中国莱姆病螺旋体FP1外膜蛋白A的克隆表达及其抗原性的初步研究

目的　克隆并表达中国莱姆病螺旋体阿弗西尼疏螺旋体基因种参照菌株FP1的外膜蛋白A(OspA) ,并对其抗原性进行初步研究和基因序列分析 ,为研制中国莱姆病疫苗提供资料。方法设计引物 ,用聚合酶链反应 (PCR)从莱姆病螺旋体FP1全基因组DNA中扩增出OspA编码基因 ,经酶切、连接 ,插入原核表达载体pET 11d ,构成重组质粒pET 11d ospA ,在大肠杆菌BL2 1(DE3)中表达 ,表达产物用SDS PAGE、Westernblot分析 ,并进行基因序列测定。结果　rOspA在宿主菌内获得高效、稳定表达 ;相对分子质量 (Mr)约为 31× 10 3,Westernblot显示其与抗OspA的多抗有良好的免疫反应性 ;经测序显示该rOspA的基因片段长度为 783bp ,编码 2 6 1个氨基酸 ,与欧洲标准菌株Pko和瑞士标准菌株VS4 6 1的OspA的DNA碱基序列比较 ,同源性均为 94 %。结论　在国内首次成功地对中国莱姆病螺旋体阿弗西尼疏旋体基因种的代表菌株FP1的OspA基因进行了克隆和表达。并且证实rOspA有良好的抗原性 ,可进一步对其免疫保护性进行研究 ,以便为研制有效的莱姆病疫苗提供数据。     

中国莱姆病螺旋体PD91外膜蛋白A肽段的克隆表达及其免疫保护性的初步研究

目的:克隆并表达中国莱姆病螺旋体基因型代表菌株伽氏疏螺旋体( Borrelia garinii, B. garinii)PD91外膜蛋白A(OspA)的126~274 aa肽段,并对其免疫保护性进行初步研究。 方法:采用聚合酶链反应(PCR)扩增 B. garinii PD91的126~274 aa OspA肽段基因,克隆至原核表达载体pET-30a上,构建pET-30a-OspA-pep重组质粒,转入大肠埃希菌感受态细胞BL21(DE3)中,利用IPTG诱导表达,表达产物用Ni-IDA树脂层析纯化,采用Western blot分析其免疫原性。将不同剂量的重组OspA-pep(rOspA-pep)蛋白(20、30、40、50、60、80、100 μg)免疫新西兰家兔,采用间接免疫荧光法(IFA)检测免疫前后的抗体滴度,选取产生抗体滴度最高的剂量组为最佳剂量组。用最佳剂量组的免疫兔血清进行体外中和试验以检测rOspA-pep蛋白免疫后血清抗体的体外杀菌能力,同时用最佳剂量的rOspA-pep蛋白免疫新西兰家兔,观察其抗体滴度变化。 结果:重组质粒pET-30a-OspA-pep构建成功并在宿主菌体内高效表达。Western blot表明rOspA-pep蛋白与 B. garinii PD91的多抗有明显的免疫应答。IFA检测结果表明rOspA-pep蛋白免疫后的兔血清IgG抗体滴度明显升高(最高可达1∶2 480),40 μg为rOspA-pep的最佳免疫剂量。体外中和试验结果表明该剂量rOspA-pep蛋白免疫家兔后产生的抗体对10 6个/ml的 B. garinii和阿弗西尼疏螺旋体( Borrelia afzelii, B. afzelii)型代表菌株PD91和FP1的中和率达100%,对10 7个/ml的FP1的中和率为100%,对10 7个/ml的PD91的中和率为60%。用40 μg rOspA-pep在1 d和30 d免疫新西兰家兔2次后,其抗体达到高峰,持续时间为3~4个月,之后抗体滴度逐渐下降。 结论:中国莱姆病螺旋体基因型代表菌株 B. garinii PD91的126~274 aa OspA肽段具有较好的免疫原性,其诱导的抗体有较好的体外中和能力,可作为中国二代亚单位疫苗的候选成分。     

中国莱姆病伽氏疏螺旋体蛋白免疫印迹法诊断标准的研究

目的研究中国莱姆病伽氏疏螺旋体基因型蛋白免疫印迹法（WB）的阳性诊断标准。方法采用中国莱姆病螺旋体伽氏疏螺旋体基因型参照菌株PD91作抗原，建立WB检测方法。采用Gel—Pro凝胶分析软件进行结果分析。病例组和对照组各组蛋白条带的频数分布比较采用x^2检验或Fisher’s精确检验，WB检测莱姆病阳性诊断标准确定采用ROC曲线。结果共检测127例莱姆病病例和504例对照的血清标本，经统计学分析得出我国莱姆病伽氏疏螺旋体检测中WB阳性诊断标准为：对于IgG P83／100、P58、P39、P30、OspC、P17、P66、OspA中至少有一条蛋白条带显色即可诊断为阳性，此标准敏感度为73．2％，特异度为99．4％；对于IgM P83／100、P58、OspA、P30、OspC、P17、P41中至少有一条蛋白条带显色则可诊断为阳性，此标准敏感度为50．4％，特异度为93．1％。结论建立了中国莱姆病伽氏疏螺旋体基因型WB阳性诊断标准，用于莱姆病的WB诊断具有较好的特异性。     

莱姆病螺旋体60kD抗原的基因克隆及表达

利用质粒ｐＡＴ１５３为载体，构建了莱姆螺旋体全细胞ＤＮＡ基因文库，用菌落原位固相酶斑法从文库中初选出一株表达６０ｋＤ抗原的克隆子，命名为ｐＬＷ２２７。经免疫印迹分析、核酸杂交、连续亚克隆分析，证明此重组菌株表达了莱姆螺旋体６０ｋＤ抗原，编码该抗原的基因表达单位为２．２ｋｂ或更小，位于莱姆螺旋体的染色体上。该抗原的表达为进一步研究莱姆病的致病机理打下了基础。     

离体免疫制备莱姆病螺旋体单克隆抗体的初步研究

制备单克隆抗体采用离体免疫时间短、抗原用量少 ,并有不受有毒有害物质限制、诱发抗体生成所需时间短等优点。目前 ,未见有鼠 -鼠离体免疫制备单克隆抗体的详细报道。本文试图探索离体免疫制备抗莱姆病螺旋体的单克隆抗体。材料和方法抗原 :从全沟硬蜱自行分离培养伯氏疏螺旋体株Ｈ 0 1,菌体经超声破碎后离心 ,取上清。实验动物 :ＢＡＬＢ ｃ、Ｃ5 7 ＢＬ小鼠 ,购于中华预防医学科学院流行病学微生物学研究所动物室。试剂 :ＲＰＭＩ 16 40 (ＧＩＢＣＯＢＲＬ)、β 巯基乙醇 (2 ＭＥ)、胞壁酰二肽 ＭＤＰ (Ｓｉｇｍａ)。离体免疫基础培养…     

贝氏柯克斯体30kD外膜蛋白基因表达及免疫保护性的研究

贝氏柯克斯体可引起人类急、慢性Q热。急性Q热临床表现多种多样,主要症状为发热、头痛和肌肉酸痛,并常伴有肺炎、肝炎等。慢性Q热常发展为Q热性心内膜炎、骨髓炎。Q热疫苗是预防Q热流行的最有效手段,目前人用Q热疫苗是灭活的柯克斯体。虽然灭活疫苗的免疫保护效果很好,但注射该疫苗接种部位常出现不良反应。研究发现,在贝氏柯克斯体的Ⅰ相和Ⅱ相中含有相对分子质量(Mr)为30×10 3 的外膜蛋白,该蛋白具有良好的免疫原性和免疫反应性〔1〕。我们采用分子生物学技术,研制出贝氏柯克斯体Mr30×10 3 重组外膜蛋白,用重组蛋白免疫小鼠,对其免…     

肠致病性大肠杆菌外膜蛋白免疫保护性研究

目的：观察免疫家兔对肠致病性大肠杆菌外膜蛋白的免疫应答。方法：以提纯的兔源致病性大肠杆菌WF0305菌株的外膜蛋白（Outer membrane protein，OMP）皮下注射免疫组家兔，对照组仅注射佐剂，于免疫前及免疫后38天分别收集血清，以间接ELISA法检测血清中的抗体效价，同时用免疫血清与菌株做玻板凝集试验和保护力试验；采用MTT法检测OMP对体外脾淋巴细胞特异性增殖反应。结果：免疫组的家兔在三免后免疫血清中抗OMP的抗体效价可达1∶25600；免疫血清均能与WF0305菌株、WF0408菌株、WF0504菌株发生直接凝集反应，且效价均能达到1∶16以上，小鼠的半数保护量均在0.03-0.06ml之间。采用MTT法检测OMP对体外脾淋巴细胞特异性增殖反应，证明OMP对脾淋巴细胞的增殖有明显增强作用（P〈0.01）。结论：OMP可诱导兔对大肠杆菌的特异性体液免疫和细胞免疫应答，说明OMP具有较好的免疫原性，且对大肠杆菌感染有一定的保护力。     

问号钩端螺旋体铁离子调节蛋白A的免疫原性及保守性研究

目的克隆表达和鉴定问号钩端螺旋体（L．interrogans，简称钩体）黄疸出血群赖型赖株中铁离子调节蛋白A（IRPA），研究IRPA的免疫原性和在不同钩体菌种中的保守性，探讨其在致病和疫苗研究中的意义。方法生物信息学软件分析预测IRPA的特征。构建原核表达质粒pQE31-IRPA，经IPTC诱导后用SDS-PAGE及Western blot鉴定表达情况。用表达的重组蛋白免疫BALB／c小鼠，Western blot检测其免疫原性和在不同血清型钩体中的保守性。ELISA和Western blot检测钩体全菌兔抗血清中的IRPA抗体。结果生物信息学预测结果显示，IRPA是可能位于外膜的脂蛋白，在钩体内有多个蛋白可能与之相互作用。成功克隆表达了重组质粒pQE31-IRPA，重组蛋白能刺激BALB／c小鼠产生抗体（效价为1：32000），并能与相应抗体反应，具有良好的免疫原性。在15株不同血清型的问号钩体及1株双曲钩体（L．biflex）中均可检测到重组蛋白的表达，并在钩体全菌兔抗血清中检测到其抗体。结论IRPA具有良好的免疫原性和保守性，为进一步研究其在钩体病中的作用机制及其作为候选基因疫苗的研究奠定了基础。     

中国强毒力赖型钩端螺旋体外膜蛋白基因的克隆表达及对大肠杆菌活力的影响

目的:构建表达致病性赖型 017株钩端螺旋体OmpL1外膜蛋白的真核-原核穿梭表达载体,并在原核细胞中表达出目的蛋白。方法:用PCR方法从 017株钩体基因组中钓出OmpL1蛋白基因,酶切纯化后与质粒pBK-CMV连接,通过电泳、限制性内切酶分析及PCR鉴定筛选出正确重组质粒。重组质粒在大肠杆菌中诱导表达后提取总蛋白以SDS-PAGE检测表达情况,同时监测目的蛋白表达前后各时段宿主菌OD600值的变化。结果:筛选出 5株带重组质粒菌,其中有 4株能在大肠杆菌中表达 37kD的特异蛋白,而且随着该外源蛋白的表达,宿主菌生长各时段的OD600值下降。结论:成功地构建了钩体OmpL1蛋白的穿梭表达质粒,并在大肠杆菌中表达出OmpL1融合蛋白,该异源蛋白的表达导致宿主菌的活力降低。本工作为OmpL1蛋白用于钩体病诊断、疫苗研制和致病机制的研究奠定了基础.     

Plasmid DNA and protein vaccination of mice to the outer surface protein A of Borrelia burgdorferi leads to induction of T helper cells with specificity for a major epitope and augmentation of protective IgG antibodies in vivo

Plasmid DNA-based vaccination is an efficient way to evoke various forms of protective immunity in laboratory animals. Our previous experiments have shown that mice immunized with either plasmid DNA encoding the outer surface lipoprotein A (pOspA) of Borrelia burgdorferi or the respective lipoprotein (Lip-OspA) produce protective antibodies against subsequent challenge with virulent spirochetes. In the present study, we compared the specificity and function of T cells generated in AKR/N mice previously immunized to either pOspA or Lip-OspA. T cell populations derived by either of the two protocols consistently responded by proliferation in vitro to one (residues 186–203; B4) out of a panel of 27 overlapping 20-mer peptides spanning the entire OspA molecule of strain ZS7. B4 was shown to express allele-specific ligand motifs for I-E^k. Most of the other peptides produced variable and much less pronounced or marginal proliferative T cell responses. T cells reactive to B4 as well as to some minor epitopes were CD4⁺CD8⁻ T cells which produced IFN-γ but no detectable IL-4 upon antigen stimulation in vitro Priming of AKR/N mice with B4 but not with inactive peptides of OspA led to an enhanced production of IgG antibodies, mainly of the IgG1 isotype, including those to a prominent protective epitope (LA-2) upon subsequent challenge with Lip-OspA or intact spirochetes. The data demonstrate that both plasmid DNA and protein immunization with OspA results in T cell responses with specificity for a dominant OspA epitope and suggest that priming of mice with immunodominant peptides accelerates the appearance of protective antibodies in vivo. The identification of T helper cell epitopes relevant for the induction of protective antibodies will also facilitate the design of more potent vaccines against Lyme disease.     

中国莱姆病螺旋体鞭毛蛋白中央区的基因克隆和表达

目的　对中国莱姆病螺旋体Borreliagarinii基因种参照菌株PD91的鞭毛蛋白中央区的编码基因进行克隆表达 ,对重组鞭毛蛋白作为莱姆病血清学诊断抗原进行初步的研究 ,并进行基因序列和氨基酸序列分析。方法　设计引物 ,用PCR技术获得PD91的鞭毛蛋白中央区的编码基因片段 ,经酶切、连接 ,插入质粒pET 30a中 ,构成重组质粒pET30a mfla,转化到大肠杆菌BL2 1,提取质粒进行酶切、DNA测序和氨基酸序列分析鉴定 ,诱导表达 ,筛选高效表达株 ,应用SDS PAGE和Westernblot鉴定重组蛋白及其抗原性。结果　成功地获得了鞭毛蛋白中央区的编码基因片段和基因重组 ,重组蛋白在宿主菌BL2 1中高效表达。Westernblot结果显示重组鞭毛蛋白的中央区与PD91的鞭毛蛋白具有相同的抗原性。经测序显示该中央区基因片段为鞭毛蛋白基因的 4 0 9～ 786bp ,与北美莱姆病螺旋体标准株B31的DNA碱基序列比较分析 ,同源性 92 %。结论　成功地对中国莱姆病螺旋体Borreliagarinii基因种的鞭毛蛋白中央区进行了克隆表达 ,并证实具有抗原性 ,为我国莱姆病血清学诊断研究提供资料。     

Heterogeneity of Borrelia burgdorferi sensu lato demonstrated by an ospA-type-specific PCR in synovial fluid from patients with Lyme arthritis

Borrelia burgdorferi sensu lato, the etiological agent of Lyme borreliosis, has been divided into three genospecies: B. burgdorferi sensu stricto (OspA-type 1), B. afzelii (OspA-type 2) and B. garinii (OspA-type 3–7). Whereas in Europe B. afzelii (OspA-type 2) is predominant among human skin isolates and B. garinii (OspA-type 3–7) among human CSF isolates, some previous serological studies suggested that Lyme arthritis is also associated with B. burgdorferi sensu stricto in Europe. In the present study we designed ospA type-specific PCRs and identified four different ospA types associated with Lyme arthritis. Our study group consisted of 20 patients with positive serology (ELISA and immunoblotting) and clinical criteria for Lyme arthritis. B. burgdorferi DNA was detected in 13 patients and in none of 10 control patients from synovial fluid. We identified ospA-type 1 (26.6%), ospA-type 2 (33.3%), ospA-type 4 (6.6%) and ospA-type 5 (33.3%). Our conclusion is that in Europe B. burgdorferi sensu lato strains causing Lyme arthritis are considerably heterogeneous and that there is no prevalence of certain genospecies or OspA-types among this strains. Received: 14 May 1998     

Expression of outer surface proteins A and C of Borrelia burgdorferi in Ixodes ricinus ticks removed from humans

A total of 131 Ixodes ricinus (51 females, 1 male and 79 nymphs) removed from persons living in Southern Germany were investigated by immunofluorescence assay for the presence of Borrelia burgdorferi with a polyvalent rabbit immune serum and monoclonal antibodies specific for outer surface proteins (Osp) A or C. Borreliae were detectable in 48 (36.6%) of the ticks. Infection rates of these adults and nymphs were significantly higher than infection rates of unfed ticks from Southern Germany. Borreliae in 31.3% (n = 15) of the infected ticks expressed solely OspA, solely OspC in 12.5% (n = 6), and both OspA and OspC in 39.6% (n = 19) of ticks, while in 16.7% (n = 8) of ticks neither were expressed. Presentation of OspC by B. burgdorferi in I. ricinus was correlated with tick weight: in females, OspC was detectable only in ticks with a minimum weight of about 3.5 mg, and in nymphs weighing at least 1 mg. These results indicate that in I. ricinus removed from humans OspC is up-regulated during the blood meal of the tick, but in most ticks OspA is still detectable and might even be present in the absence of OspC expression in the midgut and salivary glands of nearly fully engorged nymphal ticks. Furthermore, we found strong evidence that borreliae expressing solely OspA while in the salivary glands can cause Lyme borreliosis. Our findings indicate that during tick feeding, humans are exposed to borreliae that may express either OspA or OspC or both, or lack both OspA and C. These findings suggests that, at the minimum, both OspA and C should be considered as vaccine candidates for prophylaxis of Lyme borreliosis in Europe. Received: 28 July 1998     

Differential expression of outer surface proteins A and C by individual Borrelia burgdorferi in different genospecies

Previous studies with different Borrelia burgdorferi sensu stricto (s.s.) strains revealed that temperature as well as cocultivation with tick cells modulates the expression of outer surface proteins (Osp) A and C. We investigated the effects of temperature and of interaction with tick cells in culture on the expression of OspA and OspC of the B. afzelii clones cPKo97 and cPKo345 in comparison to the B. burgdorferi s.s. strain N40. To follow the dynamics of Osp expression of single borreliae we used indirect immunofluorescence microscopy with double staining of OspA and OspC. Clone PKo345 always showed expression of only OspA, regardless the conditions it was subjected to. Sequencing of the ospC gene disclosed a insertion leading to a stop codon after base 222 and inability to produce OspC. In cPKo97 and N40 OspC is down-regulated at lower temperatures and up-regulated at higher temperatures, which was especially pronounced on cocultivation with tick cells. Borreliae adherent to tick cells showed greater OspA expression compared to the nonadherent ones, an indication that OspA might play a role as adhesin for tick cells. Interestingly, cPKo97 and N40 displayed different patterns of Osp expression: cPKo97 simultaneously presents OspA and OspC on single borreliae, while N40 has either OspA or OspC on single cells. Adaptation of OspC expression in cPKo97 seems to occur by up- or down-regulation of this protein on single borreliae, as shown by alternating intensities of OspC expression at different temperatures. In contrast, N40 seem to consist of two subsets of borreliae one expressing only OspA and the other only OspC, and change in temperature results in growth benefit for one of these subtypes. Our findings indicate that, regarding OspA and OspC expression, response to temperature and cocultivation with tick cells of B. afzelii is comparable to B. burgdorferi s.s., but the mode of regulation seems phenotypically different. Further European isolates should be investigated for OspA and OspC regulation, especially in the face of vaccine development for the European situation. Received: 19 July 2000     

Osp17, a novel immunodominant outer surface protein of Borrelia afzelii: recombinant expression in Escherichia coli and its use as a diagnostic antigen for serodiagnosis of Lyme borreliosis

Western blot analyses of the human humoral response of patients with Lyme borreliosis have shown that a 17-kDa protein is an immunodominant protein in late disease. Immune electron microscopy with a monoclonal antibody against this protein revealed that the 17-kDa protein is abundantly expressed on the surface of Borrelia afzelii strain PKo. Therefore, the protein has been renamed outer surface protein (Osp)17. Recombinant Osp17 of strain PKo was expressed in Escherichia coli and purified by chromatography. Immunoblot analysis of human sera showed a comparable sensitivity with recombinant and natural proteins. The DNA sequences of the osp17 genes from different B. afzelii strains were determined. The DNA sequences of the different osp17 homologues (six isolates from skin, three isolates from CSF and one isolate from synovial fluid) had high sequence identities of at least 94%. Using a polyclonal antibody against recombinant Osp17, it was shown that Osp17 expression varied considerably among the investigated B. afzelii strains. As previously also observed for OspA- and OspC-encoding genes, the osp17 gene is present in strains not expressing the respective protein. It has been shown that OspA and OspC expression varies in different environments such as tick and vertebrate host. Studies are underway to examine whether this is also true for Osp17. For diagnostic purposes the use of recombinant Osp17 has the advantage that the amount of Osp17 antigen can be easily standardized for immunoblotting, and that this antigen can be used in a protein-specific enzyme-linked immunosorbent assay. Received: 18 December 1998     

Loss of pathogenic potential after cloning of the low-passage Borrelia burgdorferi ZS7 tick isolate: a cautionary note

To study clonal polymorphism of Borrelia burgdorferi antigens in the course of an experimental infection sequence, the low-passage tick isolate ZS7 was cloned by two rounds of agar subsurface plating. The resulting clones showed a variable pathogenic potential after experimental infection of C.B-17.scid mice. The test clone 4.2.II, selected for virulence by two passages in immunodeficient scid mice, failed to establish a successful infection in immunocompetent AKR/N mice, indicating the loss of pathogenicity traits required for evasion of the specific immune response. Cloning of natural or clinical B. burgdorferi isolates is a prerequisite for analyzing genetic and antigenic variation of the pathogen. However, the inevitable propagation in artificial media during cloning may lead to a loss of pathogenic features rendering the subsequent experimental infection of animals impossible. A combined procedure of in vitro cloning and in vivo selection also does not solve the dilemma because B. burgdorferi variants arise by recombinatorial processes in the pathogen's dynamic genome during the course of infection. Consequently, the resulting bacterial isolates from infected animal tissues represent again non-clonal, heterogeneous B. burgdorferi populations. In principle, cloning of a B. burgdorferi population is the appropriate method to analyze the polymorphism of individual molecules during infection. As a caveat, however, one has to envisage that during propagation of individual clones in vitro and in vivo independent genetic variations (epigenetic or mutational) may occur with consequences on the virulence of the clones. This may complicate the delineation of a clear correlation between the antigenic polymorphism observed and the change of virulence. Received: 4 October 1999     

Characterization of Borrelia burgdorferi sensu lato strains isolated from Ixodes ricinus in Mecklenburg-Vorpommern,Germany

Epidemiological studies in Mecklenburg-Vorpommern have shown a high prevalence ofBorrelia burgdorferi-infected ticks. A total of 17B. burgdorferi sensu lato strains were isolated from ticks and investigated by Western blots (immunoblot) with eight monoclonal antibodies against different epitopes of the outer surface protein A (OspA). Except for one, all strains could be classified using this system. The majority of strains belonged to theB. garinii-associated OspA serotypes 3, 5 and 6. Three isolates were classified as OspA serotype 2 (B. afzelii).B. burgdorferi sensu stricto strains (Ospa serotype 1) as well asB. garinii-associated OspA serotype 4 were not present.