铜绿假单胞菌外膜蛋白Ⅰ载体疫苗构建及免疫效果研究  

Construction of Pseudomonas aeruginosa Outer Membrane Protein I Recombinant Vector Vaccine and Its Immune Efficacy Study

作　　者：张艳琪 胡基华[1] 曲晓军[1] ZHANG Yan-qi;HU Ji-hua;QU Xiao-jun(Institute of Microbiology,Heilongjiang Academy of Sciences/Innovation Research Center for Microbial Pharmacology,Harbin,Heilongjiang 150010)

机构地区：[1]黑龙江省科学院微生物研究所/微生物药物学创新研究中心,黑龙江哈尔滨150010

出　　处：《安徽农业科学》2025年第7期86-91,101,共7页Journal of Anhui Agricultural Sciences

基　　金：黑龙江省科学院科学研究基金项目(KY2022SW01)。

摘　　要：[目的]构建表达铜绿假单胞菌外膜蛋白I的重组乳酸乳球菌载体疫苗,评价构建的载体疫苗免疫效果,为研发可防治铜绿假单胞菌感染的疫苗奠定基础。[方法]根据GenBank中铜绿假单胞菌OprI基因序列设计引物,扩增、鉴定OprI基因,将OprI基因扩增产物与pMG36c质粒双酶切、连接,构建重组质粒pMG36c-OprI;对乳酸乳球菌MG1363菌株感受态细胞制备和电转化方法优化,将重组质粒电转化MG1363感受态细胞,酶切和PCR反应及测序鉴定转化情况,SDS-PAGE电泳检测OprI蛋白表达情况;采用灌胃、皮下注射和鼻腔喷雾接种小鼠方式评价载体疫苗安全性和异常毒性,采用免疫小鼠、攻毒试验评价免疫效果。[结果]重组载体疫苗构建成功;感受态细胞制备和电转化优化条件:细菌OD_(600)=0.5,复壮时间3 h,质粒添加量100 ng和转化电压2.5 kV时,转化效率可达到425 CFU/μg DNA;载体疫苗安全性、异常毒性试验合格;载体疫苗对小鼠的保护率为76.67%;免疫后,小鼠血清抗体IgG和IgE水平显著高于免疫前(P<0.01),载体疫苗免疫组水平显著高于MG1363菌株免疫组(P<0.01)。[结论]构建了安全、无异常毒性、免疫效果较好的表达铜绿假单胞菌外膜蛋白I的乳酸乳球菌重组载体疫苗,为有效防治铜绿假单胞菌感染提供了坚实的技术支撑。[Objective]Construct a recombinant lactobacillus vector vaccine expressing the outer membrane protein I(OprI)of Pseudomonas aeruginosa,evaluate the immune efficacy of the constructed recombinant vector vaccine,and lay the foundation for the development of vaccines that can prevent and treat Pseudomonas aeruginosa infection.[Method]Based on the OprI gene sequence of Pseudomonas aeruginosa in GenBank,primers were designed to amplify and identify the OprI gene.The PCR amplification product of the OprI gene was cleaved and linked to the pMG36c plasmid to construct a recombinant plasmid pMG36c-OprI;the preparation and electroconversion methods for the competent cells of Lactococcus lactis MG1363 strain were optimized.The recombinant plasmid was electroconverted into the competent cells of MG1363 strain.Single and double enzyme digestion,PCR reactions,and sequencing were used to identify the transformation of the recombinant plasmid.The expression of OprI protein was determined by SDS-PAGE electrophoresis;the safety and abnormal toxicity of the recombinant vector vaccine were evaluated by gavage,subcutaneous injection and nasal spray inoculation in mice,and the immune effect was evaluated by immunization of the recombinant vector vaccine and MG1363 strain in mice and challenge test.[Result]The recombinant vector vaccine was successfully constructed;the optimized conditions for the preparation and electroconversion of receptive cells were as follows:bacterial growth OD_(600)=0.5,cell weakening agent glycine concentration of 25 g/L,rejuvenation time of 3 hours,plasmid addition of 100 ng,and conversion voltage of 2.5 kV,the conversion efficiency could reach 425 CFU/μg DNA.The safety and abnormal toxicity tests of recombinant vector vaccines were qualified;the protection rate of recombinant vector vaccine on mice was 76.67%,and the protection rate of MG1363 strain on mice was 6.67%;after immunization with both MG1363 strain and recombinant vector vaccine,the serum IgG and IgE levels of mice were significantly higher than

关 键 词：铜绿假单胞菌 外膜蛋白I 乳酸乳球菌 重组载体疫苗 免疫效果 

分 类 号：R392[医药卫生—免疫学]