Development and Evaluation of a MAb-Based ELISA for Detection of Chlamy- dophila pneumoniae Infection with Variable Domain 2 and 3 of the Major Outer Membrane protein

Objective This paper aims to develop a monoclonal antibodies （MAbs）- based ELISA for detecting Chlamydophila pneumoniae （C. pneumonioe） antigens in humans with the variable domains （VD） 2 and 3 of the major outer membrane protein （MOMPvD2-VD~） and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. Methods MOMPvo2-vo3were overexpressed in Escherichia coil and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I （156 patients with typical respiratory illness clinically confirmed before） and Group II （57 healthy donors）. Results In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II ＂healthy donors＂, 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. Conclusion The novel MOMPvD2.VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.

Study on Outer Membrane Protein Patterns of Escherichia coli O38,O53 and O75 Isolated from Chickens

[Objective] This study aimed to investigate the outer membrane protein （OMP） patterns of Escherichia coli 038, 053 and 075 isolates from chickens. [Method] Eight pathogenic E. coil isolates with various serotypes were used as experimental materials to extract OMP by using supersonic schizolysis method and Sarcosyl. After SDS-PAGE electrophoresis, OMP patterns of the extracted products were determined based on the OMP model diagram. [Result] OMP of eight E. coil isolates with three serotypes were divided into three patterns, to be specific, 2 075 isolates respectively belonged to OMP-I and OMP-II pattern, 1 053 isolate belonged to OMP-II pattern, and 5 038 isolates belonged to OMP-I and OMP-III pattern. [Conclusion] Experimental results showed that E. coli isolates with the same serotype may belong to completely different OMP patterns, while serologically unrelated isolates may belong to the same OMP pattern. OMP of E. coil isolates with the same serotype may generate genetic differentiation; in addition, OMP of E. coli isolates with different serotypes may have different genetic correlation.

Prokaryotic Expression and Identification of Outer Membrane Protein 2 of Chlamydia trachomatis

Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 X His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coli M15, providing an efficient and simple system for assaying the immunological properties of Omp2.

Antimicrobial Susceptibility and Characterization of Outer Membrane Proteins of Aeromonas hydrophila Isolated in China

Aeromonas hydrophila isolates from clinical cases （n=43） were tested against 8 antimicrobial agents and typed by outer membrane protein （OMP） pattern by using sodium dodecyl sulfate gel electrophoresis. All isolates were resistant to ampicillin （MICs, ≥16 μg mL-1） and sulfamonomethoxine （MICs≥64 μg mLl）, but susceptible to norfloxacin （MICs,≤0.5 μg mL-1）. There was a high incidence of resistance to erythromycin （90.70%） and tylosin （93.02%）, while a low incidences of resistance to ciprofloxacin （2.33%）, enrofloxacin （2.33%） and florfenicol （4.65%）. Six different outer membrane protein patterns were found among 34 isolates by analyzing proteins in the range of 22 to 50 kDa, other than 9 isolates with their respective profiles. The strains with the similar OMP profiles had similar resistances. Compared with the other strains from the same OMP patterns, NB-1, A.Pun and MR-1 had lacked the proteins in the range of 30 to 45 kDa and their resistance to florfenicol substantially increased. It is speculated that the outer membrane protein changes might correlate with decreased susceptibility to florfenicol in the three strains. Some strains which showed completely identical OMP types had a little difference in their resistance to fluoroquinolones, indicating that there might be other factors that were involved in the antimicrobial resistance of A. hydrophila.

Determination of the genus-specific antigens in outer membrane proteins from the strains of Leptospira interrogans and Leptospira biflexa with different virulence

Objective:To determine the existence of genus-specific antigens in outer membrane proteins (OMPs) of leptospira with different virulence. Methods: Microscope agglutination test (MAT) was applied to detect the agglutination between commercial rabbit antiserum against leptospiral genus-specific TR/Patoc I antigen and 17 strains of Leptospira interrongans belonging to 15 serogroups and 2 strains of Leptospira biflexa belonging to 2 serogroups.The outer envelopes (OEs) of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain lai (56601) with strong virulence and serogroup Pomona serovar pomona strain Luo (56608) with low virulence,and L.biflexa serogroup Semaranga serovar patoc strain Patoc I without virulence were prepared by using the method reported in Auran et al.(1972).OMPs in the OEs were obtained by treatment with sodium deoxycholate. SDS-PAGE and western blot were used for analyzing the features of the OMPs on electrophoretic pattern and the immunoreactivity to the antiserum against TR/Patoc I antigen, respectively. Results:All the tested strains belonging to different leptospiral serogroups agglutinated to the antiserum against leptospiral genus-specific TR/Patoc I antigen with agglutination titers ranging from 1:256-1:512. A similar SDS-PAGE pattern of the OMPs from the three strains of leptospira with different virulence was shown and the molecular weight of a major protein fragment in the OMPs was found to be approximately 60 KDa.A positive protein fragment with approximately 32 KDa confirmed by Western blot,was able to react with the antiserum against leptospiral genus-specific TR/Patoc I antigen, and was found in each the OMPs of the three stains of leptospira.Conclusion: There are genus-specific antigens on the surface of L.interrogans and L.biflexa. The OMP with molecular weight of 32 KDa may be one of the genus-specific protein antigens of leptospira.

Immunogenicity and protective role of antigenic regions from five outer membrane proteins of Flavobacterium columnare in grass carp Ctenopharyngodon idella

Flavobacterium columnare causes columnaris disease in freshwater fi sh. In the present study, the antigenic regions of fi ve outer membrane proteins(OMPs), including zinc metalloprotease, prolyl oligopeptidase, thermolysin, collagenase and chondroitin AC lyase, were bioinformatically analyzed, fused together, and then expressed as a recombinant fusion protein in Escherichia coli. The expressed protein of 95.6 k Da, as estimated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was consistent with the molecular weight deduced from the amino acid sequence. The purifi ed recombinant protein was used to vaccinate the grass carp, C tenopharyngodon idella. Following vaccination of the fi sh their Ig M antibody levels were examined, as was the expression of I g M, Ig D and Ig Z immunoglobulin genes and other genes such as MHC Iα and MHC I I β, which are also involved in adaptive immunity. Interleukin genes( IL), including I L- 1β, IL- 8 and I L- 10, and type I and type II interferon(I FN) genes were also examined. At 3 and 4 weeks post-vaccination(wpv), signifi cant increases in Ig M antibody levels were observed in the fi sh vaccinated with the recombinant fusion protein, and an increase in the expression levels of I g M, Ig D and Ig Z genes was also detected following the vaccinations, thus indicating that an adaptive immune response was induced by the vaccinations. Early increases in the expression levels of IL and IFN genes were also observed in the vaccinated fi sh. At four wpv, the fi sh were challenged with F. column a re, and the vaccinated fi sh showed a good level of protection against this pathogen, with 39% relative percent survival(RPS) compared with the control group. It can be concluded, therefore, that the fi ve OMPs, in the form of a recombinant fusion protein vaccine, induced an immune response in fi sh and protection against F. columnare.

Immunoproteomic Analysis of Bordetella bronchiseptica Outer Membrane Proteins and Identification of New Immunogenic Proteins

Bordetella bronchiseptica is a Gram-negative pathogen that causes acute and chronic respiratory infection in a variety of animals. To identify useful antigen candidates for diagnosis and subunit vaccine of B. bronchiseptica, immunoproteomic analysis was adopted to analyse outer membrane proteins of it. The outer membrane proteins extracted from B. bronchiseptica were separated by two-dimensional gel electrophoresis and analyzed by Western blotting for their reactivity with the convalescent serum against two strains. Immunogenic proteins were identified by matrix-assisted laser desorption/ionization time of flight-mass spectrometry（MALDI-TOF-MS）, a total of 14 proteins are common immunoreactive proteins, of which 1 was known antigen and 13 were novel immunogenic proteins for B. bronchiseptica. Putative lipoprotein gene was cloned and recombinantly expressed. The recombinant protein induced high titer antibody, but showed low protective indices against challenges with HB（B. bronchiseptica strain isolated from a infected rabbit）. The mortality of mice was 80% compared to 100% of positive controls. The identification of these novel antigenic proteins is an important resource for further development of a new diagnostic test and vaccine for B. bronchiseptica.

Gene cloning and prokaryotic expression of recombinant outer membrane protein from Vibrio parahaemolyticus

Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals, The outer membrane protein of bacteria plays an important role in the infection and pathogenicity to the host. Thus, the outer membrane proteins are an ideal target for vaccines. We amplified a complete outer membrane protein gene （ompW） from E parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 （DE3） cells. The gene coded for a protein that was 42.78 kDa. We purified the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for future application of the OmpW protein as a vaccine candidate against infection by E parahaemolyticus. In addition, the purified OmpW protein can be used for further functional and structural studies.

Research progress on Helicobacter pyloriouter membrane protein

Helicobacter pylori (H pylori), one of the most common bacterial pathogens on human beings, colonizes the gastric mucosa. In its 95 paralogous gene families, there is a large outer membrane protein (OMP) family. It includes 32 members. These OMP are important for the diagnosis, protective immunity, pathogenicity of H pylori and so on. They are significantly associated with high H pylori density,the damage of gastric mucosa, high mucosal IL-8 levels and severe neutrophil infiltration. We introduce their research progress on pathogenicity.

Recombinant outer membrane protein F-B subunit of LT protein as a prophylactic measure against Pseudomonas aeruginosa burn infection in mice

AIM: To study immunogenicity of outer membrane protein F(Opr F) fused with B subunit of LT(LTB), against Pseudomonas aeruginosa(P. aeruginosa). METHODS: The Opr F, a major surface exposed outer membrane protein that is antigenically conserved in various strains of P. aeruginosa, is a promising immunogen against P. aeruginosa. In the present study recombinant Opr F and Opr F-LTB fusion gene was cloned, expressed and purified. BALB/c mice and rabbits were immunized using recombinant Opr F and Opr F-LTB and challenged at the burn site with P. aeruginosa lethal dose of 104 CFU. The protective efficacy of rabbit anti Opr F Ig G against P. aeruginosa burn infection was investigated by passive immunization. RESULTS: It has been well established that the LTB is a powerful immunomodulator with strong adjuvant activity. LTB as a bacterial adjuvant enhanced immunogenicity of Opr F and anti Opr F Ig G titer in serum was increased. Experimental findings showed significantly higher average survival rate in burned mice immunized with Opr F-LTB than immunized with Opr F or the control group. Rabbits anti Opr F Ig G brought about 75% survival of mice following challenge with P. aeruginosa. Post challenge hepatic and splenic tissues of mice group immunized with Opr F-LTB had significantly lower bacterial load than those immunized with Opr F or the control groups. CONCLUSION: These results demonstrate that LTBfused Opr F might be a potential candidate protein for a prophylactic measure against P. aeruginosa in burn infection.

30 and 32 kDa outer membrane proteins of Bordetella pertussis as a modulator on promoting degranulation of mast cells

The correlation between the activities of the outer menbrane proteins （OMPs） of Bordetella pertussis and the lgE-mediated asthma was investigated in the present study, in which the OMPs of B. pertussis and their components were prepared by detergent treatment and chromatography, and the molecular weights of the OMPs components were determined by SDS-PAGE. The amounts of total as well as the ovalbumin （OVA）-specific IgE induced by dead B. pertussis whole bacterial vaccine on guinea pigs were detected by ELISA. Meanwhile, the effect of the OMPs and their components to promote the degranulation of guinea pig mast cells was observed by using the mast cell degranulation test, and ELISA assay was used to measure the histatmine levels in the supematants from the mast cell cultures. Histamine sensitive test was used to demonstrate the effects of the OMPs and their components to increase the histamine lethal sensitivity in mice. It was found that four components with molecular weights of 30, 32, 38 and 69 kDa could be obtained from the OMPs of B. pertussts, and the dead whole bacteria vaccine of B. pertussis had the ability to increase the levels of the total as well as the OVA-specific IgE in sera of guinea pigs. The OMPs and their 30 and 32 kDa components demonstrated significantly enhancing effect on the degranulation of guinea pig mast cells, and the histamine levels in the supematants from the mast cell culture treated with OMPs and their 30 and 32 kDa components were also significantly increased. It is evident that the strong adjuvant activity and the enhancing effect to degranulation of mast cells and the release of histamine of certain outer membrane components of B. pertussis could be demonstrated as revealed by the results of the present study, suggesting the possibility of a close relationship between the infection of vaccination with B. pertussis and the IgE-mediated asthma.

SDS-PAGE analysis of whole cell protein and outer memrbane protein patterns of clinical isolates of Burkholderia pseudomallei

Objective:To investigate the banding patterns of whole cell protein(WCP) and outer membrane protein (OMP) of Burkholderia pseudomallei(B.pseudomallei) in clinical isolates from patients with melioidosis. Methods:WCP and OMP of of B.pseudomallei in 50 clinical isolates,from 47 patients with melioidosis were prepared and separated by polyacrylamide gel electrophoresis(SDS-PAGE) using 10%gels and stained with Coomassie brilliant blue.The banding patterns were compared by using a laser densitometer and dendrogram. Results:There were 6 different banding patterns of WCP and 2 types of OMP.Type 1 -5 WCP had 8 common protein bands at 19.0 - 45.0 kDa with identical OMP pattern.The banding patterns of WCP in type 6 were distinct from the others and also its OMP profile.The majority of clinical isolates(37/50,74%) were in type 1 WCP.Of the remaining isolates,8 were in type 2,2 in type 3,and one each was in type 4 to 6.There was no significant association between the WCP typing and the demographic or clinical features of the investigated patients.Conclusion:Despite the wide variation of clinical features of melioidosis,the results of this study show that B.pseudomallei had a few differences in the WCP and OMP profiles.Therefore typing of WCP and OMP,using SDS-PAGE analysis,could be an alternative method for phenotypic differentiation in clinical isolates of B.pseudomallei.

鲍曼不动杆菌OmpA蛋白的生物学特性分析

目的提取临床分离的鲍曼不动杆菌外膜蛋白A(OmpA)的基因,并进行生物学预测和分析。方法查询鲍曼不动杆菌OmpA基因序列,设计特异性PCR引物,以提取的鲍曼不动杆菌基因组DNA为模板,PCR扩增OmpA片段。回收目的片段,采用生物信息学软件分析OmpA蛋白的生物学特性。结果12株鲍曼不动杆菌多位点序列分型(MLST)序列分析有6个ST分型,分别是ST 208、ST 229、ST 191、ST 195、ST540、ST 1145,鲍曼不动杆菌OmpA基因序列全长为1070 bp的,单核苷酸多态性(SNP)位点较少,预测蛋白为跨膜蛋白,具有6种三级结构,均由β-桶状结构组成的保守结构域。结论临床分离的不同鲍曼不动杆菌株OmpA蛋白为结构相对保守的跨膜蛋白,具有维持细胞的形状和稳定性,参与细菌耐药机制及免疫原性作用。

E型沙眼衣原体MOMP基因重组腺病毒的构建及免疫原性研究

目的:构建E型沙眼衣原体(Ct)主要外膜蛋白(MOMP)基因重组腺病毒,为沙眼衣原体腺病毒疫苗的研究奠定基础。方法:根据Genebank中E型Ct MOMP基因序列设计引物,用高保真PCR方法从E型Ct基因组DNA中扩增得到MOMP基因片段,克隆至pcDNAII载体,测序后连接入腺病毒穿梭载体pDC316。穿梭载体pDC316-MOMP与含腺病毒基因组的辅助质粒PBHGlox△E1,3Cre共转染至HEK293细胞,在Cre-loxP重组酶作用下进行重组,包装成重组腺病毒颗粒,用PCR和RT-PCR方法进行鉴定。并用动物免疫试验检测重组腺病毒的免疫原性。结果:从E型Ct基因组DNA中扩增出约1.1 kb的特异MOMP基因片段,酶切鉴定及DNA序列测定证实穿梭载体pDC316-MOMP构建正确。穿梭载体pDC316-MOMP与含腺病毒基因组的辅助质粒PBHGlox△E1,3Cre共转染至293细胞,出现明显细胞病变效应。收集重组腺病毒,PCR法证实重组腺病毒含有MOMP基因,RT-PCR证实重组腺病毒在293细胞能表达MOMP基因。重组腺病毒免疫小鼠可诱导特异性抗体产生,证明重组腺病毒具有良好免疫原性。结论:成功构建了E型沙眼衣原体MOMP基因重组腺病毒,该重组腺病毒可诱导小鼠产生特异性抗体。

基于不同抗原的犬布鲁氏菌病免疫层析胶体金抗体试纸条的制备和比较

为建立针对犬布鲁氏菌病的免疫层析胶体金快速检测方法,利用从犬种布鲁氏菌菌株提纯的膜蛋白和可溶性蛋白,原核表达纯化的外膜蛋白Omp31和BP26以及基于B细胞抗原肽的融合蛋白F14,分别制备了试纸条,然后采用犬布鲁氏菌病阳性和阴性血清,分别对上述试纸条进行敏感性和特异性比较。结果显示:Omp31抗原的敏感性最高,其次是F14和BP26,而全菌膜蛋白和全菌可溶性蛋白的敏感性较差;特异性上,使用由5种抗原制备的胶体金试纸条检测30份犬布鲁氏菌病阴性血清,结果均为阴性;5种试纸条与沙门氏菌、大肠杆菌(O157)、大肠杆菌(H7)、霍乱弧菌、副溶血弧菌、军团菌阳性血清均不发生交叉反应。另外,由F14和BP26制备的试纸条可识别羊布鲁氏菌病阳性血清,而由Omp31、全菌膜蛋白和全菌可溶性蛋白制备的试纸条均不能识别牛、羊布鲁氏菌病阳性血清。综上所述,Omp31、F14和BP26可作为制备犬布鲁氏菌病免疫层析胶体金检测卡的备选抗原。本研究为粗糙型布鲁氏菌引起的布鲁氏菌病的诊断提供了技术支撑。

猪增生性肠病间接ELISA抗体检测试剂盒的研制与应用

【目的】研制一种检测胞内劳森菌(Lawsonia intracellularis,LI)抗体的间接ELISA试剂盒,为LI的监测和疫苗评价提供工具。【方法】表达并纯化LI的外膜蛋白Omp2,以其为包被抗原建立检测LI抗体的间接ELISA方法,优化ELISA反应条件;用建立的ELISA方法检测稀释的LI阳性血清来评价试剂盒的敏感性,检测其他病原的阳性血清来评价试剂盒的特异性;试制3批试剂盒,制定试剂盒的敏感性和特异性检验标准,测定试剂盒在4℃的保存期;用试制的试剂盒检测LI阴性和阳性质控血清,研究试剂盒的成立条件;通过对不同时间和猪场采集的1 000份临床猪血清样本的检测来评价该试剂盒的实用性;用本试剂盒与进口商品化试剂盒平行检测LI阳性和阴性血清各50份以评价试剂盒的符合率。【结果】成功表达并纯化了Omp2蛋白,蛋白纯度为90.31%,在Western blot试验中可以与LI阳性血清发生反应。ELISA最佳反应条件为:Omp2以200 ng/孔4℃包被12-16 h;血清1﹕50稀释,37℃孵育30 min;羊抗猪Ig G-HRP 1﹕20 000稀释,37℃孵育30 min;TMB底物37℃显色15 min,用0.5 mol·L^(-1)的硫酸终止反应。该试剂盒检测LI阳性血清的最高检出倍数为8,敏感性较好;试剂盒检测E.coli、App、Mhp、SS2、PRRSV、PRV的结果均为阴性,特异性较好。制定了试剂盒的敏感性和特异性检验标准。试剂盒在4℃可保存15个月。试剂盒的成立条件为:阳性标准血清OD_(450nm)≥1.25,阴性标准血清OD_(450nm)<0.3。试制的试剂盒批内、批间重复性试验变异系数均小于10%。与国外商品化ELISA检测试剂盒对比的符合率达86%。用试制的ELISA试剂盒检测1 000份华东部分地区临床猪血清样品,其中LI阳性检出率为59.90%,表明LI在华东地区猪群中普遍存在。【结论】本研究开发的LI间接ELISA抗体检测试剂盒特异性高,灵敏度高,与商品试剂盒符合率高,可用于临床LI抗体的检测。

幽门螺杆菌东亚型菌株GZ7/cagA^(+)和GZ7/ΔcagA源外膜囊泡的蛋白组学比较

目的通过分离、鉴定和比较细胞毒素相关基因A蛋白(cagA)、阳性幽门螺杆菌(H.pylori)、东亚型菌株GZ7/cagA^(+)及其cagA敲除菌株GZ7/ΔcagA来源的外膜囊泡(OMVs)中的差异表达蛋白(DEPs),分析cagA基因对OMVs中蛋白表达的影响。方法采用超速离心法分别提取GZ7/ΔcagA和GZ7/cagA^(+)的OMVs,通过透射电镜和纳米颗粒追踪技术鉴定其形态和粒径,使用Western blot技术验证两组OMVs中cagA蛋白的表达,分析OMVs的蛋白质组学;对蛋白组学数据进行质控分析和主成分分析鉴定后,以上调蛋白倍数变化(FC)>2.0、下调蛋白FC<0.5,FDR≤0.05为筛选条件筛选DEPs,利用OmicsBean在线工具、Gene Ontology和KOBAS对DEPs进行生物信息学分析;采用免疫荧光鉴定OMVs细胞在细胞中的定位,实时无标记细胞分析仪检测细胞活性。结果通过电镜和粒径证实成功分离纯化了OMVs;蛋白质组分析发现,GZ7/cagA^(+)-OMVs组与GZ7/ΔcagA-OMVs组比较有79个DEPs,其中38个蛋白下调、41个蛋白上调;生物信息学分析显示,DEPs主要与丙酮酸代谢、丙酸代谢、糖酵解/糖异生及柠檬酸循环等代谢途径有关;免疫荧光和实时无标记细胞分析证实H.pylori来源的OMVs能进入细胞并定位在线粒体并抑制细胞增殖。结论cagA能影响H.pylori分泌的OMVs中蛋白质的成分,DEPs可能促进cagA^(+)H.pylori在胃黏膜上的定植及致病性。

沙眼衣原体D型MOMP基因的克隆和序列分析

目的 从D型沙眼衣原体培养物中克隆主要外膜蛋白(MOMP)基因。方法 利用细胞培养法扩增沙眼衣原体后,提取基因组为模板,经聚合酶链反应(PCR)扩增全长MOMP基因,并用酶切、PCR扩增及序列测定等方法对重组质粒进行鉴定。结果 成功扩增了MOMP基因,并插入克隆载体 pUCmT中。结论 MOMP基因的克隆为进一步开展沙眼衣原体的疫苗研究奠定了基础。

钩端螺旋体OmpR家族相关TCS 鉴定及功能初探

目的建立钩端螺旋体(钩体)OmpR家族双组分系统(TCS)的鉴定方法,初步探讨OmpR家族TCS在钩体感染人巨噬细胞过程中对钩体主要外膜蛋白(OMPs)表达水平的调控作用。方法采用生物信息学技术预测致病性钩体赖株TCS的组氨酸激酶(HK)和应答调节蛋白(RR)OmpR功能结构域。采用激酶活性检测试剂盒和细菌双杂交技术进一步验证各HK激酶活性以及各HK与RR的相互作用。采用qRT-PCR法检测HK抗血清封闭前后钩体主要OMPs基因mRNA水平的变化。结果钩体56601株中LA2828、LA1710含有HATPase_c激酶催化结构域,LA2827、LA1709含有Response_reg与OmpR功能结构域,提示其分别可构成HK/OmpR-TCS。体外重组HK-2828和HK-1710蛋白均具有激酶活性,且分别与OmpR-2827和OmpR-1709存在直接的相互作用。HK-2828抗体封闭处理可使感染过程中显著下降的外膜蛋白编码基因lipL32、lipL41和ompL1的mRNA水平明显回升。结论成功构建钩体OmpR家族-TCS鉴定及功能探究体系,为阐明感染过程中问号钩体OMPs表达改变调控机制,以及制定基因工程疫苗OMPs抗原筛选新策略提供依据。

梅毒螺旋体外膜蛋白功能的研究进展

梅毒是一种严重影响人类健康的慢性传播性疾病,是由梅毒螺旋体感染所引起。梅毒螺旋体外膜蛋白(Omp)是一类具有关键性功能的蛋白。Omp在梅毒螺旋体的免疫原性、黏附宿主细胞以及转运营养物质等方面具有非常重要的作用。本文就梅毒螺旋体主要外膜蛋白的结构、功能等特性做一综述。