本文選題：沙眼衣原體 + 質(zhì)粒蛋白��； 參考：《中南大學(xué)》2008年博士論文

【摘要】： 沙眼衣原體(Chlamydia trachomatis,Ct)泌尿生殖道感染是世界范圍內(nèi)嚴(yán)重危害公眾健康的一種細(xì)菌感染性疾病。Ct的生殖道感染常引起女性盆腔炎、宮頸炎等,常引起男性尿道炎、睪丸炎和前列腺炎等。由于無(wú)癥狀性衣原體感染的普遍存在,致使該疾病不易被發(fā)現(xiàn)而得不到及時(shí)治療,導(dǎo)致Ct在宿主體內(nèi)持續(xù)存在并引起不孕、異位妊娠等嚴(yán)重并發(fā)癥,故闡明Ct致病機(jī)制,尋找免疫優(yōu)勢(shì)抗原基因研制疫苗,是預(yù)防、控制Ct感染的關(guān)鍵。 質(zhì)粒蛋白與衣原體毒力直接相關(guān),O'Connell等報(bào)道質(zhì)粒缺失Ct不引起生殖道的病變,說(shuō)明質(zhì)粒蛋白在衣原體致病中發(fā)揮重要作用。包涵體(Inclusion,InC)膜蛋白能介導(dǎo)衣原體與宿主細(xì)胞的相互作用,鑒定新的InC蛋白可為衣原體與宿主細(xì)胞相互作用機(jī)制提供重要信息。因此,為闡明Ct質(zhì)粒蛋白致病機(jī)理,尋找新的膜定位分子以及研制有效的Ct疫苗,我們進(jìn)行了以下研究: 一、沙眼衣原體質(zhì)粒蛋白定位與生物學(xué)特性分析及質(zhì)粒蛋白pORF5核酸疫苗免疫效果研究 研究目的 構(gòu)建Ct 8種質(zhì)粒蛋白原核表達(dá)載體pGEX-6p-pCTs,制備單克隆抗體和多克隆抗體,旨在分析8種質(zhì)粒蛋白在衣原體感染細(xì)胞中的定位,探討衣原體致病機(jī)理;構(gòu)建真核表達(dá)載體pcDNA3.1-pORF5,與免疫佐劑pcDNA3.1-IL-12以鼻粘膜方式聯(lián)合免疫小鼠,建立小鼠生殖道感染模型,觀察生殖道局部病理變化以及其誘導(dǎo)小鼠產(chǎn)生體液免疫和細(xì)胞免疫應(yīng)答水平,為研制新型的Ct核酸疫苗提供實(shí)驗(yàn)依據(jù)。 研究方法 1、從GenBank中查詢Ct質(zhì)粒蛋白基因序列,設(shè)計(jì)并合成8對(duì)特異性引物,PCR擴(kuò)增8個(gè)質(zhì)粒蛋白全長(zhǎng)基因片段,構(gòu)建pGEX-6p-pCTs原核表達(dá)重組體;重組體經(jīng)IPTG誘導(dǎo)在E.coliXL1Blue中表達(dá)融合蛋白GST-pCTs,Glutathione Sepharose 4B Beads親和層析純化融合蛋白;純化的融合蛋白免疫BALB/c鼠,制備多克隆抗體,采用雜交瘤技術(shù)制備單克隆抗體;間接免疫熒光試驗(yàn)檢測(cè)8種質(zhì)粒蛋白在感染細(xì)胞中的定位及pORF5在感染細(xì)胞中的表達(dá)模式及存在方式;ELISA分析比較8種質(zhì)粒蛋白的免疫原性及pORF5質(zhì)粒蛋白體外刺激Raw264.7細(xì)胞產(chǎn)生細(xì)胞因子水平。 2、將pORF5基因亞克隆至真核表達(dá)載體構(gòu)建pcDNA3.1-pORF5重組體,以BALB/c為免疫動(dòng)物,采用鼻粘膜免疫方式進(jìn)行免疫。將小鼠分為pcDNA3.1空質(zhì)粒對(duì)照組、PBS對(duì)照組、pcDNA3.1-IL12對(duì)照組、pcDNA3.1-pORF5單獨(dú)免疫組、pcDNA3.1-pORF5與pcDNA3.1-IL12聯(lián)合免疫組;ELISA檢測(cè)血清和生殖道局部抗體滴度和細(xì)胞因子產(chǎn)生水平;MTT法檢測(cè)脾淋巴細(xì)胞特異性增殖反應(yīng);建立Ct鼠肺炎型(MoPn)生殖道感染模型,檢測(cè)衣原體生殖道攻擊后其清除情況、體重變化及局部組織病理學(xué)變化,綜合分析pORF5聯(lián)合疫苗免疫保護(hù)效果。 研究結(jié)果 1、以D型Ct基因組為模板PCR擴(kuò)增得到了8種質(zhì)粒蛋白全長(zhǎng)基因片段;構(gòu)建了質(zhì)粒蛋白原核表達(dá)系統(tǒng)pGEX-6p-pCTs,原核表達(dá)重組體均在E.coli中表達(dá)出相應(yīng)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親和層析純化得到了高純度的8種質(zhì)粒融合蛋白;制備了8種融合蛋白的多克隆抗體,各抗體效價(jià)均在1:6400以上;獲得了17株特異性且抗體分泌穩(wěn)定的雜交瘤細(xì)胞株。 2、8種質(zhì)粒蛋白能與Ct生殖道感染患者血清發(fā)生免疫反應(yīng),但反應(yīng)強(qiáng)度、反應(yīng)頻率不同,在所檢測(cè)的15個(gè)病人血清中,pORF5識(shí)別所有病人的血清,且免疫反應(yīng)最強(qiáng);N端缺失66氨基酸的F6片段在與患者血清ELISA反應(yīng)中,免疫反應(yīng)強(qiáng)度與pORF5全長(zhǎng)基本相似。 3、pORF5在感染細(xì)胞中的分布模式與CPAF一致,分布于宿主細(xì)胞胞漿中,但也少量分布在EB、RB上,其余7種質(zhì)粒蛋白與MOMP的分布模式相同,分布于衣原體菌體上;pORF5在Ct感染后12h就有表達(dá),常以單體、三聚體、多聚體的形式存在;胞漿表達(dá)pORF5不影響其后Ct的感染(p0.05)。 4、pORF5蛋白誘導(dǎo)Raw264.7細(xì)胞產(chǎn)生TNF-α、IL-6、IL-8的水平隨著pORF5濃度的升高而增多,當(dāng)pORF5蛋白的濃度升為10μg/mL時(shí),TNF-α、IL-6、IL-8的產(chǎn)生量分別為1658.87±255.34pg/mL、7511.55±720.13 pg/mL、4643.20±412.24 pg/mL。 5、pORF5單獨(dú)免疫組、pORF5與IL-12聯(lián)合免疫組血清抗體隨著接種次數(shù)的增多,滴度逐漸升高。第一次免疫后第3w部分小鼠血清中可明顯檢測(cè)到特異性抗體,第6w特異性抗體升高顯著,血清抗體持續(xù)升高,直至第9w抗體產(chǎn)生水平基本穩(wěn)定,聯(lián)合免疫組血清抗體總量及IgG2a抗體升高顯著,對(duì)照組的抗體未出現(xiàn)明顯變化;同時(shí),pORF5聯(lián)合免疫組生殖道局部粘膜抗體顯著高于單獨(dú)免疫組。5組中以聯(lián)合免疫組IFN-γ產(chǎn)生水平最高,淋巴細(xì)胞增殖反應(yīng)最強(qiáng)。 6、pORF5單獨(dú)免疫組、pORF5與IL-12聯(lián)合免疫組MoPn感染后體重下降較慢,恢復(fù)較快,分別在接種后第15d、12d體重恢復(fù)正常,3組對(duì)照組在接種MoPn后第21天體重恢復(fù)正常。pORF5單獨(dú)組、pORF5與IL-12聯(lián)合免疫組清除Ct的速度較早,分別在接種MoPn后第24d、18d完全清除,而對(duì)照組在接種MoPn后第30d完全清除。 7、3組對(duì)照組小鼠輸卵管壺腹部、峽部腫脹,管壁血管擴(kuò)張充血,管腔內(nèi)充滿透明液體,輸卵管單側(cè)或雙側(cè)積水;免疫組化結(jié)果顯示粘膜柱狀上皮細(xì)胞成矮柱狀,頂部纖毛顯著減少或消失,漿膜層毛細(xì)血管擴(kuò)張,大量淋巴細(xì)胞、漿細(xì)胞浸潤(rùn);pORF5單獨(dú)免疫組出現(xiàn)輸卵管管壁增厚,炎性細(xì)胞浸潤(rùn),聯(lián)合免疫組病理改變最輕;空質(zhì)粒組,IL-12組、PBS組小鼠炎癥積分分別為3.0±0.4、2.6±0.4、2.4±0.3明顯高于pORF5單獨(dú)免疫(1.5±0.3)、pORF5與IL-12聯(lián)合免疫小鼠(0.8±0.2)。 結(jié)論 1、成功地構(gòu)建了8種質(zhì)粒蛋白pGEX-6p-pCTs原核表達(dá)載體,并均在E.coli XL1Blue中表達(dá)出相應(yīng)的融合蛋白;成功地制備了17株分泌質(zhì)粒蛋白的單克隆抗體雜交瘤細(xì)胞株,所分泌的單克隆抗體具有特異性。 2、8種質(zhì)粒融合蛋白具有較好的免疫原性和免疫反應(yīng)性。 3、在自然感染狀態(tài)下,Ct 8種質(zhì)粒蛋白基因均被激活產(chǎn)生內(nèi)源性靶蛋白,其中,pORF5免疫原性最強(qiáng);pORF5為構(gòu)象依賴性抗原;在天然狀態(tài)下以單體、三聚體、多聚體等多種形式存在宿主細(xì)胞中。 4、首次證實(shí)pORF5為一種分泌性蛋白,這是繼CPAF以來(lái)迄今所發(fā)現(xiàn)的衣原體第二種分泌性蛋白,而其它7種蛋白均位于衣原體菌體上。 5、pORF5能刺激RAW264.7細(xì)胞產(chǎn)生TNF-α、IL-6、IL-8等前炎癥細(xì)胞因子,并具有劑量依賴性。 6、首次將pORF5核酸疫苗與IL-12聯(lián)合免疫小鼠,該聯(lián)合疫苗能刺激機(jī)體產(chǎn)生體液免疫應(yīng)答和細(xì)胞免疫應(yīng)答,減少輸卵管炎性病理改變,顯著改變衣原體感染的正常進(jìn)程,發(fā)揮了免疫保護(hù)作用。 二、沙眼衣原體預(yù)測(cè)的包涵體膜蛋白定位及特性研究 研究目的 構(gòu)建50種預(yù)測(cè)的Ct包涵體膜(InC)蛋白基因原核表達(dá)載體pGEX-6p-InCs,制備多克隆抗體,分析50種預(yù)測(cè)的InC蛋白在衣原體感染細(xì)胞中的定位及生物學(xué)特性,為闡明Ct致病機(jī)理提供實(shí)驗(yàn)依據(jù)。 研究方法 1、從GenBank中查詢預(yù)測(cè)的Ct InC基因序列,設(shè)計(jì)并合成50對(duì)特異性引物;PCR擴(kuò)增50個(gè)膜蛋白基因片段,構(gòu)建pGEX-6p-InCs原核表達(dá)重組體,重組體經(jīng)IPTG誘導(dǎo)在E.coli XL1Blue中表達(dá)融合蛋白GST-InCs;Glutathione Sepharose 4B Beads親和層析純化融合蛋白,Western-blot分析鑒定表達(dá)產(chǎn)物。 2、純化的融合蛋白與弗氏佐劑充分乳化后,腹腔免疫3～6周齡的BALB/c鼠,制備多克隆抗體,ELISA鑒定50種預(yù)測(cè)的InC免疫原性;間接免疫熒光實(shí)驗(yàn)分析預(yù)測(cè)的膜蛋白在感染細(xì)胞中的分布;同時(shí),檢測(cè)Ct在自然感染狀態(tài)下,InCs的表達(dá)情況及機(jī)體針對(duì)InC蛋白所產(chǎn)生抗體滴度水平。 3、將免疫優(yōu)勢(shì)InC基因分成N、C片段,重新克隆構(gòu)建pGEX-6p-InC/N重組體;所獲得的片段分別在E.coli XL1Blue誘導(dǎo)表達(dá)GST融合蛋白;Western blot鑒定其免疫原性部位,確定免疫優(yōu)勢(shì)表位。 4、將50種預(yù)測(cè)的膜蛋白基因克隆到pDSRed-C1真核表達(dá)載體中,構(gòu)建真核表達(dá)載體重組體pDSRed-C1-InCs,重組體轉(zhuǎn)染Hela細(xì)胞表達(dá)RFP融合蛋白,該RFP融合蛋白用于檢測(cè)小鼠產(chǎn)生的抗融合蛋白抗體的特異性及分析胞漿表達(dá)的RFP-InCs蛋白對(duì)衣原體感染的影響。 研究結(jié)果 1、成功地構(gòu)建了pGEX-6p-InCs原核表達(dá)載體,并均在E.coliXL1Blue中表達(dá)出相應(yīng)的重組融合蛋白;Glutathione Sepharose 4B親和層析純化得到了較高純度的融合蛋白;重組融合蛋白具有較強(qiáng)的免疫原性,能刺激小鼠產(chǎn)生較高滴度的抗體。 2、成功地構(gòu)建了pDSRed-C1-InCs真核表達(dá)載體,各基因均在HeLa細(xì)胞胞漿部位表達(dá)靶蛋白;胞漿表達(dá)CT119能顯著抑制Ct感染率,在轉(zhuǎn)染陽(yáng)性的HeLa細(xì)胞中衣原體的感染率為29.3±9.5%,而在轉(zhuǎn)染陰性的HeLa細(xì)胞中衣原體的感染率為64.1±15.4%。而其它49種預(yù)測(cè)的InC蛋白不影響衣原體的感染率(P0.05)。 3、CT089、CT115、CT116、CT118、CT119、CT147、CT223、CT225、CT226、CT228、CT229、CT442、CT529、CT618、CT813等15種包涵體蛋白與病人血清發(fā)生較強(qiáng)的免疫反應(yīng),為免疫優(yōu)勢(shì)抗原。進(jìn)一步分析15種包涵體蛋白的免疫原性部位,發(fā)現(xiàn)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分布模式與CT119相似,定位于包涵體膜上,為InC蛋白;CT058、CT192、CT195、CT383、CT484、CT565、CT850分布模式與MOMP、HSP60相似,為衣原體菌體蛋白。 結(jié)論 1、首次證實(shí)CT225、CT228、CT358、CT440為包涵體膜蛋白,CT058、CT192、CT195、CT383、CT484、CT565、CT850為衣原體菌體蛋白。 2、多數(shù)InC蛋白具有較強(qiáng)的免疫原性,可作為疫苗的侯選抗原。 3、InC蛋白的免疫原性主要定位于C端。 4、胞漿表達(dá)的RFP融合蛋白,除了CT119外,其它49種預(yù)測(cè)InC蛋白不影響衣原體的感染。
[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.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類號(hào)】：R374

【共引文獻(xiàn)】

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