain CAU0792(1.8×10^(10) cfu/mouse)and the survival rate and protection rate were calculated.The results showed that the protective rates of inactivated vaccine group,inulin quaterna

关 键 词：铜绿假单胞菌 菊粉季铵盐 纳米DNA疫苗 免疫效果 

分 类 号：S852.61[农业科学—基础兽医学]