沙眼衣原体质粒蛋白及预测的包涵体膜蛋白定位与生物学特性研究

发布时间：2018-05-17 12:50

本文选题：沙眼衣原体 + 质粒蛋白　；参考：《中南大学》2008年博士论文

【摘要】： 沙眼衣原体(Chlamydia trachomatis,Ct)泌尿生殖道感染是世界范围内严重危害公众健康的一种细菌感染性疾病。Ct的生殖道感染常引起女性盆腔炎、宫颈炎等,常引起男性尿道炎、睾丸炎和前列腺炎等。由于无症状性衣原体感染的普遍存在,致使该疾病不易被发现而得不到及时治疗,导致Ct在宿主体内持续存在并引起不孕、异位妊娠等严重并发症,故阐明Ct致病机制,寻找免疫优势抗原基因研制疫苗,是预防、控制Ct感染的关键。质粒蛋白与衣原体毒力直接相关,O'Connell等报道质粒缺失Ct不引起生殖道的病变,说明质粒蛋白在衣原体致病中发挥重要作用。包涵体(Inclusion,InC)膜蛋白能介导衣原体与宿主细胞的相互作用,鉴定新的InC蛋白可为衣原体与宿主细胞相互作用机制提供重要信息。因此,为阐明Ct质粒蛋白致病机理,寻找新的膜定位分子以及研制有效的Ct疫苗,我们进行了以下研究: 一、沙眼衣原体质粒蛋白定位与生物学特性分析及质粒蛋白pORF5核酸疫苗免疫效果研究研究目的构建Ct 8种质粒蛋白原核表达载体pGEX-6p-pCTs,制备单克隆抗体和多克隆抗体,旨在分析8种质粒蛋白在衣原体感染细胞中的定位,探讨衣原体致病机理;构建真核表达载体pcDNA3.1-pORF5,与免疫佐剂pcDNA3.1-IL-12以鼻粘膜方式联合免疫小鼠,建立小鼠生殖道感染模型,观察生殖道局部病理变化以及其诱导小鼠产生体液免疫和细胞免疫应答水平,为研制新型的Ct核酸疫苗提供实验依据。研究方法 1、从GenBank中查询Ct质粒蛋白基因序列,设计并合成8对特异性引物,PCR扩增8个质粒蛋白全长基因片段,构建pGEX-6p-pCTs原核表达重组体;重组体经IPTG诱导在E.coliXL1Blue中表达融合蛋白GST-pCTs,Glutathione Sepharose 4B Beads亲和层析纯化融合蛋白;纯化的融合蛋白免疫BALB/c鼠,制备多克隆抗体,采用杂交瘤技术制备单克隆抗体;间接免疫荧光试验检测8种质粒蛋白在感染细胞中的定位及pORF5在感染细胞中的表达模式及存在方式;ELISA分析比较8种质粒蛋白的免疫原性及pORF5质粒蛋白体外刺激Raw264.7细胞产生细胞因子水平。 2、将pORF5基因亚克隆至真核表达载体构建pcDNA3.1-pORF5重组体,以BALB/c为免疫动物,采用鼻粘膜免疫方式进行免疫。将小鼠分为pcDNA3.1空质粒对照组、PBS对照组、pcDNA3.1-IL12对照组、pcDNA3.1-pORF5单独免疫组、pcDNA3.1-pORF5与pcDNA3.1-IL12联合免疫组;ELISA检测血清和生殖道局部抗体滴度和细胞因子产生水平;MTT法检测脾淋巴细胞特异性增殖反应;建立Ct鼠肺炎型(MoPn)生殖道感染模型,检测衣原体生殖道攻击后其清除情况、体重变化及局部组织病理学变化,综合分析pORF5联合疫苗免疫保护效果。研究结果 1、以D型Ct基因组为模板PCR扩增得到了8种质粒蛋白全长基因片段;构建了质粒蛋白原核表达系统pGEX-6p-pCTs,原核表达重组体均在E.coli中表达出相应的融合蛋白,各融合蛋白主要以可溶性的形式存在;采用Glutathione Sepharose 4B Beads亲和层析纯化得到了高纯度的8种质粒融合蛋白;制备了8种融合蛋白的多克隆抗体,各抗体效价均在1:6400以上;获得了17株特异性且抗体分泌稳定的杂交瘤细胞株。 2、8种质粒蛋白能与Ct生殖道感染患者血清发生免疫反应,但反应强度、反应频率不同,在所检测的15个病人血清中,pORF5识别所有病人的血清,且免疫反应最强;N端缺失66氨基酸的F6片段在与患者血清ELISA反应中,免疫反应强度与pORF5全长基本相似。 3、pORF5在感染细胞中的分布模式与CPAF一致,分布于宿主细胞胞浆中,但也少量分布在EB、RB上,其余7种质粒蛋白与MOMP的分布模式相同,分布于衣原体菌体上;pORF5在Ct感染后12h就有表达,常以单体、三聚体、多聚体的形式存在;胞浆表达pORF5不影响其后Ct的感染(p0.05)。 4、pORF5蛋白诱导Raw264.7细胞产生TNF-α、IL-6、IL-8的水平随着pORF5浓度的升高而增多,当pORF5蛋白的浓度升为10μg/mL时,TNF-α、IL-6、IL-8的产生量分别为1658.87±255.34pg/mL、7511.55±720.13 pg/mL、4643.20±412.24 pg/mL。 5、pORF5单独免疫组、pORF5与IL-12联合免疫组血清抗体随着接种次数的增多,滴度逐渐升高。第一次免疫后第3w部分小鼠血清中可明显检测到特异性抗体,第6w特异性抗体升高显著,血清抗体持续升高,直至第9w抗体产生水平基本稳定,联合免疫组血清抗体总量及IgG2a抗体升高显著,对照组的抗体未出现明显变化;同时,pORF5联合免疫组生殖道局部粘膜抗体显著高于单独免疫组。5组中以联合免疫组IFN-γ产生水平最高,淋巴细胞增殖反应最强。 6、pORF5单独免疫组、pORF5与IL-12联合免疫组MoPn感染后体重下降较慢,恢复较快,分别在接种后第15d、12d体重恢复正常,3组对照组在接种MoPn后第21天体重恢复正常。pORF5单独组、pORF5与IL-12联合免疫组清除Ct的速度较早,分别在接种MoPn后第24d、18d完全清除,而对照组在接种MoPn后第30d完全清除。 7、3组对照组小鼠输卵管壶腹部、峡部肿胀,管壁血管扩张充血,管腔内充满透明液体,输卵管单侧或双侧积水;免疫组化结果显示粘膜柱状上皮细胞成矮柱状,顶部纤毛显著减少或消失,浆膜层毛细血管扩张,大量淋巴细胞、浆细胞浸润;pORF5单独免疫组出现输卵管管壁增厚,炎性细胞浸润,联合免疫组病理改变最轻;空质粒组,IL-12组、PBS组小鼠炎症积分分别为3.0±0.4、2.6±0.4、2.4±0.3明显高于pORF5单独免疫(1.5±0.3)、pORF5与IL-12联合免疫小鼠(0.8±0.2)。结论 1、成功地构建了8种质粒蛋白pGEX-6p-pCTs原核表达载体,并均在E.coli XL1Blue中表达出相应的融合蛋白;成功地制备了17株分泌质粒蛋白的单克隆抗体杂交瘤细胞株,所分泌的单克隆抗体具有特异性。 2、8种质粒融合蛋白具有较好的免疫原性和免疫反应性。 3、在自然感染状态下,Ct 8种质粒蛋白基因均被激活产生内源性靶蛋白,其中,pORF5免疫原性最强;pORF5为构象依赖性抗原;在天然状态下以单体、三聚体、多聚体等多种形式存在宿主细胞中。 4、首次证实pORF5为一种分泌性蛋白,这是继CPAF以来迄今所发现的衣原体第二种分泌性蛋白,而其它7种蛋白均位于衣原体菌体上。 5、pORF5能刺激RAW264.7细胞产生TNF-α、IL-6、IL-8等前炎症细胞因子,并具有剂量依赖性。 6、首次将pORF5核酸疫苗与IL-12联合免疫小鼠,该联合疫苗能刺激机体产生体液免疫应答和细胞免疫应答,减少输卵管炎性病理改变,显著改变衣原体感染的正常进程,发挥了免疫保护作用。二、沙眼衣原体预测的包涵体膜蛋白定位及特性研究研究目的构建50种预测的Ct包涵体膜(InC)蛋白基因原核表达载体pGEX-6p-InCs,制备多克隆抗体,分析50种预测的InC蛋白在衣原体感染细胞中的定位及生物学特性,为阐明Ct致病机理提供实验依据。研究方法 1、从GenBank中查询预测的Ct InC基因序列,设计并合成50对特异性引物;PCR扩增50个膜蛋白基因片段,构建pGEX-6p-InCs原核表达重组体,重组体经IPTG诱导在E.coli XL1Blue中表达融合蛋白GST-InCs;Glutathione Sepharose 4B Beads亲和层析纯化融合蛋白,Western-blot分析鉴定表达产物。 2、纯化的融合蛋白与弗氏佐剂充分乳化后,腹腔免疫3～6周龄的BALB/c鼠,制备多克隆抗体,ELISA鉴定50种预测的InC免疫原性;间接免疫荧光实验分析预测的膜蛋白在感染细胞中的分布;同时,检测Ct在自然感染状态下,InCs的表达情况及机体针对InC蛋白所产生抗体滴度水平。 3、将免疫优势InC基因分成N、C片段,重新克隆构建pGEX-6p-InC/N重组体;所获得的片段分别在E.coli XL1Blue诱导表达GST融合蛋白;Western blot鉴定其免疫原性部位,确定免疫优势表位。 4、将50种预测的膜蛋白基因克隆到pDSRed-C1真核表达载体中,构建真核表达载体重组体pDSRed-C1-InCs,重组体转染Hela细胞表达RFP融合蛋白,该RFP融合蛋白用于检测小鼠产生的抗融合蛋白抗体的特异性及分析胞浆表达的RFP-InCs蛋白对衣原体感染的影响。研究结果 1、成功地构建了pGEX-6p-InCs原核表达载体,并均在E.coliXL1Blue中表达出相应的重组融合蛋白;Glutathione Sepharose 4B亲和层析纯化得到了较高纯度的融合蛋白;重组融合蛋白具有较强的免疫原性,能刺激小鼠产生较高滴度的抗体。 2、成功地构建了pDSRed-C1-InCs真核表达载体,各基因均在HeLa细胞胞浆部位表达靶蛋白;胞浆表达CT119能显著抑制Ct感染率,在转染阳性的HeLa细胞中衣原体的感染率为29.3±9.5%,而在转染阴性的HeLa细胞中衣原体的感染率为64.1±15.4%。而其它49种预测的InC蛋白不影响衣原体的感染率(P0.05)。 3、CT089、CT115、CT116、CT118、CT119、CT147、CT223、CT225、CT226、CT228、CT229、CT442、CT529、CT618、CT813等15种包涵体蛋白与病人血清发生较强的免疫反应,为免疫优势抗原。进一步分析15种包涵体蛋白的免疫原性部位,发现除了CT223,CT529、CT618抗原表位定位于N端外,其余InC蛋白的抗原表位定位于C端。 4、CT225、CT228、CT358、CT440分布模式与CT119相似,定位于包涵体膜上,为InC蛋白;CT058、CT192、CT195、CT383、CT484、CT565、CT850分布模式与MOMP、HSP60相似,为衣原体菌体蛋白。结论 1、首次证实CT225、CT228、CT358、CT440为包涵体膜蛋白,CT058、CT192、CT195、CT383、CT484、CT565、CT850为衣原体菌体蛋白。 2、多数InC蛋白具有较强的免疫原性,可作为疫苗的侯选抗原。 3、InC蛋白的免疫原性主要定位于C端。 4、胞浆表达的RFP融合蛋白,除了CT119外,其它49种预测InC蛋白不影响衣原体的感染。
[Abstract]:Chlamydia trachomatis (Ct) genitourinary tract infection is a worldwide bacterial infection of the public health. The genital tract infection of.Ct often causes female pelvic inflammation, cervicitis, and so on. It often causes male urethritis, orchitis and prostatitis. The prevalence of asymptomatic Chlamydia infection is common. The disease is not easily found and can not be found in time, which leads to the persistent existence of Ct in the host and causes severe complications such as infertility and ectopic pregnancy. Therefore, it is the key to prevent and control Ct infection by clarifying the pathogenesis of Ct and searching for the immune dominant antigen gene.
The plasmid protein is directly related to the virulence of Chlamydia. O'Connell and other reports that the deletion of plasmid Ct does not cause the disease of the genital tract. It shows that the plasmid protein plays an important role in the pathogenesis of Chlamydia. The inclusion body (Inclusion, InC) membrane protein can mediate the interaction between Chlamydia and host cells, and the new InC protein can be identified as the chlamydia and the host cell. The interaction mechanism provides important information. Therefore, in order to elucidate the pathogenesis of Ct plasmid protein, search for new membrane localization molecules and develop effective Ct vaccines, we have conducted the following studies:
First, the location and biological characteristics of Chlamydia trachomatis plasmid protein and the immunogenicity of plasmid DNA pORF5 DNA vaccine.
research objective
Ct 8 plasmid protein prokaryotic expression vector pGEX-6p-pCTs was constructed to prepare monoclonal antibody and polyclonal antibody. The aim of this study was to analyze the location of 8 plasmids in Chlamydia infected cells, explore the pathogenesis of Chlamydia, construct eukaryotic expression vector pcDNA3.1-pORF5, and immunize mice with immune adjuvant pcDNA3.1-IL-12 in nasal mucosa. To establish a mouse reproductive tract infection model, observe the local pathological changes of the reproductive tract, and to induce the level of humoral and cellular immune responses in mice, and provide experimental basis for the development of a new type of Ct nucleic acid vaccine.
research method
1, query Ct plasmid protein gene sequence from GenBank, design and synthesize 8 pairs of specific primers, PCR amplification of 8 full length gene fragment of plasmid protein, and construct pGEX-6p-pCTs prokaryotic expression recombinant. The recombinant body is induced by IPTG to express fusion protein GST-pCTs in E.coliXL1Blue, Glutathione Sepharose 4B Beads affinity chromatography purification fusion protein; pure fusion protein is purified by Glutathione Sepharose 4B; BALB/c mice were immunized with fusion protein to prepare polyclonal antibodies and to prepare monoclonal antibodies by hybridoma. Indirect immunofluorescence test was used to detect the localization of 8 plasmids in infected cells and the expression pattern and existence mode of pORF5 in infected cells. The immunogenicity of the 8 plasmids and pORF5 plasmid protein were compared by ELISA analysis. In vitro stimulation of Raw264.7 cells produces cytokine levels.
2, the pORF5 gene was subcloned to the eukaryotic expression vector to construct pcDNA3.1-pORF5 recombinant, and BALB/c was used as immune animal and immunized by nasal mucosa. The mice were divided into pcDNA3.1 empty plasmid control group, PBS control group, pcDNA3.1-IL12 control group, pcDNA3.1-pORF5 single immunization group, pcDNA3.1-pORF5 and pcDNA3.1-IL12 combined immune group, ELIS, ELIS. A detected the titer of serum and reproductive tract local antibody and the level of cytokine production; MTT assay was used to detect the specific proliferation of splenic lymphocytes; establish a Ct rat pneumonia type (MoPn) reproductive tract infection model, detect the clearance of Chlamydia after genital attack, body weight change and local histopathological changes, and comprehensive analysis of the pORF5 joint vaccine immunization Protect the effect.
Research results
1, 8 kinds of full length gene fragments of plasmid protein were amplified with the D Ct genome as template PCR, and the plasmid protein prokaryotic expression system pGEX-6p-pCTs was constructed. The prokaryotic expression recombinant expressed the corresponding fusion protein in E.coli, and the fusion proteins were deposited mainly in soluble form; Glutathione Sepharose 4B Beads affinity chromatography was used. 8 plasmid fusion proteins with high purity were purified and polyclonal antibodies of 8 kinds of fusion proteins were prepared. The titers of each antibody were above 1:6400, and 17 specific hybridoma cell lines with stable antibody secretion were obtained.
The 2,8 plasmids can react with the sera of the patients with Ct genital tract infection, but the reaction intensity and frequency are different. In the serum of the 15 patients detected, pORF5 recognizes all the patients' serum and the immune response is the strongest; the F6 fragment of the 66 amino acid missing from the N terminal is in the response to the patient's serum, the immune response intensity and the pORF5 full length base. This is similar.
3, the distribution pattern of pORF5 in infected cells was consistent with CPAF, distributed in the cytoplasm of host cells, but also distributed in EB, RB, and the other 7 plasmids were the same distribution pattern as MOMP; pORF5 was expressed in 12h after Ct infection, often in the form of monomers, trimers and polymers; cytoplasm expressed pORF5 Infection (P0.05) that affects the subsequent Ct.
4, pORF5 protein induced Raw264.7 cells to produce TNF- alpha, IL-6, IL-8 increased with the increase of pORF5 concentration. When the concentration of pORF5 protein increased to 10 mu, the production of TNF- alpha, IL-6, IL-8 was 1658.87 + 720.13 255.34pg/mL, 7511.55 + 720.13, 4643.20 + 412.24.
5, pORF5 alone immune group, pORF5 and IL-12 combined immunization group antibody with the increase of the number of inoculation, the titer gradually increased. After the first immunization, the serum of part 3W can detect specific antibodies, 6W specific antibody increased significantly, serum antibody continued to rise, until the level of 9W antibody production was basically stable, combined immunization The serum antibody and IgG2a antibody increased significantly in the epidemic group, and the antibody in the control group had no obvious change. At the same time, the local mucosal antibody in the pORF5 combined immunization group was significantly higher than that in the.5 group of the single immunization group, with the highest level of IFN- gamma production in the combined immunization group and the strongest lymphocyte proliferation reaction.
6, pORF5 alone immune group, pORF5 and IL-12 combined immune group MoPn infection after MoPn decreased slowly, recovered faster, after inoculation, 15d, 12D weight resumed normal, the 3 groups in the twenty-first day after the inoculation of MoPn to restore normal.PORF5 alone group, pORF5 and IL-12 combined immune group to clear the Ct rate earlier, respectively after inoculation MoPn after vaccination, respectively, MoPn after MoPn MoPn, Marxism respectively after MoPn MoPn MoPn after MoPn MoPn, respectively, MoPn after MoPn, MoPn, MoPn, respectively. The control group was completely removed, while the control group was completely removed after 30d was inoculated with MoPn.
In group 7,3, the control group was swollen in the ampulla of the oviduct, isthmus, swelling of the isthmus, dilatation and congestion of the vessel wall, full of transparent liquid in the lumen, unilateral or bilateral water in the tubal. The immunohistochemical results showed that the columnar epithelium of the mucous membrane became short columnar, the cilium at the top was reduced or disappeared, the serous capillaries expanded, a large number of lymphocytes and plasma cells infiltrated. 5 the tubal wall thickening, inflammatory cell infiltration and the lightest pathological changes in the combined immune group were found in the individual immunization group. The inflammatory scores in the empty plasmid group, IL-12 group and PBS group were 3 + 0.4,2.6 + 0.4,2.4 + 0.3 respectively higher than pORF5 alone (1.5 + 0.3), and pORF5 and IL-12 combined immunized mice (0.8 + 0.2).
conclusion
1, 8 plasmids protein pGEX-6p-pCTs prokaryotic expression vector was successfully constructed, and the corresponding fusion protein was expressed in E.coli XL1Blue. The monoclonal antibody hybridoma cell line of 17 plasmid proteins was successfully prepared, and the secreted monoclonal antibody was specific.
The fusion protein of 2,8 plasmid has good immunogenicity and immunoreactivity.
3, under natural infection, the 8 plasmid protein genes of Ct were activated to produce endogenous target proteins, in which the pORF5 immunogenicity was the strongest, pORF5 was conformation dependent antigen, and in natural state, there were host cells in a variety of forms, such as monomers, trimers and polymers.
4, pORF5 was first confirmed as a secretory protein, which was the second secretory protein of Chlamydia found so far since CPAF, and the other 7 proteins were located on the Chlamydia.
5, pORF5 stimulates RAW264.7 cells to produce proinflammatory cytokines such as TNF-, IL-6, IL-8 and so on, and dose dependently.
6, the combination of pORF5 nucleic acid vaccine and IL-12 immunized mice for the first time. The combined vaccine can stimulate the body's humoral and cellular immune responses, reduce the pathological changes of tubal inflammation, significantly change the normal process of Chlamydia infection, and play the role of immune protection.
Two, the location and characterization of inclusion body membrane proteins predicted by Chlamydia trachomatis.
research objective
50 kinds of Ct inclusion body membrane (InC) protein gene prokaryotic expression vector pGEX-6p-InCs was constructed, and polyclonal antibody was prepared. The location and biological characteristics of the 50 predicted InC proteins in Chlamydia infected cells were analyzed to provide experimental basis for elucidating the pathogenesis of Ct.
research method
1, query and predict the Ct InC gene sequence from GenBank, design and synthesize 50 pairs of specific primers; PCR amplification of 50 membrane protein gene fragments, construct pGEX-6p-InCs prokaryotic expression recombinant, IPTG inducement in E.coli XL1Blue to express the fusion protein GST-InCs; Glutathione Sepharose 4B affinity chromatography purification fusion protein, Blot analysis identified the expression products.
2, after the purified fusion protein was fully emulsified with Freund's adjuvant, the intraperitoneal immunized BALB/c mice of 3~6 weeks old were prepared to prepare polyclonal antibodies. ELISA was used to identify the 50 predicted InC immunogenicity; indirect immunofluorescence assay was used to predict the distribution of membrane proteins in the infected cells; meanwhile, the expression of InCs and the body in the natural infection state of the Ct and the body were detected. The level of antibody titer produced by InC protein.
3, the immune dominant InC gene was divided into N, C fragment and recloned to construct pGEX-6p-InC/N recombinant. The obtained fragments were induced to express GST fusion protein in E.coli XL1Blue, Western blot identified the immunogenicity site and determined the immune dominance epitopes.
4, 50 kinds of predicted membrane protein genes were cloned into the pDSRed-C1 eukaryotic expression vector, and the eukaryotic expression body weight group pDSRed-C1-InCs was constructed. The recombinant plasmid was transfected with Hela cells to express RFP fusion protein. The RFP fusion protein was used to detect the specificity of anti fusion protein antibody produced in mice and the analysis of cytoplasmic expression of RFP-InCs protein to Chlamydia. The influence of infection.
Research results
1, the pGEX-6p-InCs prokaryotic expression vector was successfully constructed and the corresponding recombinant fusion protein was expressed in E.coliXL1Blue; the purified fusion protein was purified by Glutathione Sepharose 4B affinity chromatography, and the recombinant fusion protein had strong immunogenicity and could stimulate the antibody of high titer in mice.
2, the pDSRed-C1-InCs eukaryotic expression vector was successfully constructed. Each gene expressed the target protein in the cytoplasm of HeLa cells. Cytoplasmic expression of CT119 could significantly inhibit the infection rate of Ct. The infection rate of Chlamydia in the transfected HeLa cells was 29.3 + 9.5%, while the infection rate of Chlamydia in the negative transfected HeLa cells was 64.1 + 15.4%. and the other 49 was 49. The predicted InC protein did not affect Chlamydia infection rate (P0.05).
3, CT089, CT115, CT116, CT118, CT119, CT147, CT223, CT225, CT226, CT228, CT229, CT442, CT118, etc. The epitopes of the InC protein are located at the C end.
4, CT225, CT228, CT358, CT440 distribution pattern is similar to CT119, located on the inclusion body membrane, InC protein, CT058, CT192, CT195, CT383, CT484, CT565, similar, as the Chlamydia bacterial protein.
conclusion
1, for the first time, CT225, CT228, CT358 and CT440 were identified as inclusion body membrane proteins, CT058, CT192, CT195, CT383, CT484, CT565, and CT850 as chlamydial proteins.
2, most of the InC proteins have strong immunogenicity and can be used as vaccine candidate antigens.
3, the immunogenicity of InC protein is mainly located at the C end.
4, cytoplasmic expression of RFP fusion protein, except CT119, the other 49 predicted InC protein did not affect Chlamydia infection.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R374

【共引文献】