YIKE安徽医科大学学报Acta Universitatis Medicinalis AnhuiActa Universitatis Medicinalis Anhui安徽医科大学学报1000-149234-1065/RAnhui Lianzhong Printing Limited CompanyEditorial Board of Acta Universitatis Medi-cinalis Anhui Meishan Road , Hefei 230032《安徽医科大学学报》编辑部安徽省合肥市安徽医科大学校内老图书馆三楼1000–1492（2026）03–0518–0610.19405/j.cnki.issn1000–1492.2026.03.018V180李文桂-R 759R 392.11A基础医学研究屎肠球菌介导的铜绿假单胞菌重组Ef-PA0057疫苗的构建及其保护力观察Construction of Enterococcus faecium -based recombinant Ef-PA0057 vaccine of Pseudomonas aeruginosa andits protection in mice李文桂LiWengui欧兴坤OuXingkun何爱琳HeAilin重庆医科大学附属第一医院传染病寄生虫病研究所，重庆市传染病与 寄生虫病学重点实验室，重庆400016Chongqing Key Laboratory of Infectious and Parasitic Diseases， Institute of Infections and Parasitic Diseases， The First Affiliated Hospital， Chongqing Medical University， Chongqing400016Zhu Huaqing， E-mail：aydzhq@126.comWang Yi， E-mail ： wangyi@ahmu.edu.cnLi Wengui， E-mail： cqliwengui@163.com
To construct an Enterococcus faecium （Ef）-based recombinant Ef-PA0057 vaccine of Pseudomonas aeruginosa（Pa）and to study its protective immune mechanism in mice.
Methods
The PA0057 gene was cloned from the genomic DNA of PA01 strain ATCC9027 by PCR and inserted into pGEX-1λT to construct pGEX-PA0057. The recombinant plasmid was electroporated into TX0016 strain to construct rEf-PA0057 vaccine. The plasmid was extracted from rEf for PCR. The rEf vaccine was expressed through IPTG induction， and the expression of protein was analyzed by SDS-PAGE and Western blot. BALB/c mice were immunized intragastrically with 5×108 CFU rEf-PA0057 vaccine 3 times per week for 3 weeks. 4 weeks after the first immunization， mice were challenged intranasally with 5×107 CFU of PA01 strain. 2 weeks after challenge， mice were sacrificed， and their lungs were separated. Bacteria in lungs were incubated and colonies were counted. Sera were collected at 0， 4， and 6 weeks after the first immunization. The IgG and its subclasses， and IgE were detected by ELISA.
Results
The 900 bp PA0057 gene was successfully cloned by PCR. PCR showed that PA0057 gene was amplified when the extracted plasmid from rEf as template； the relative molecular mass （Mr） of the expressed PA0057-GST fusion protein was approximately 58 ku， detected by SDS-PAGE. The amount of the expressed protein was 18% of the total bacterial proteins. Western blot showed that the target protein could be recognized by Pa sera. The colony numbers of lung tissue in rEf-PA0057 vaccine group， blank vector group and Ef control group were （0.297±0.011）×108 CFU， （7.576±0.206）×108 CFU and （7.551±0.185）×108 CFU， respectively， the difference was statistically significant （P0.01）. The levels of IgG， IgG1， IgG2b， IgG3 and IgE increased. At the same time point， there was a significant difference compared with the two control groups （P0.01）.
Conclusion
The rEf-PA0057 vaccine is successfully constructed. It may induce mice to produce humoral response against challenge with PA01.
屎肠球菌铜绿假单胞菌PA0057疫苗体液免疫Enterococcus faeciumPseudomonas aeruginosaPA0057vaccinehumoral immunity国家自然科学基金项目3067183530200239国家自然科学基金项目（编号：30671835、30200239）National Natural Science Foundation of China3067183530200239Fund programs National Natural Science Foundation of China （Nos. 30671835，30200239）version1.0.0.25071structure-time2026-05-25T09:36:44word-sourceFX
Construction and identification of rEf-PA0057 vaccine
A：Identification of PCR product of PA0057 gene；M：DNA marker；1-6：PCR products of PA0057；B：Double endonuclease identification of pGEX-PA0057；M：DNA marker；1/2：pGEX-1λT vector；3/4：endonuclease products of pGEX-PA0057；C：PCR identification of target gene of rEf-PA0057 vaccine；M：DNA marker；1-4：PCR products of pGEX-PA0057；D：SDS-PAGE analysis detected the expression product of rEf-PA0057 vaccine；M：Protein marker；1：rEf-RhlR without culture；2-8：rEf-PA0057 cultured for 1，3，5，7，9，11 and 13 h， respectively；E：Western blot detected the expression product of rEf-PA0057 vaccine；M：Protein marker；1：Control；2：PA0057 protein.
Bacterial load in the lungs of mice following immunization with the rEf-PA0057 vaccine and challenge with PA01 strain （<italic>n</italic>=7，<inline-formula><alternatives><mml:math id="M6"><mml:mover accent="true"><mml:mi>x</mml:mi><mml:mo>¯</mml:mo></mml:mover></mml:math><graphic specific-use="big" xlink:href="alternativeImage/D58D7858-C5E9-491b-918A-5F1B3F1B3190-M005.jpg"><?fx-imagestate width="1.35466671" height="2.03200006"?></graphic><graphic specific-use="small" xlink:href="alternativeImage/D58D7858-C5E9-491b-918A-5F1B3F1B3190-M005c.jpg"><?fx-imagestate width="1.35466671" height="2.03200006"?></graphic></alternatives></inline-formula>±<italic>s</italic>）
Humoral immune response following immunization with rEf-PA0057 and challenge with PA01
A：Sera IgG detection in mice following immunization with rEf-PA0057 and challenge with PA01；B：Sera IgG1 detection in mice following immunization with rEf-PA0057 and challenge with PA01；C：Sera IgG2b detection in mice following immunization with rEf-PA0057 and challenge with PA01；D：Sera IgG3 detection in mice following immunization with rEf-PA0057 and challenge with PA01；E：Sera IgE detection in mice following immunization with rEf-PA0057 and challenge with PA01；a：before immunization；b：4 weeks after first immunization；c：2 weeks after challenge；**P0.01 vs before immunization；##P0.01 vs 4 weeks after first immunization.
Li Wengui, Ou Xingkun, He Ailin. Construction of Enterococcus faecium -based recombinant Ef-PA0057 vaccine of Pseudomonas aeruginosa andits protection in mice[J]. Acta Universitatis Medicinalis Anhui, 2026, 61(03): 518-523.