Expression and purification of the recombinant outermembrane protein Tp0453 of Treponema pallidum and its characterization of immuno-competence

ELISA test has been shown to have some advan tages in relation to the tests used for the diagnosisof syphilis because of its easy and quick perfor mance and result readings. With recent develop ment of the gene engineering technology and eluci dation of the whole genome of Nichols strain ofTreponema pallidum, new protein coding openreading frames (ORFs) are available for testing,and the study on the serological tests based on therecombinant protein have been become the focus ofinter…     

Serological diagnosis of syphilis: enzyme-linked immunosorbent assay to measure antibodies to individual recombinant Treponema pallidum antigens

We standardized an indirect ELISA for measurement of serum antibody levels to four individual treponemal recombinant proteins that have been commonly used in a number of commercial EIAs, mostly as a mixture of antigens. When tested with 127 syphilis-negative and 37 secondary syphilis sera, ELISA O.D.s obtained for each of the four antigens clearly distinguished between these two groups of samples. Sensitivity and specificity of 100% was obtained with the current set of samples. Further evaluations with sera from different stages of syphilis can help to define the applications of this ELISA test for each of the four antigens studied.     

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.     

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.     

化学发光法检测受血者梅毒螺旋体特异性抗体的方法学评价

目的 通过与梅毒螺旋体微量血凝试验(TPHA)和蛋白印迹试验法(WB)比较评估梅毒血清学筛查法化学发光法(CLIA)的性能.方法 回顾性研究18 494例受血者血清标本CLIA和TPHA检测梅毒螺旋体特异性抗体结果,对CLIA和(或)TPHA结果阳性177例标本用WB检测确认.同时用CLIA、TPHA和WB检测了81例各期梅毒患者血清、55例有潜在干扰的患者血清和250例阴性对照血清梅毒抗体.结果 以WB检测结果为金标准,CLIA方法的灵敏度为98.4％,显著高于TPHA(94.4％)(x2=5.76,P＜0.05);CLIA方法特异性为100％,高于TPHA方法特异性(99.7％),但差异无统计学意义(x2=1.0,P＞0.05).结论 CLIA法具有高度敏感性和特异性,适合于临床实验室进行梅毒筛查.     

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.     

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。     

Reactivity of antibodies from syphilis patients to a protein array representing the Treponema pallidum proteome

To identify antigens important in the human immune response to syphilis, the serum antibody reactivity of syphilitic patients was examined with 908 of the 1,039 proteins in the proteome of Treponema pallidum subsp. pallidum using a protein array enzyme-linked immunosorbent assay. Thirty-four proteins exhibited significant reactivity when assayed with human sera from patients in the early latent stage of syphilis. A subset of antigens identified were further scrutinized for antibody reactivity at primary, secondary, and latent disease stages, and the results demonstrate that the humoral immune response to individual T. pallidum proteins develops at different rates during the time course of infection.     

Protection against syphilis correlates with specificity of antibodies to the variable regions of Treponema pallidum repeat protein K

Syphilis has been recognized as a disease since the late 1400s, yet there is no practical vaccine available. One impediment to the development of a vaccine is the lack of understanding of multiple reinfections in humans despite the development of robust immune responses during the first episode. It has been shown that the Treponema pallidum repeat protein K (TprK) differs in seven discrete variable (V) regions in isolates and that the antibody response during infection is directed to these V regions. Immunization with TprK confers significant protection against infection with the homologous strain. We hypothesize that the antigenic diversity of TprK is involved in immune evasion, which contributes to the lack of heterologous protection. Here, using the rabbit model, we show a correlation between limited heterologous protection and tprK diversity in the challenge inoculum. We demonstrate that antibody responses to the V regions of one TprK molecule show limited cross-reactivity with heterologous TprK V regions.     

Genetics of Treponema: characterization of Treponema hyodysenteriae and its relationship to Treponema pallidum.

Saturation reassociation assays with 125I-labeled treponemal DNAs show that Treponema hyodysenteriae is genetically unrelated to T. pallidum (Nichols), T. phagedenis biotype Reiter, and T. refringens biotype Noguchi. Pathogenic and nonpathogenic isolates of T. hyodysenteriae exhibited 28% sequence homology and had an extremely low guanine-plus-cytosine content (25.8%).     

Overproduction and purification of Treponema pallidum recombinant-DNA-derived proteins TmpA and TmpB and their potential use in serodiagnosis of syphilis.

We report the construction of expression plasmids carrying two Treponema pallidum genes encoding for the 42-kilodalton membrane protein TmpA (treponemal membrane protein A) and the 34-kilodalton membrane protein TmpB. Using the leftward promoter of bacteriophage lambda, which is controlled by a thermosensitive repressor, we obtained a high level of heat-inducible synthesis of TmpA and TmpB in Escherichia coli K-12. Both proteins were purified to near homogeneity, and the presence of antibodies to TmpA and TmpB in human sera was determined by an enzyme-linked immunosorbent assay. Whereas in all 44 serum samples from untreated patients in the secondary and early latent stages of syphilis, high levels of anti-TmpA antibodies were detected, only 34 serum samples contained anti-TmpB antibodies. As has been previously observed for TmpA, a correlation was found between the presence of anti-TmpB antibodies and anti-cardiolipin antibodies, suggesting that the level of antibodies to TmpB drops soon after successful antibiotic treatment. We concluded that, in contrast to TmpA, TmpB is not suitable for serodiagnostic purposes as a single antigen, because a significant fraction of sera from syphilitic patients was nonreactive with TmpB.     

Sensitivity and specificity of monoclonal antibodies directed against antigenic determinants of Treponema pallidum Nichols in the diagnosis of syphilis.

Murine anti-Treponema pallidum monoclonal antibodies were employed in studies on sensitivity and specificity of binding to examine their potential for use in the detection of low numbers of pathogenic treponemes present in various body fluids. Monoclonal antibodies were used as a primary antibody source in a solid-phase immunoblot assay system. All monoclonal antibodies assayed were capable of detecting ca. 1.0 X 10(3) to 2.5 X 10(3) treponemes. Of 13 monoclonal antibodies examined, 3 were able to detect 10(3) virulent treponemes, and 1 of these antibodies was able to reveal the presence of as few as 500 organisms. Western blot analyses showed that all anti-T. pallidum monoclonal antibodies exhibiting high sensitivities for the detection of T. pallidum cells were directed against an abundant, 47,000-dalton surface-exposed antigen of the organism (S. A. Jones, K. S. Marchitto, J. N. Miller, and M. V. Norgard, Abstr. Annu. Meet. Am. Soc. Microbiol. 1984, B173, p. 46; K. S. Marchitto, S. A. Jones, and M. V. Norgard, Abstr. Annu. Meet. Am. Soc. Microbiol. 1984, B182, p. 48). Differences in binding properties of the various monoclonal antibodies were most likely a reflection of differential binding affinities or their specificities for different epitopes on the 47,000-dalton surface antigen. With two possible exceptions, the monoclonal antibodies tested reacted specifically with T. pallidum, either purified or found within a high-contaminating tissue background, and not with Treponema phagedenis biotype Reiter, Haemophilus ducreyi, Neisseria gonorrhoeae, herpes simplex virus type 2, or normal rabbit testicular tissue. The high sensitivity and specificity exhibited by these anti-T. pallidum monoclonal antibodies make them excellent candidates for employment in new syphilis or other treponemal diagnostic tests designed to detect very low numbers of pathogenic treponemes in lesion exudates or other body fluids.     

A new attempt to distinguish serologically the subspecies of Treponema pallidum causing syphilis and yaws.

In an effort to serologically differentiate syphilis from yaws, 69 monoclonal antibody species raised against Treponema pallidum subsp. pallidum were tested by immunoblotting for their reactivity with Treponema pallidum subsp. pertenue. All monoclonal antibodies reacted with antigens with the same molecular weight of both subspecies. Furthermore, no differences in reactivity between sera from yaws patients and from syphilis patients were found by Western blot (immunoblot) analysis of cell lysates of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue. We tried to exploit the only known molecular difference between the subspecies. The subunits of the 190-kilodalton multimeric proteins TpF1 and TyF1 of T. pallidum subsp. pallidum and T. pallidum subsp. pertenue, respectively, have previously been shown to differ in one amino acid residue at position 40. In this study, no difference was found in immunoreactivity of TpF1 or TyF1 with either syphilis sera or yaws sera. Synthetic peptides based on the sequence of TpF1 and of TyF1 were used in an enzyme-linked immunosorbent assay with syphilis sera and yaws sera. Again, no difference in reactivity between the T. pallidum subsp. pallidum- and T. pallidum subsp. pertenue-derived peptides was observed.     

Characterization of the proteoglycans synthesized by rabbit testis in response to infection by Treponema pallidum.

Organ cultures of syphilitic and normal rabbit testes were incubated with 35S-sulfate for labeling of proteoglycans. Syphilitic rabbit testes synthesized three macromolecular fractions (I, II, and III) which were not detected in extracts of normal uninfected tissue. The three fractions comprised a larger (approximately 10(6) mol wt) chondroitin sulfate/dermatan sulfate proteoglycan (Fraction I), a smaller (approximately 10(5) mol wt) chondroitin sulfate/dermatan sulfate proteoglycan (Fraction II), and a putative sulfated glycoprotein of Mr 40 kd (Fraction III). The glycosaminoglycan chains of both proteoglycans eluted with a Kav of 0.45 on Sepharose CL-6B, consistent with a molecular weight of 25,000. The smaller proteoglycan was not a cleavage product of the larger species. Erythromycin had no significant effect on the synthesis of any of the three macromolecules. In contrast, the synthesis of both proteoglycans was totally inhibited by a 2-hour preincubation with cycloheximide, which suggests that the constitutive "pools" of the two core proteins were small. The putative sulfated 40-kd glycoprotein was insensitive to a 2-hour preincubation with cycloheximide.     

Spirochetemia due to Treponema pallidum using polymerase-chain-reaction assays in patients with early syphilis: prevalence,associated factors and treatment response

Between 2009 and 2013, polymerase-chain-reaction assay was used to detect Treponema pallidum in the blood samples collected from 296 patients with early syphilis (241 being HIV infected) and 102 patients (34.5%) had spirochetemia. The presence of spirochetemia was associated with lower CD4 counts (per 10-cell/mm³ decrease, adjusted odds ratio (AOR), 1.020; 95% CI, 1.006–1.036) and secondary syphilis (AOR, 4.967; 95% CI, 2.016–12.238). Patients with early latent syphilis were less likely to achieve serological response compared with those with primary or secondary syphilis (AOR, 0.317; 95% CI, 0.142–0.708). However, serological response was not affected by presence of spirochetemia or antibiotic regimens.     

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…     

Effect of passive immunization with purified specific or cross-reacting immunoglobulin G antibodies against Treponema pallidum on the course of infection in guinea pigs.

Whole immune serum or highly purified immunoglobulin G (IgG) antibodies to Treponema pallidum exhaustively adsorbed with three strains of nonpathogenic treponemes (TPI-IgG) were used for passive immunization of inbred strain 2 guinea pigs before and after intradermal challenge with 3.4 x 10(7) virulent T. pallidum Nichols organisms. Before challenge, control animals received a similarly purified IgG fraction containing either a cocktail of antibodies against three nonpathogenic treponemes (NPTI-IgG) or IgG prepared from normal guinea pig serum (NGPS-IgG). The purified fractions contained both IgG1 and IgG2 isotypes. The antibody levels (detected by fluorescent treponemal antibody test and enzyme-linked immunosorbent assay) and molecular specificities (immunoblot) of sera obtained from recipient animals before infection reflected those of the purified fractions used for immunization. Three protocols of passive immunization were used. Whole immune serum containing specific and cross-reacting antibodies afforded better protection than TPI-IgG even though asymptomatic animals were not fully protected. A single intradermal injection (0.1 ml) of TPI-IgG or NPTI-IgG into one hind leg 22 h before infection at the same site provided relatively higher protection than multiple intravenous injections (total, 15 ml) of the respective individual preparations. Since purified NGPS-IgG injected in the same animals, into the opposite hind leg, failed to protect against the challenging infection, it is reasonable to assume that specific and cross-reacting antitreponemal antibodies of the IgG1 subclass, which in guinea pigs are homocytotropic, play a relevant role in local protection.     

Characterization of the 35-kilodalton Treponema pallidum subsp. pallidum recombinant lipoprotein TmpC and antibody response to lipidated and nonlipidated T. pallidum antigens.

The gene encoding the 35-kDa immunogenic Treponema pallidium subsp. pallidum (T. pallidum) membrane protein C, TmpC, was cloned, sequenced, and expressed in Escherichia coli. The deduced amino acid sequence carries on N-terminal signal sequence with a four-amino-acid motif, which is characteristic for bacterial lipoproteins. Metabolic labeling with radioactive palmitic acid of E. coli expressing TmpC revealed incorporation of the fatty acid into the antigen. The antigen was overproduced, purified to near homogeneity and used in an enzyme-linked immunosorbent assay (ELISA) to evaluate its potential for the serodiagnosis of syphilis. Although all sera from untreated secondary syphilis patients were reactive in this TmpC ELISA, only a minority of the serum samples from untreated patients in the primary or early latent stage of the disease contained significant anti-TmpC antibodies. To study the influence of the lipid moiety on the antigenic properties of the TmpC, TmpA, and TpD lipoproteins, plasmids encoding nonlipidated forms of these antigens were constructed. In addition, a plasmid expressing a lipidated form of the otherwise non-lipid-modified antigen TmpB was constructed. Immunization and absorption experiments with these lipidated and nonlipidated antigens showed that antibodies against the lipid moiety of lipoproteins could not be detected on immunoblots, neither in sera from infected rabbits nor in sera from animals immunized with the lipoproteins. In addition, we were unable to demonstrate cross-reactivity between antibodies against the T. pallidum lipoproteins and those reactive to the Venereal Diseases Research Laboratories test, suggesting that antibodies reactive to the Venereal Diseases Research Laboratories test are unrelated to antilipoprotein antibodies.