铜绿假单胞菌flgE和oprL基因联合DNA疫苗的免疫效果  

Immune efficacy of combination DNA vaccine of flgE and oprL genes from Pseudomonas aeruginosa

作　　者：宫强[1] 翟文汉 李雅静[1] 牛明福[1] 秦翠丽[1] 侯颖[1] GONG Qiang;ZHAI Wen-han;LI Ya-jing;NIU Ming-fu;QIN Cui-li;HOU Ying(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：《中国兽医科学》2021年第6期715-721,共7页Chinese Veterinary Science

基　　金：河南省自然科学基金项目(162300410068)。

摘　　要：为测试铜绿假单胞菌flgE和oprL基因的联合DNA疫苗在小鼠体内的免疫效果,本试验分别构建铜绿假单胞菌flgE和oprL基因的DNA疫苗pflgE和pOPRL,并制备了联合DNA疫苗pflgE+pOPRL,同时设灭活疫苗作为阳性对照,pcDNA3.1(+)空载体和PBS分别作为阴性对照,免疫BALB/c小鼠,间接ELISA检测免疫后小鼠血清特异性抗体水平,MTT法检测免疫小鼠脾淋巴细胞增殖情况(SI值),双抗夹心ELISA测定脾淋巴细胞分泌的IFN-γ和IL-2的水平,通过攻毒试验并计算各疫苗的保护率。间接ELISA结果显示,以铜绿假单胞菌裂解液为包被抗原检测pflgE+pOPRL组小鼠的抗体水平显著高于pflgE组和pOPRL组(P<0.05),但以菌毛蛋白和外膜蛋白为包被抗原时pflgE+pOPRL组小鼠的抗体水平则分别低于pflgE组(P<0.05)和pOPRL组(P<0.05)。pflgE+pOPRL组的SI值及IFN-γ和IL-2的分泌水平与灭活疫苗组相当,且显著高于两种单价DNA疫苗组(P<0.05)。攻毒试验结果显示,各疫苗均可为小鼠提供一定的保护力,其中灭活疫苗保护率最高(83.3%),联合DNA疫苗的保护率为72.2%,pflgE和pOPRL疫苗则较低,分别为55.6%和50%。结论,以铜绿假单胞菌flgE和oprL基因构建的DNA疫苗,尤其是二价组合DNA疫苗,具有一定的应用前景。In order to detect the immune efficacy of a bivalent combination DNA vaccine of Pseudomonas aeruginosa,two recombinant plasmids pflgE and pOPRL were constructed,and the bivalent combination DNA vaccine pflgE+pOPRL were prepared.Subsequently,the BALB/c mice were immunized with these DNA vaccines,and inactivated vaccines was as positive controls,and pcDNA3.1(+)and PBS were as negative controls,respectivly.Then the level of serum antibody were detected by indirect ELISA,and the lymphocyte proliferation assay was detected by MTT,and the concentrations interferon-γ(IFN-γ),interleukin-2(IL-2)and interleukin-4(IL-4)were detected by double antibody sandwich ELISA,respectively.Lastly,the protection rates were assessed after challenging with virulent P.aeruginosa.ELISA indicated that,the antibody levels in the pflgE+pOPRL group were higher than that of in the pflgE group and pOPRL group(P<0.05)when P.aeruginosa lysis solution was adopted as a coating antigen.However,when the fimbirae protein and the outer membrane protein were the coating antigen,antibody levels in the pflgE+pOPRL group were lower than that of the pflgE group and pOPRL group(P<0.05),respectively.The stimulation index(SI)values,IFN-γand IL-2 concentrations in the pflgE+pOPRL group were equal to that of the inactivated vaccine group and significantly higher than that of the pflgE group and pOPRL group(P<0.05).The protection provided by pflgE+pOPRL was lower than that of the inactivated vaccine but higher than that of the two monovalent DNA vaccines.The protection rate of inactivated vaccine,pflgE+pOPRL,pflgE,and pOPRL were 83.3%,72.2%,55.6%and 50%,respectively.In conclusion,DNA vaccines constructed with the flgE and oprL genes of P.aeruginosa,particularly the divalent combination DNA vaccine,present a certain application prospect.

关 键 词：铜绿假单胞菌 pflgE pOPRL 联合DNA疫苗 免疫效果 

分 类 号：S855.1[农业科学—临床兽医学]