铜绿假单胞菌oprI基因DNA疫苗及其重组亚单位疫苗免疫效果的评估  

Evaluation of the Immune effectiveness for Pseudomonas aeruginosa oprI gene DNA vaccine and its recombinant subunit vaccine

作　　者：祝世纪 宫强[1] 田佳雨 李雅静[1] ZHU Shi-ji;GONG Qiang;TIAN Jia-yu;LI Ya-jing(Henan University of Science and TechnologyHenan 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》2024年第3期285-291,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：河南省自然科学基金面上项目(242300420145)。

摘　　要：为评估铜绿假单胞菌opr I基因DNA疫苗和重组亚单位疫苗对小鼠的免疫效果,本实验将铜绿假单胞菌外膜蛋白编码基因opr I克隆至真核表达载体p CAGGS-HA中构建重组质粒p CAGGS-opr I,经酶切鉴定正确后获得DNA疫苗。同时将opr I基因克隆至原核表达载体p ET32a中构建重组质粒p ET32a-oprI,经酶切鉴定正确后转入大肠杆菌,经IPTG诱导重组Opr I蛋白(rOprI)的表达,采用亲和层析法纯化后以SDS-PAGE检测,结果显示正确表达了r Opr I,且获得了其纯化蛋白,利用其制备重组亚单位疫苗。分别以DNA疫苗和重组亚单位疫苗免疫BALB/c小鼠,并以铜绿假单胞菌的灭活疫苗和外膜蛋白疫苗为对照,采用间接ELISA法检测免疫后不同时间小鼠血清中的抗体水平和血清中IFN-γ、IL-2和IL-4的含量;初免42 d后以铜绿假单胞菌(1.25×109 cfu/mL,100μL/只)对各组小鼠进行攻毒试验,通过测定各疫苗对小鼠的保护率,评估疫苗的保护效果。结果显示,各疫苗诱导的免疫小鼠血清抗体水平和细胞因子含量均显著高于阴性对照组(P<0.05),且重组亚单位疫苗诱导小鼠的抗体水平和IL-4含量均显著高于DNA疫苗(P<0.05),而IFN-γ和IL-2含量则与DNA疫苗无显著差异(P>0.05)。小鼠攻毒试验结果显示,DNA疫苗组、重组亚单位疫苗组、外膜蛋白疫苗组和灭活疫苗组小鼠获得的免疫保护率分别为45%、55%、70%和95%。上述结果首次表明以铜绿假单胞菌opr I基因制备的DNA疫苗和r Opr I重组亚单位疫苗均可诱导小鼠产生体液和细胞免疫应答,并可为小鼠提供对铜绿假单胞菌攻毒的免疫保护效果,但二者的免疫效果还需进一步提高。本研究为铜绿假单胞菌疫苗的研究提供了参考依据。To detect the immune effectiveness of Pseudomonas aeruginosa opr I gene DNA vaccine and its recombinant subunit vaccine.The target gene fragment was cloned into the eukaryotic expression vector p CAGGS-HA to construct recombinant plasmid p CAGGS-opr I.After enzyme digestion,the DNA vaccine was obtained.At the same time,the opr I gene fragment was cloned into the prokaryotic expression vector p ET32a to construct recombinant plasmid p ET32a-opr I.Then the recombinan plasmid was identified by digestion and transferred into Escherichia coli induced with IPTG.After the recombinant protein wa expressed and purified by affinity chromoatography,the recombinant subunit vaccine was prepared and tested with SDS-PAGE BALB/c mice were vaccinated with DNA vaccine,recombinant subunit vaccine,and outer membrane vaccine and inactivated vaccines as the controls.Then sera were collected every 7 days,and the levels of antibody,interferon-γ(IFN-γ),interleukin-2(IL-2)and interleukin-4(IL-4)concentrations were detected by indirect ELISA.The mice were challenged with virulen Pseudomonas aeruginosa following primary immunication for 42 days,and protective efficacy of vaccines were evaluated.The results showed that the serum antibody and cytokine levels in the four vaccine groups were significantly higher than those in the negative control group(P<0.05).The serum antibodies level and IL-4 concentration in the recombinant subunit vaccine group were higher than those in the DNA vaccine group(P<0.05).No significant differences were detected for the concentrations of IFN-γand IL-2 between the DNA vaccine group and recombinant subunit vaccine group(P>0.05).The protective efficacy rates of DNA vaccine group,recombinant protein vaccine group,outer membrane vaccine group and inactive vaccine group were 45%,55%70%and 95%,respectively.The results indicated for the first time that both the DNA vaccine and recombinant subunit vaccine based on the opr I gene of Pseudomonas aeruginosa could induce immune response and provide immune protection from

关 键 词：铜绿假单胞菌 oprI基因 DNA疫苗 重组亚单位疫苗 免疫效果 

分 类 号：S852.61[农业科学—基础兽医学]