【作者】 李蓉；

【导师】 刘鹏；

【作者基本信息】 南华大学 ， 基础医学（病原生物学）， 2025， 硕士

【摘要】 背景:微小脲原体(Ureaplasma parvum,Up)是一种缺乏细胞壁的机会致病菌,目前在临床上感染人群类型愈来愈广,其中育龄期妇女与孕妇检出率较高,可导致非淋菌性尿道炎、盆腔炎、早产及新生儿呼吸道感染等多种疾病。研究表明Up可附着于红细胞、人上皮细胞、He La细胞等多种细胞表面,病原体表面抗原成分有助于其感染与入侵宿主细胞,这些表面蛋白不仅参与病原体的黏附、侵袭和代谢过程,还在免疫逃逸和致病性方面发挥重要作用。目前对Up毒力因子的相关研究较少,尤其是黏附相关蛋白,并且尚无对抗原蛋白的系统性分析。目的:通过对Up抗原蛋白进行免疫蛋白组学分析,揭示有价值的潜在性候选抗原,为Up的治疗方案提供多个针对性靶点,同时在检测诊断中提供重要候选分子。从筛选出的抗原膜蛋白中挑取EF-Tu验证其黏附功能,为深入探究Up致病机制提供重要理论依据。方法:1.分离培养Up临床菌株,查阅文献获取可用来鉴定Up的管家基因Fts H序列,设计构建引物,在Up中克隆出相应基因片段测序鉴定,经NCBI blast比对后构建系统进化树;2.提取Up总蛋白、胞浆蛋白、膜蛋白,使用总蛋白免疫新西兰兔制备抗总蛋白多克隆抗体,同时使用未免疫前的兔阴性血清作为对照,利用免疫沉淀实验洗脱三组蛋白中的抗原成分,并进行银染分析;3.质谱鉴定三组洗脱液中的抗原成分,对所有抗原成分进行生信分析,分析鉴定结果后挑选出膜蛋白中相应抗原成分;4.从挑选出的结果中选取EF-Tu基因序列,构建pET-30a/EF-Tu质粒,通过原核系统表达获得蛋白EF-Tu;采用Ni-NTA纯化出EF-Tu;5.使用EF-Tu蛋白免疫新西兰兔,制备抗EF-Tu的多克隆抗体;通过Western blot检测EF-Tu在Up上的亚定位;6.验证EF-Tu黏附功能,检测不同浓度的EF-Tu与Up对SV-HUC-1细胞的黏附能力;EF-Tu抗体抑制Up黏附SV-HUC-1的能力;以及EF-Tu与Up共同竞争黏附SV-HUC-1的水平;结果:1.NCBI blast比对分离出的临床菌株序列后,发现该菌株与CP021987.1:497528-497922 Ureaplasma parvum strain hebnu3h04亲缘关系较近,将分离菌株命名为Ureaplasma parvum strain hyzyy 23。2.银染分析结果显示,实验组各泳道在分子量60kD与100kD处的蛋白条带要多于对照组,表明有差异蛋白的存在。经免疫蛋白组学鉴定后,筛选出Up抗原膜蛋白成分主要有多重带状抗原(Multiple banded antigen)、延伸因子(Elongation factor Tu)、脲酶类蛋白(Urease subunit alpha、Urease complex component Ure G)、细胞分裂蛋白(Cell division protein Fts H)、分子伴侣蛋白(Chaperone protein Dna K)及Ⅲ型限制性内切酶Res亚基家族(Type III restriction enzyme,res subunit family),挑取EF-Tu验证其对SV-HUC-1细胞的黏附功能。3.SDS-PAGE和Western blot结果显示成功表达并纯化出EF-Tu;免疫新西兰兔4次后,ELISA检测抗EF-Tu血清效价达到1280000,表明成功制备EF-Tu抗血清;Western blot结果显示EF-Tu分布在Up的胞膜和胞浆中,这与我们抗原组学鉴定结果中,胞浆与胞膜抗原中均存在EF-Tu结果相一致。4.间接免疫荧光结果显示:EF-Tu和Up均具有黏附SV-HUC-1的能力;EF-Tu抗体能抑制部分Up对SV-HUC-1的黏附;同时EF-Tu和Up对SV-HUC-1黏附功能存在竞争结合。结论:1.经鉴定,Up抗原膜蛋白成分主要有多重带状抗原(Multiple banded antigen)、延伸因子(Elongation factor Tu)、脲酶系列(Urease subunit alpha、Urease complex component Ure G)、细胞分裂蛋白(Cell division protein Fts H)、分子伴侣蛋白(Chaperone protein Dna K)、Ⅲ型限制性内切酶Res亚基家族(Type III restriction enzyme,res subunit family),这些抗原成分在Up入侵宿主细胞及感染过程中均有重要作用。2.EF-Tu作为一个多功能兼职蛋白,还可能介导Up的黏附过程。更多还原

【Abstract】 Background:Ureaplasma parvum(Up),an opportunistic pathogen characterized by the absence of a cell wall,has been increasingly identified in clinical populations,particularly among women of childbearing age and pregnant women.It is implicated in a range of diseases,including non-gonococcal urethritis,pelvic inflammatory disease,preterm labor,and neonatal respiratory infections.Research indicates that Up can adhere to the surfaces of various cell types,such as red blood cells,human epithelial cells,and He La cells.The surface antigens of this pathogen are pivotal in the infection and invasion of host cells,these surface proteins are integral not only to the processes of adhesion,invasion,and metabolism of the pathogen but also to immune evasion and pathogenicity.Presently,research on Up virulence factors,particularly adhesion-related proteins,remains limited,and there has been no comprehensive analysis of antigenic proteins.Object:The identification of valuable candidate antigens through immunoproteomics analysis of Up presents significant molecular candidates to provide multiple targeted targets for the treatment of Up,and provide multiple targeted targets for the treatment plan of Up,at the same time offering important candidate molecules in detection and diagnosis.Furthermore,we aimed to verify the adhesive function of EF-Tu,selected from the identified antigenic membrane proteins,to provide a critical theoretical foundation for exploring the pathogenic mechanisms of Up.Methods:1.Clinical strains were isolated and cultured.The housekeeping gene fts H sequence used for Up identification was obtained from the literature,and primers were designed and constructed.The corresponding sequence was cloned and sequenced in Up,and a phylogenetic tree was constructed following NCBI blast alignment.2.Total protein,cytoplasmic protein,and membrane proteins of Up were extracted.Polyclonal antibodies against total protein were prepared by immunizing New Zealand rabbits with total protein;pre-immune rabbit serum served as a negative control.Antigen components from the three fractions were eluted by immunoprecipitation and analyzed by silver staining.3.The antigenic components in the eluates were identified using mass spectrometry,and bioinformatics analysis was performed on all antigenic components.Antigenic components in the membrane protein fraction were selected based on the results of the analysis.4.The EF-Tu gene sequence was selected from the results,and the pET-30a/EF-Tu plasmid was constructed.The protein EF-Tu was expressed using a prokaryotic system and purified using Ni-NTA.5.Polyclonal antibodies against EF-Tu were prepared by immunizing New Zealand rabbits with the EF-Tu protein,and the subcellular localization of EF-Tu in Up was detected by western blotting.6.The adhesive function of EF-Tu was verified,The adhesion ability of different concentrations of EF-Tu and Up to SV-HUC-1 cells was assessed.The ability of EF-Tu antibodies to inhibit Up adhesion to SV-HUC-1 and the competitive adhesion between EF-Tu and Up to SV-HUC-1 was also evaluated.Results:1.The NCBI blast alignment of the clinical strain sequences demonstrated a close phylogenetic relationship with CP021987.1:497528-497922 Ureaplasma parvum strain hebnu3h04.Therefore,the strain was designated as Ureaplasma parvum strain hyzyy 23.2.Silver staining analysis revealed that the experimental group lanes exhibited more protein bands at molecular weights of 60 kDa and 100 kDa compared to the control group,suggesting the presence of differential proteins.Immunoproteomics identified Up antigenic membrane protein components,including Multiple banded antigen,Elongation factor,Urease-related proteins,Cell division protein,Chaperone protein,and the Type III restriction endonuclease Res subfamily.EF-Tu,recognized as a multifunctional protein,was highlighted for further investigation.3.SDS-PAGE and Western blotting results confirmed the successful expression and purification of EF-Tu.Following four immunizations,the titer of anti-EF-Tu serum reached 1,280,000,indicating the successful preparation of anti-EF-Tu serum.Western blotting results demonstrated that EF-Tu was distributed in both the cytoplasm and membrane of Up,consistent with the immunoproteomics results.4.The indirect immunofluorescence results demonstrated that both EF-Tu and Up could adhere to SV-HUC-1 cells.Anti-EF-Tu antibodies partially inhibited Up adhesion to SV-HUC-1,and a competitive relationship was observed between EF-Tu and Up in adhering to SV-HUC-1 cells.Conclusions:1.The major antigenic membrane protein components of Up were identified as Multiple banded antigen,Elongation factor,Urease-related proteins,Cell division protein,Chaperone protein dna K,and the Type III restriction endonuclease Res subfamily.These antigens play significant roles in Up invasion of host cells and infection process.2.EF-Tu,as a multifunctional protein,may also mediate the adhesion process of Up.更多还原