梅毒螺旋体Tp0751黏附蛋白的表达、纯化及免疫活性研究

目的 表达梅毒螺旋体黏附蛋白Tp0751,纯化表达产物并进行免疫活性分析,为探索Tp0751重组蛋白在梅毒致病过程中的作用奠定基础.方法 通过生物信息学分析,去除Tp0751信号肽序列,构建原核表达体进行诱导表达;Ni亲和层析柱纯化重组蛋白,Western blot检测其免疫反应性,用重组蛋白免疫新西兰家兔,评价其免疫原性.结果 成功构建了pET-28a(+)-0751原核表达载体,经表达、纯化后获得了相对分子质量约为26×103的融合蛋白;Western blot检测其能与梅毒患者阳性血清发生特异性反应;利用纯化的Tp0751重组蛋白免疫新西兰家兔,能诱导家兔产生特异性免疫应答,ELISA法测定免疫血清中特异性抗体滴度在1∶10 240以上.结论 重组表达的Tp0751黏附蛋白具有良好的免疫活性,为进一步研究其在梅毒致病过程中的作用和生物学功能奠定了基础.     

The Treponema pallidum outer membrane protein Tp92 activates endothelial cells via the chemerin/CMKLR1 pathway

Endothelium damage caused by Treponema pallidum is the key step in the systemic dissemination and pathophysiology of syphilis, particularly cardiovascular syphilis and neurosyphilis. However, the molecular mechanisms supporting endothelium damage of syphilis are undefined. The outer membrane proteins were thought to be involved. Tp92 was first identified as an outer membrane protein of T. pallidum. Homologous proteins to Tp92 play important roles in cell attachment, inflammation, and tissue destruction in other bacterial species. In this study, we investigated the effect of Tp92 on endothelial cells activation. The data showed that Tp92 induced chemerin production in activated endothelial cells. Endothelial cell-derived chemerin upregulated the expression of TNF-α and ICAM-1 in endothelial cells via CMKLR1. In addition, endothelial cell-derived chemerin promoted THP-1-derived macrophage migration towards endothelial cells. These findings suggest that Tp92 may play an important role in mediating endothelial cell activation by inducing the secretion of chemerin.     

梅毒螺旋体Tp0136活性肽段的可溶性表达、纯化及鉴定

目的 筛选梅毒螺旋体特异性抗原Tp0136的活性肽段,可溶性表达和纯化该肽段,并鉴定其免疫活性,探索Tp0136活性肽段在早期梅毒诊断中的价值.方法 通过生物信息学方法对Tp0136亲水性、B细胞表位和二级结构等进行分析,筛选出Tp0136活性肽段(Tp0136B)替代全蛋白.将Tp0136B基因插入到pET22b(+)上,在E.coli BL21中表达.镍离子亲和色谱纯化表达产物,Western blot检测其免疫反应性,免疫日本大耳白兔评价其免疫原性,免疫双扩检测其效价,以重组Tp0136B蛋白为包被抗原的间接ELISA检测早期梅毒血清抗体.结果 重组工程菌可溶性表达相对分子质量约为28×103的rTp0136B,表达率为21%,制备得到纯度大于98%的rTp0136B.纯化的rTp0136B能诱导大耳白兔产生特异性免疫应答,免疫双扩测得其效价为1:16.Western blot检测重组蛋白能与兔抗Tp0136多克隆抗体发生特异性反应.间接ELISA检测正常人血清均为阴性,而早期梅毒血清抗体的阳性率为85.5%.结论 重组表达的Tp0136活性肽段具有良好的免疫活性,预示其在早期梅毒血清学诊断中具有良好的前景.

Abstract：

Objective To express and purify recombinant Tp0136 epitope fragment, and study the immunity activity. Methods The Tp0136 selective fragment(Tp0136B) gene was devised by the surface property analysis, solvent-accessible suface calculateions, secondary structure function region analysis, and was inserted between the sites of Nde Ⅰ and Not Ⅰ in pET22b ( + ) . The recombinant plasmid was expressed in E. coli BI21. After nickel ion metal affinity chromatography, the antigenic and immune reactivity of rTp0136B was confirmed. Then indirect ELISA with the rTp0136B as coating antigen was performed to detect the anti-Tp0136 antibody in sera from 100 normal human controls and 131 primary syphilis patients. Results The rTp0136B was soluble expressed with a molecular weight of about 28 000 and was obtained with a purity of ＞98% by chromatography. Western blot proved that the rTp0136B could specifically react with anti-Tp0136 polyclonal antibody. Specific humoral response was elicited by the recombinant protein in Japan negative. The positive detection rate in sera from primary syphilis patients was 85.5%. Conclusion This result suggested that the recombinant Tp0136 epitope fragments have a satisfactory immunocompetence,which may have applications in the serodiagnosis of primary syphilis.     

Construction of the Eukaryotic Expression Vector for Outer Membrane Protein Tp92 from Treponema pallidum and Its Preliminary Study on the Immune Responses in New Zealand Rabbits

Treponema pallidum subsp. pallidum is the causative a gents of syphilis. Presently, the annual infection rate ofdomestic and international syphilis remains rather high [1,2]. Apart from the serious nature of the disease itself, anumber of studies sugg…     

重组梅毒螺旋体蛋白Tp47通过诱导uPA增加血管内皮细胞通透性

目的探讨重组梅毒螺旋体蛋白Tp47(rTpp47)对单核-巨噬细胞系THP-1合成尿激酶型纤溶酶原激活物(uPA)的调控及其对人脐静脉/血管内皮细胞(HUVECs)通透性的影响。方法用rTpp47刺激THP-1细胞24 h后,分别收集细胞培养上清和THP-1细胞,用ELISA和Western blot检测THP-1细胞表达的uPA含量;用THP-1细胞培养上清刺激人单层血管内皮细胞后,使用FITC-葡聚糖评价单层内皮细胞通透性的变化,用Western blot检测uPA对HUVECs细胞紧密连接蛋白claudin-5表达的影响以及PKC信号通路是否参与rTpp47诱导的THP-1细胞表达uPA。结果重组蛋白rTpp47刺激THP-1细胞合成和分泌的uPA显著高于对照组(P<0.05,P<0.001);用rTpp47与THP-1细胞共培养24 h后收集的细胞培养上清刺激单层血管内皮细胞12和24 h,实验组血管内皮细胞相对通透性显著高于对照组(P<0.05,P<0.000 1);uPA活性抑制剂阿米洛利(amiloride)抑制了rTpp47刺激THP-1细胞分泌...     

Eukaryotic expression of outer membrane protein Gpd from Treponema pallidum and preliminary studies on its immune response in rabbits

Treponema pallidum(Tp) ,the causative agent ofsyphilis,causes a chronic ,lifelong infection withpossible serious complications if untreated. Re-cently,the annual incidence of syphilis has beenkeeping high at home and abroad [1 ,2] . Apartfromthe serious nature of the disease itself , anumber of studies suggest that syphilis infectionmayincrease the risk of acquisition and transmis-sionof human immunodeficiency virus(HIV) [3 ,4] ,so syphilis still remains a public health con-cern worldwide . …     

梅毒螺旋体抗原基因的克隆、表达及其在临床检验中的应用

目的　克隆、表达并纯化梅毒螺旋体相对分子质量为 47× 10 3 抗原 ,并检测梅毒患者的血清标本。方法　应用基因扩增技术分离此蛋白的全长基因 ,采用基因工程的方法 ,应用融合表达载体pGEX 2T ,重组、克隆得到带有梅毒螺旋体 47× 10 3 特异性抗原基因的重组菌株 ,经大肠杆菌表达系统获得重组融合蛋白。再经亲和层析获得纯品 ,并联合应用ELISA检测梅毒患者的血清标本 30份 ,同时检测非梅毒患者的血清标本 30份。结果　纯化后获得了梅毒螺旋体相对分子质量为 47× 10 3 的特异性抗原。ELISA检测梅毒患者血清结果均阳性。非梅毒患者血清均阴性 ,无非特异性交叉反应。结论　此项方法具有操作简便、准确的特点 ,为临床检测梅毒开辟了新的领域     

梅毒螺旋体TpN15抗原基因的扩增、克隆及表达

目的用基因工程技术表达梅毒螺旋体TpN15重组抗原,为进一步研制梅毒预防性疫苗和诊断试剂盒打下基础.方法采用PCR技术扩增梅毒螺旋体TpN15基因,然后克隆到原核表达载体pET28b中表达TpN15重组抗原蛋白.结果成功的构建了pET28b-TpN15重组表达载体,重组的TpN15蛋白在大肠杆菌BL21中得到稳定表达.结论梅毒螺旋体TpN15基因在大肠杆菌中的成功表达为建立梅毒血清学诊断的新方法奠定了基础.     

Serodiagnosis of syphilis: antibodies to recombinant Tp0453, Tp92, and Gpd proteins are sensitive and specific indicators of infection by Treponema pallidum

Syphilis serodiagnosis relies on a combination of nonspecific screening tests (antilipoidal antibodies) and Treponema pallidum-specific tests (anti-T. pallidum antibodies). We studied a group of six recombinant T. pallidum antigens for their sensitivities and specificities with sera from individuals with syphilis (n = 43), relapsing fever (n = 8), Lyme disease (n = 8), and leptospirosis (n = 9) and from uninfected individuals (n = 15). Three recombinant proteins, Tp0155, Tp0483, and Tp0751, demonstrated sensitivity values that ranged from 28 to 42%. In contrast, three other recombinant proteins exhibited the following sensitivity and specificity values: Tp0453, 100% sensitivity and 100% specificity; Tp92 (Tp0326), 98% sensitivity and 97% specificity; and Gpd (Tp0257), 91% sensitivity and 93% specificity. Tp0453, Tp92, and Gpd also were recognized by sera from individuals with early primary syphilis that were nonreactive with the antilipoidal Venereal Disease Research Laboratory test. The reactivities of syphilis patient sera with Tp0453, Tp92, and Gpd were proportional to the titers of these sera with the treponemal test MHA-TP (microhemagglutination assay for T. pallidum). Thus, the recombinant T. pallidum antigens Tp0453, Tp92, and Gpd show promise as diagnostic antigens in the enzyme-linked immunosorbent assay-based assay.     

Identification and purification of a recombinant Treponema pallidum basic membrane protein antigen expressed in Escherichia coli.

A recombinant plasmid designated pLVS3 previously was described that harbored a 14-kilobase insert of Treponema pallidum genomic DNA. Escherichia coli maxicells programmed with this plasmid synthesized three treponemal protein antigens of molecular weights 39,000, 35,000, and 25,000 (39K, 35K, and 25K proteins, respectively). In this study, a detailed deletion analysis of pLVS3 demonstrated that the genetic information for all three protein antigens is contained within a 1.5-kilobase EcoRI-HpaI restriction fragment. The DNA sequence of this fragment revealed a single open reading frame of 361 codons that most likely encodes a signal peptide-bearing precursor to the 39K protein that can be transiently detected in E. coli maxicells. Evidence indicated that the 35K and 25K protein antigens are derivatives of the larger protein and are only produced in maxicells. A significant elevation in expression of the 39K treponemal protein antigen in E. coli was obtained by using the E. coli lpp and lac promoters and a genetic construction in which the signal peptide and first four residues of the "mature" 39K protein were replaced by six amino acids encoded by the vector. This hybrid protein exhibited an unusually high pI, which greatly facilitated its purification to homogeneity. By using antibody prepared against the hybrid protein, the native treponemal protein counterpart, also of molecular weight 39,000, was identified as a membrane component of T. pallidum. Since the native protein also exhibited a net positive charge, it has been designated the T. pallidum basic membrane protein.     

Specific and sensitive diagnosis of syphilis using a real-time PCR for Treponema pallidum

A real-time PCR assay with a Taqman probe was developed that targeted the polA gene of Treponema pallidum. The test was validated using an analytical panel (n = 140) and a clinical panel of genital samples (n = 112) from patients attending a sexually transmitted infections clinic. High sensitivities and specificities of 94-100% were achieved using two real-time PCR platforms, the Rotor-Gene and the iCycler. The assay can be completed within 2 h, enabling reporting in <8 h. This fast and robust assay is suitable for implementation in routine laboratories for diagnosing primary syphilis.     

幽门螺杆菌Lpp20重组蛋白的表达、纯化及其免疫活性初步研究

目的表达幽门螺杆菌(Helicobacter pylori,H.pylori)脂蛋白Lpp20基因的重组蛋白,并检测其免疫活性。方法应用PCR技术从H.pylori标准株26695染色体DNA中扩增出Lpp20的编码基因片段,将其克隆至表达载体pGEX-6P-2上,在大肠杆菌(Escherichia coli,E.coli)BL21中表达并进行GST亲和层析纯化,用Western blot方法检测纯化产物重组Lpp20(recombinantLpp20,rLpp20)的免疫反应性。免疫C57BL/6小鼠后,以rLpp20为包被抗原建立间接ELISA法对免疫小鼠血清进行特异性抗体检测;ELISA双抗体夹心法检测小鼠脾淋巴细胞培养上清中IFN-γ、IL-2、IL-4水平;MTT比色法检测小鼠脾淋巴细胞增殖反应等指标以分析rLpp20的免疫活性。结果扩增的lpp20全长528 bp,与GenBank上公布的H.pylori 26695株lpp20序列作BLAST比较,结果完全一致;成功构建了pGEX-6P-2/Lpp20重组质粒,表达的rLpp20 M_r 43 000,纯化产物可被鼠抗H.pylori抗体识别;以...     

Evaluation of recomWell Treponema, a novel recombinant antigen-based enzyme-linked immunosorbent assay for the diagnosis of syphilis

Objective   To evaluate the diagnostic performance of an enzyme immunosorbent assay (recomWell Treponema) for the diagnosis of syphilis. The novel recombinant antigens Tpn47, TpN17 and TpN15 were utilized.
Methods   A total of 782 human serum specimens, belonging to four different categories (blood donors, n  = 200; routine laboratory screening for syphilis, n  = 400; syphilis patients, n  = 122; potential cross-reactors, n  = 60), were evaluated to compare the sensitivity and specificity of the recomWell Treponema kit with a standard whole Treponema pallidum cell lysate antigen-based ELISA (Syphilis Screening) and with micro-haemagglutination (MHA-TP).
Results   The overall specificity and sensitivity of the recomWell Treponema IgG was 98.9% and 98.3%, respectively. The specificity and sensitivity of Syphilis Screening ELISA was 98.7% and 98.3%, respectively. The agreement between recomWell Treponema and Syphilis Screening was 100%, 97.8%, 95.9% and 95% among the blood donor specimens, screening samples, syphilis specimens and the potential cross-reactors, respectively. Values of concordance varying from 96.7% to 98.3% were found in the different groups of sera between recomWell Treponema and MHA-TP. In addition, recomWell Treponema demonstrated a good diagnostic performance when used to detect the IgM to T. pallidum . No false-positive sera were identified and, in 17/19 samples from primary infection, an IgM immune response was found.
Conclusions   recomWell Treponema was shown to be a highly specific and sensitive method in all stages of syphilis screening and it can be considered as alternative to other ELISA tests based on native antigen preparations.     

Magnitude and trend of HIV and Treponema pallidum infections among blood donors in Offinso-North District,Ghana: a nine-year retrospective,cross-sectional study

IntroductionBlood transfusion poses a high public health risk to recipients; hence no effort recommended to eradicate or minimize the danger of transmitting the infections.Reproductive Biology should be underestimated at minimizing the risk of TTIs. This study determined the prevalence and trend of HIV and syphilis infections in voluntary blood donors.MethodA retrospective analysis of secondary data from consecutive prospective voluntary blood donors who accessed Nkenkaasu District Hospital''s Blood Bank from January 2010 to December 2018 was conducted.ResultCumulatively, HIV and Treponema pallidum seropositivity identified in the present study was high (19.1%, [95% C.I (0.026–0.028)]). The prevalence of HIV and syphilis infections were 10.9% (95% C.I (0.098–0.120)) and 8.9% (95% C.I (0.073–0.92)) respectively. Prospective female blood donors were less likely to test positive for T. pallidum than males (OR 0.511, [0.340 – 0.769], p=0.001), but the infection was similar among different ages. The data showed downward trend for both HIV and T. pallidum seropositivity, (slope=-2.9467, p<0.0001) and (slope=-0.7117, p<0.0001) respectively.ConclusionSeroprevalence of HIV and Treponema pallidum were high, and their individual or combined seropositivity pose a significant threat to the safety of blood. Extensive and continuous screening for high-risk behaviours and infectious markers before blood donation is therefore Unit, Department of Obstetrics and Gynaecology, College of Medicine, Pan African University of Life and Earth Sciences Institute (PAULESI), University of Ibadan, Ibadan, Nigeria.     

HIV-1 gp41蛋白的表达及其免疫反应性

目的高效表达HIV-1gp41基因片段,满足生产HIV体外检测试剂的需要。方法以含HIV-1gp41基因的质粒X1为模板,采用PCR方法扩增出gp41基因,克隆入E1载体构建重组表达质粒E1—1,转化E．coliBL21DE3感受态细胞,经异丙基-D-D-硫代半乳糖苷（IPTG）诱导后,使用镍螯合琼脂糖亲和层析柱（Ni．NTAAgaros）纯化重组蛋白,通过ELISA法检测重组蛋白的免疫反应性和特异性。结果SDS．PAGE鉴定结果表明,重组E1-1蛋白获得了正确的表达;纯化的重组E1-1蛋白纯度〉95％;ELISA检测结果表明,重组E1-1蛋白具有较优的免疫反应性和特异性。结论成功表达出具有较高免疫反应性的重组E1—1蛋白,可应用于HIV体外检测试剂。     

沙眼衣原体CPAF免疫活性区域重组蛋白的表达及在临床诊断中的应用

目的分析沙眼衣原体(Ct)蛋白酶样活性因子(CPAF)免疫优势区的免疫活性,探讨其在Ct感染诊断中的应用价值。方法构建原核表达重组质粒pGEX-6p-2/CPAF′,异丙基硫代半乳糖苷(IPTG)诱导表达,用该纯化的表达产物包被酶标板,建立间接ELISA检测病人血清中的Ct特异性IgM和IgG抗体,同时与晶美公司Ct ELISA试剂盒检测结果进行对比分析。结果含pGEX-6p-2/ CPAF′的大肠杆菌BL21大量表达相对分子质量(M_r)约为43×10~3的目的蛋白,用该重组蛋白包被酶标板,建立间接ELISA检测Ct IgM、IgG阴性和阳性参考血清各50份,符合率均为100%;同时检测250份临床血清标本,与晶美公司ELISA试剂盒检测结果的符合率IgM为96.8%,IgG为98.4%。结论表达的Ct CPAF免疫优势区(AA_(200)～AA_(338))重组蛋白具有良好的免疫反应活性,可望用于Ct感染的临床诊断。     

TpN17重组抗原的表达及在梅毒血清学诊断中的初步应用

目的用基因工程技术表达梅毒螺旋体TpN17重组抗原,并建立操作简便、特异性好、敏感性高的梅毒血清抗体间接酶联免疫吸附试验(ELISA)检测。方法采用PCR技术扩增梅毒螺旋体TpN17基因,再进行T-A克隆及测序,然后亚克隆到原核表达载体中,以亲和层析法纯化重组蛋白。将重组蛋白包被于反应板,建立检测血清中梅毒抗体的ELISA间接检测法,利用该法与梅毒螺旋体血凝试验(TPHA)、甲苯胺红不加热血清试验(TRUST)同时检测血清样品,对其结果进行比较研究。结果TpN17重组蛋白在大肠杆菌BL21中得到稳定表达,并成功建立了检测血清中梅毒抗体的ELISA间接检测法及试剂。对135例梅毒可疑患者血清进行检测,ELISA、TPHA和TRUST的检出阳性率分别为82.96%、98.52%和71.11%,而TRUST测出的8例可疑样品及31例阴性样品中,TPHA结果全部阳性、而ELISA只有2例阴性。结论TpN17重组抗原ELISA试剂在特异性和敏感性上明显优于TRUST法。     

Cell and Tissue Interactions of Treponema pallidum in Primary and Secondary Syphilitic Skin Lesions: An Ultrastructural Study of Serial Sections

There are limited reports on the ultrastructure of syphilis skin lesions. The aim of this study has been to perform an electron microscopic investigation of the morphology and the tissue distribution of treponemes in primary and secondary cutaneous lesions. Three cases of primary syphilitic chancre and one case of secondary syphilis were included. Prominent epidermal abnormalities in the primary chancre and a perivascular inflammatory infiltrate in the secondary lesion were found by light microscopy. Ultrastructurally, spirochetes were located mainly in the blood vessel walls and dermal tissue of the chancre lesions. In the secondary syphilis case, spirochetes were more abundant between epidermal keratinocytes. Most of them adjusted to the intercellular spaces. Occasionally, the electron microscopy images were highly suggestive of an intracellular location. Both the ultrastructural and immunohistochemical examination of the primary and secondary syphilis lesions showed a paradoxical distribution of the causative microorganisms compared to the light microscopic changes. In addition, the ultrastructural findings strongly suggest that Treponema pallidum subspecies pallidum invades tissues, not only through an intercellular, but also through a transcellular pathway.     

梅毒螺旋体黏附素Tp0751 核酸菌影的构建及免疫原性研究

目的:构建梅毒螺旋体(Tp)黏附素Tp0751 的重组真核大肠埃希菌菌影(EBG)并检测其在免疫鼠中的免疫原性,为探讨新型梅毒疫苗奠定基础。方法:构建pcDNA3.1(+) / Tp0751 真核表达载体,将其装载入已构建的空EBG 中,形成重组核酸菌影pcD/ Tp0751-BG,计算装载率;将核酸菌影转染鼠源性巨噬细胞RAW264.7,Western blot(WB)鉴定目的蛋白表达。将雌性BALB/ c 鼠随机分为A(PBS)、B(空EBG)、C(空pcDNA3.1)三个对照组和D(pcD/ Tp0751)、E(pcD/ Tp0751-BG)、F(pcD/ Tp0751-BG+rTp0751)三个实验组,各组间隔两周肌注免疫共三次,检测特异性血清IgG 及生殖道黏膜SIgA、小鼠脾细胞增殖水平和分泌IFN-γ水平。结果:重组真核质粒对菌影的装载率为76.1%;WB 显示此转染细胞能有效表达重组目的蛋白。D、E、F 实验组小鼠特异性血清IgG 与生殖道SIgA 效价均随免疫次数增加而增加,各时间点均显著高于三个对照组(P<0.01),于末次加免后第8 周达到峰值,此时F 组IgG 与SIgA 效价分别为1 :102 400 与1 :12 800;首次加免2 周后,E、F 组均显著高于D 组(P<0.01);末次加免2 周后,F 组显著高于E 组(P<0.01)。末次加免后第8 周,D、E、F 组的刺激指数(SI)值与IFN-γ水平均分别显著高于三个对照组(P<0.01);E、F 组均分别显著高于D 组(P<0.01);F 组分别均高于E 组(P<0.05)。结论:Tp0751 真核质粒菌影具有良好的免疫原性,在小鼠体内诱生了有效的系统和黏膜的体液应答以及系统细胞免疫应答;异源加免较同源加免免疫效果更好。     

重组大肠杆菌不耐热肠毒素B亚单位的表达、纯化及黏膜佐剂活性研究

目的　对大肠杆菌不耐热肠毒素B亚单位(LTB)进行基因克隆、重组表达、蛋白纯化和黏膜佐剂活性分析。方法　应用PCR技术从产肠毒素大肠杆菌基因组中扩增出不耐热肠毒素B基因,将此构建于原核表达载体pET 11C,并在大肠杆菌BL21(DE3)中表达,然后采用D(+) immobilizedgalactose亲和层析柱对重组蛋白进行纯化。以纯化后重组LTB为佐剂,与幽门螺杆菌重组尿素酶B亚单位蛋白(rUreB)共同口服和滴鼻免疫BALB c小鼠,分析重组LTB的黏膜佐剂活性。结果　PCR扩增出390 bp LTB基因,与GenBank公布的核苷酸序列的相似性为99.5%。重组LTB蛋白的表达率为6%,以胞周质分泌形式表达,经亲和层析后蛋白纯度大于95%。该重组蛋白保持了与神经节苷脂GM1结合的生物学活性和良好的免疫原性及免疫反应性。动物试验表明,重组LTB与rUreB抗原共同口服和滴鼻免疫小鼠后的特异性血清IgG及鼻腔、气管、胃黏膜和小肠黏膜sIgA水平显著高于单独rUreB抗原免疫组(P<0.05)。结论　所获得的重组LTB具有较好的黏膜佐剂活性,为其在黏膜疫苗中的应用奠定基础。