粪肠球菌介导的铜绿假单胞菌重组疫苗诱导小鼠的保护力及细胞免疫应答  

Cellular immune response and immunoprotection induced by Enterococcus faecalis-mediated vaccine against Pseudomonas aeruginosa in mice

作　　者：欧兴坤 李文桂[1] OU Xing-kun;LI Wen-gui(Institute of Infections and Parasitic Diseases,the First Affiliated Hospital,Chongqing Medical University,Chongqing 400016,China)

机构地区：[1]重庆医科大学附属第一医院传染病寄生虫病研究所,重庆400016

出　　处：《中国病原生物学杂志》2022年第4期430-434,共5页Journal of Pathogen Biology

基　　金：重庆市科委地方病重大专项基金项目(No.2008AB5055,2008AA5008,2008AB5054)。

摘　　要：目的研究基于粪肠球菌(Ef)载体的铜绿假单胞菌(Pa)重组疫苗rEf(pGEX-LasR)诱导小鼠的保护力及细胞免疫应答。方法分别将5×10^(9)CFU的疫苗株、空载体株和Ef株菌液口服灌胃免疫BALB/c鼠,每周3次,连续3周;初次免疫后4周用5×10^(7)CFU的Pa01株菌液滴鼻攻击小鼠,2周后杀鼠剖检;菌落计数检测小鼠肺细菌负荷,MTT法检测小鼠脾细胞增殖水平,荧光激活细胞分选仪(FACS)检测小鼠脾细胞CD4^(+)/CD8^(+)T细胞亚群和脾细胞凋亡水平;采用PaAg体外刺激培养小鼠脾细胞48 h,PCR扩增IL-2、IFN-γ、IL-12、IL-17和Fox3^(+)基因。结果疫苗组、空载体组和Ef对照组小鼠肺细菌负荷分别为(7.616±0.010)lgCFU/g、(8.876±0.011)lgCFU/g和(8.902±0.010)lgCFU/g,疫苗组显著降低(P<0.01);与空载体组和Ef对照组比较,疫苗组小鼠脾细胞CD4^(+)T细胞亚群显著增加(P<0.01),脾细胞增殖活性增强(P<0.01)、凋亡率减低(P<0.01);疫苗组小鼠脾细胞DNA可扩增出IL-2、IFN-γ、IL-12、IL-17和Fox3^(+)基因条带,空载体组和对照组未扩增出条带。结论Pa重组疫苗rEf(pGEX-LasR)可诱导小鼠产生细胞免疫应答,促进小鼠脾细胞IL-2、IFN-γ、IL-12、IL-17和Fox3^(+)基因的表达,降低Pa01攻击小鼠后的肺细菌负荷。Objective To study the cell-mediated immunity and protective efficacy induced by Enterococcus faecalis-mediated Pseudomonas aeruginosa LasR vaccine in mice.Methods BALB/c mice,6-8 weeks old,were divided into 3 groups(7 mice/group),and immunized 3 times a week for 3 weeks with live E.faecalis containing pGEX-LasR,E.faecalis containing blank vector,and E.faecalis at a dose of 5×10~9 CFUs/mouse in a volume of 100μl.4 weeks post first immunization,groups were challenged by nasal drip with 10μl of P.aeruginosa bacterial suspension at a concentration of 1×10~6CFU/μl.2 weeks post challenge,mice were sacrificed,then lungs were collected for bacterial loads investigation and spleens were isolated for cytological-testing.The splenocyte CD4^(+)/CD8^(+)T cell subsets and splenocyte apoptosis were detected with fluorescence activating cell sorter,splenocyte proliferation was determined with MTT assay.Splenocytes were isolated and stimulated in vitro with PaAg,then the IL-2,IFN-γ,IL-12,IL-17 and Fox3^(+)genes were amplified using PCR.Results The bacterial loads of vaccine group,blank vector group and E.faecalis group were(7.616±0.010)lgCFU/g,(8.876±0.011)lgCFU/g and(8.902±0.010)lgCFU/g.There was significant reduction in bacterial loads of vaccine group versus control groups(P<0.01).It was observed also that the percentage of CD4^(+)T cell and proliferative activity of splenocytes in vaccine group increased(P<0.01 for all),and the rate of apoptosis of splenocytes decreased(P<0.01).The IL-2,IFN-γ,IL-12,IL-17 and Fox3^(+)genes were amplified from genomic DNA of splenocyte in vaccine group.Conclusion The rEf(pGEX-LasR)vaccine against P.aeruginosa can induce protection and cell immune resonpse in mice,it may cause an increase in expression of IL-2,IFN-γ,IL-12,IL-17 and Fox3^(+)genes.

关 键 词：铜绿假单胞菌 载体疫苗 LasR蛋白 粪肠球菌 细胞免疫 

分 类 号：R378.99[医药卫生—病原生物学]