衣原体质粒蛋白在感染细胞中的定位及生物学特性初步研究
作者:
李忠玉;吴移谋;钟光明
作者机构:
[李忠玉; 吴移谋; 钟光明] 南华大学医学院病原生物学研究所;[李忠玉; 吴移谋; 钟光明] University;[李忠玉; 吴移谋; 钟光明] of;[李忠玉; 吴移谋; 钟光明] Texas;[李忠玉; 吴移谋; 钟光明] Health
会议名称:
中华医学会2010’全国临床微生物及感染免疫学术研讨会
会议时间:
2010-7-30
会议地点:
上海
会议主办单位:
中华医学会
会议论文集名称:
中华医学会2010’全国临床微生物及感染免疫学术研讨会论文集
语种:
中文
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淋球菌porB和大肠杆菌ltB融合基因的构建、表达及其免疫活性
作者:
戴志兵;胡四海;陈敏;陆春雪;王玉峰;...
期刊:
微生物学报 ,2010年50(4):517-523 ISSN:0001-6209
通讯作者:
Dai, Z.
作者机构:
[戴志兵; 胡四海; 陈敏; 陆春雪; 王玉峰; 张愉快; 余敏君; 朱翠明; 李忠玉] 南华大学,病原生物学研究所;[刘清南] 南华大学,心血管疾病研究所
关键词:
淋病奈瑟菌;孔蛋白B;大肠杆菌不耐热肠毒素B亚单位;基因融合;原核表达;免疫活性
摘要:
[目的]构建融合基因原核表达载体pET-30a/1tB-porB,并表达重组融合蛋白LTB- PorB,鼻饲途径免疫雌性BALB/c小鼠,分析重组融合蛋白的免疫活性,为研制抗淋病蛋白疫苗提供实验依据.[方法]构建大肠杆菌不耐热肠毒素B亚单位(LTB)与淋球菌外膜孔蛋白B(PorB)融合基因及LTB、PorB单基因pET-30a原核表达载体,在大肠杆菌BL21中表达重组蛋白;鼻饲途径免疫雌性BALB/c小鼠,检测体液免疫和细胞免疫水平.[结果]在大肠杆菌BL21中获得高效表达的重组蛋白;经鼻饲免疫小鼠后,重组融合蛋白LTB-PorB组生殖道黏膜产生的PorB特异性slgA水平随免疫时间呈上升趋势,第42天A_(450)值达0.66,明显高于对照组(P<0.01),效价高达1:1280;血清中产生的PorB特异性IgG第28天达最高,A_(450)值为0.60,明显高于LTB和蛋白溶解液(Solution Buffer)对照组(P<0.01),效价高达1:2560,但与PorB对照组血清IgG水平(A_(450):0.57)无明显差异(P>0.05).LTB-PorB组脾淋巴细胞刺激指数明显高于LTB和Solution Buffer对照组(P<0.05),但脾淋巴细胞诱生的IFN-γ水平与对照组无明显差异(P>0.05).[结论]重组融合蛋白LTB-PorB通过鼻饲途径免疫雌性BALB/c小鼠后,能诱导产生高水平的体液免疫和一定水平的细胞免疫.首次证实黏膜佐剂LTB可辅佐PorB诱导小鼠产生高水平的生殖道粘膜免疫.
语种:
中文
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Serodiagnosis of Chlamydia pneumoniae Infection Using Three Inclusion Membrane Proteins
作者:
Chen Hongliang;Zhou Zhou;Hu Zhan;Zeng Yanhua;Li Zhongyu;...
期刊:
Journal of Clinical Laboratory Analysis ,2010年24(1):55-61 ISSN:0887-8013
通讯作者:
Wu Yimou
作者机构:
[Wu Yimou; Zhou Zhou; Zeng Yanhua; Hu Zhan; Chen Hongliang; Li Zhongyu] Univ S China, Coll Med, Inst Pathogen Biol, Hengyang 421001, Hunan, Peoples R China.;[Lin Yingbiao; Dai Guozhi] ChenZhou 1 Peoples Hosp, Clin Lab, Chenzhou, Peoples R China.;[Wu Yimou] Univ S China, Coll Med, Inst Pathogen Biol, Changsheng Rd, Hengyang 421001, Hunan, Peoples R China.
通讯机构:
[Wu Yimou] U;Univ S China, Coll Med, Inst Pathogen Biol, Changsheng Rd, Hengyang 421001, Hunan, Peoples R China.
关键词:
Chlamydophila pneumoniae;serodiagnosis;recombinant antigens;ELISA
摘要:
The Chlamydia pneumoniae genome-ncoded open reading frames Cpn0146, Cpn0147, and Cpn0308 were expressed as recombinant proteins for detecting C. pneumoniae-specific antibodies in samples from three groups of individuals including 183 with C. pneumoniae-associated respiratory infection (group 1), 60 healthy blood donors (group 11), and 32 with no known respiratory infection (group 111). The recombinant Cpn0146 was recognized by 71 (38.8% positive recognition rate), 15 (25%) and 1 (3.1%), Cpn0147 by 75 (40.9%), 14 (23.3%), and 2 (6.3%), and Cpn0308 by 82 (44.8%), 16 (26.7%), and 0 (0%) samples from groups I, II, and IIIs, respectively. The positive recognition rates with any of the three antigens were significantly higher in group I than those in groups II and III, suggesting that more individuals from group I were likely infected with C. pneumoniae. This conclusion was confirmed with a commercially available whole organism-based ELISA kit (Savyon Diagnostics Ltd., Ashdod, Israel), which detected C. pneumoniae antibodies in 98 (64.1%), 26 (43.3%), and 4 (12.5%) samples from group I, II, and III, respectively. Comparing to the commercial kit, the recombinant antigen-based detection assays displayed >97% of detection specificity and >87% of sensitivity, suggesting that these recombinant antigens can be considered alternative tools for aiding in serodiagnosis of C. pneumoniae infection. J. Clin. Lab. Anal. 24:55-61, 2010. (C) 2010 Wiley-Liss, Inc.
语种:
英文
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鹦鹉嗜衣原体禽鸟株的分离鉴定及小鼠呼吸道感染模型的建立
作者:
唐国芳;陈丽丽;刘良专;李忠玉;王绍胜;...
作者机构:
湖南省南华大学病原生物学研究所
会议名称:
2010中国人兽共患病学术交流会
会议时间:
2010-11-12
会议地点:
湖南衡阳
会议论文集名称:
2010中国人兽共患病学术交流会论文集
语种:
中文
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利用多媒体技术,改革医学微生物学实验教学
作者:
李忠玉;吴移谋;胡四海;张艳
期刊:
山西医科大学学报 ,2009年11(3):346-348 ISSN:1007-6611
作者机构:
南华大学医学院微生物学与免疫学教研室,衡阳,421001
关键词:
医学微生物学;多媒体技术;实验教学
摘要:
运用多媒体技术,打破医学微生物学实验课程传统的教学模式,改革实验教学,是提高实验教学质量的关键。结合医学微生物学实验的教学特点,分析了运用多媒体技术进行教学改革的优势和应注意的问题,为实验教学改革提供一些经验。
语种:
中文
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CT358蛋白在沙眼衣原体感染细胞中的定位及特性分析
作者:
Li Zhong-Yu;Wu Yi-Mou;Huang Qiu-Lin;Wang Shi-Ping* ;Zhong Guang-Ming
期刊:
生物化学与生物物理进展 ,2009年36(5):549-555 ISSN:1000-3282
通讯作者:
Wang Shi-Ping
作者机构:
[Wu Yi-Mou; Li Zhong-Yu] Univ S China, Dept Microbiol & Immunol, Hengyang 421001, Peoples R China.;[Wang Shi-Ping] Cent S Univ, Xiangya Sch Med, Inst Schistososmiasis Res, Changsha 410078, Hunan, Peoples R China.;[Huang Qiu-Lin] Univ S China, Affiliated Hosp 1, Hengyang 421001, Peoples R China.;[Zhong Guang-Ming] Univ Texas Hlth Sci Ctr San Antonio, San Antonio, TX 78229 USA.
通讯机构:
[Wang Shi-Ping] C;Cent S Univ, Xiangya Sch Med, Inst Schistososmiasis Res, Changsha 410078, Hunan, Peoples R China.
关键词:
沙眼衣原体;包涵体膜蛋白
摘要:
确定沙眼衣原体CT358蛋白在衣原体感染细胞中的位置并初步鉴定其生物学功能.采用PCR方法从D型沙眼衣原体的基因组中扩增CT358基因,并克隆入pGEX和pDSRedC1表达载体中,将重组质粒pGEX-CT358转化到XL1-blue宿主菌,并诱导表达融合蛋白GST-CT358.纯化后的CT358融合蛋白免疫小鼠制备抗体,应用间接免疫荧光技术对CT358蛋白在衣原体感染细胞内的定位及表达模式进行分析.同时,pDSRedC1-CT358重组质粒瞬时转染HeLa细胞,观察CT358蛋白对衣原体感染的影响.实验结果证明CT358蛋白为沙眼衣原体包涌体膜蛋白.该蛋白质在衣原体感染12 h后就表达定位于包涵体膜上,直至持续到整个感染周期,转基因在胞浆表达的CT358融合蛋白不影响其后的衣原体感染.该研究为深入研究衣原体与宿主细胞间相互作用提供了新的线索,并可为衣原体性的治疗、预防提供新方向.
语种:
中文
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Antibodies from women urogenitally infected with C. trachomatis predominantly recognized the plasmid protein pgp3 in a conformation-dependent manner
作者:
Li, Zhongyu;Zhong, Youmin;Lei, Lei;Wu, Yimou;Wang, Shiping;...
期刊:
BMC Microbiology ,2008年8(1):1-13 ISSN:1471-2180
通讯作者:
Zhong, G
作者机构:
[Li, Zhongyu; Zhong, Youmin; Lei, Lei; Zhong, Guangming] Univ Texas Hlth Sci Ctr San Antonio, Dept Microbiol & Immunol, San Antonio, TX 78229 USA.;[Li, Zhongyu; Wang, Shiping] Cent S Univ, Dept Parasitol, Xiangya Med Sch, Changsha 410078, Hunan, Peoples R China.;[Li, Zhongyu; Wu, Yimou] Univ S China, Dept Microbiol & Immunol, Hengyang 421001, Hunan, Peoples R China.
通讯机构:
[Zhong, G ] ;Univ Texas Hlth Sci Ctr San Antonio, Dept Microbiol & Immunol, 7703 Floyd Curl Dr, San Antonio, TX 78229 USA.
关键词:
Fusion Protein;Human Antibody;Chlamydial Infection;Major Outer Membrane Protein;Cryptic Plasmid
摘要:
Background. C. trachomatis organisms carry a cryptic plasmid that encodes 8 open reading frames designated as pORF1 to 8. It is not clear whether all 8 pORFs are expressed during C. trachomatis infection in humans and information on the functionality of the plasmid proteins is also very limited. Results. When antibodies from women urogenitally infected with C. trachomatis were reacted with the plasmid proteins, all 8 pORFs were positively recognized by one or more human antibody samples with the recognition of pORF5 protein (known as pgp3) by most antibodies and with the highest titers. The antibody recognition of the pORFs was blocked by C. trachomatis-infected HeLa but not normal HeLa cell lysates. The pgp3 fusion protein-purified human IgG detected the endogenous pgp3 in the cytosol of C. trachomatis-infected cells with an intracellular distribution pattern similar to that of CPAF, a chlamydial genome-encoded protease factor. However, the human antibodies no longer recognized pgp3 but maintained recognition of CPAF when both antigens were linearized or heat-denatured. The pgp3 conformation is likely maintained by the C-terminal 75% amino acid sequence since further deletion blocked the binding by the human antibodies and two conformation-dependent mouse monoclonal antibodies. Conclusion. The plasmid-encoded 8 proteins are both expressed and immunogenic with pgp3 as the most immunodominant antigen during chlamydial infection in humans. More importantly, the human anti-pgp3 antibodies are highly conformation-dependent. These observations have provided important information for further understanding the function of the plasmid-encoded proteins and exploring the utility of pgp3 in chlamydial diagnosis and vaccination. © 2008 Li et al; licensee BioMed Central Ltd.
语种:
英文
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肺炎嗜衣原体的一种分泌型蛋白在临床诊断中的应用价值
作者:
郑江花;吴移谋;丁滔;李忠玉;陈丽丽;...
作者机构:
[郑江花; 吴移谋; 丁滔; 李忠玉; 陈丽丽; 刘双全] 湖南省衡阳市南华大学医学院病原生物学研究所;[郑江花; 吴移谋; 丁滔; 李忠玉; 陈丽丽; 刘双全] 南华大学附属一医院;[郑江花; 吴移谋; 丁滔; 李忠玉; 陈丽丽; 刘双全] 南华大学附属二医院
会议名称:
中华医学会第七次全国检验医学学术会议
会议时间:
2008-05
会议地点:
中国山东济南
关键词:
肺炎嗜衣原体;衣原体蛋白酶样活性因子;重组蛋白;抗原性;临床诊断
摘要:
目的表达、纯化肺炎嗜衣原体(Cpn)蛋白酶样活性因子(CPAF)免疫优势区重组蛋白,建立间接ELISA法,探讨其在Cpn感染临床诊断中的应用价值。方法构建CPAF免疫优势区基因重组质粒.诱导表达并纯化重组蛋白,分析其抗原特异性:间接ELISA法检测Cpn参考血清、临床血清标本中的特异性IgM抗体,以及呼吸道感染患者痰咽拭子中的CDp抗原;检测沙眼衣原体(Chlamydia trachomatis.
语种:
中文
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Characterization of fifty putative inclusion membrane proteins encoded in the Chlamydia trachomatis genome
作者:
Li, Zhongyu;Chen, Chaoqun;Chen, Ding;Wu, Yimou;Zhong, Youmin;...
期刊:
Infection and Immunity ,2008年76(6):2746-2757 ISSN:0019-9567
通讯作者:
Zhong, G
作者机构:
[Li, Zhongyu; Chen, Ding; Zhong, Youmin; Zhong, Guangming] Univ Texas Hlth Sci Ctr San Antonio, Dept Microbiol & Immunol, San Antonio, TX 78229 USA.;[Li, Zhongyu] Cent S Univ, Xiangya Med Sch, Dept Parasitol, Changsha 410083, Peoples R China.;[Li, Zhongyu; Wu, Yimou; Chen, Chaoqun] Univ S China, Dept Microbiol & Immunol, Hengyang, Hunan, Peoples R China.;[Zhong, Guangming] Univ Texas Hlth Sci Ctr San Antonio, Dept Microbiol & Immunol, 7703 Floyd Curl Dr, San Antonio, TX 78229 USA.
通讯机构:
[Zhong, G] U;Univ Texas Hlth Sci Ctr San Antonio, Dept Microbiol & Immunol, 7703 Floyd Curl Dr, San Antonio, TX 78229 USA.
关键词:
Bacterial Proteins;Chlamydia trachomatis;Gene Expression Profiling;Gene Expression Regulation, Bacterial;membrane proteins
摘要:
Although the Chlamydia trachomatis genome is predicted to encode 50 inclusion membrane proteins, only 18 have been experimentally localized in the inclusion membrane of C. trachomatis-infected cells. Using fusion proteins and anti-fusion protein antibodies, we have systematically evaluated all 50 putative inclusion membrane proteins for their localization in the infected cells, distribution patterns, and effects on subsequent chlamydial infection when expressed ectopically, as well as their immunogenicity during chlamydial infection in humans. Twenty-two of the 50 proteins were localized in the inclusion membrane, and 7 were detected inside the inclusions, while the location of the remaining 21 was not defined. Four (CT225, CT228, CT358, and CT440) of the 22 inclusion membrane-localized proteins were visualized in the inclusion membrane of Chlamydia-infected cells for the first time in the current study. The seven intra-inclusion-localized proteins were confirmed to be chlamydial organism proteins in a Western blot assay. Further characterization of the 50 proteins revealed that neither colocalization with host cell endoplasmic reticulum nor inhibition of subsequent chlamydial infection by ectopically expressed proteins correlated with the inclusion membrane localization. Interestingly, antibodies from women with C. trachomatis urogenital infection preferentially recognized proteins localized in the inclusion membrane, and the immunodominant regions were further mapped to the region predicted to be on the cytoplasmic side of the inclusion membrane. These observations suggest that most of the inclusion membrane-localized proteins are both expressed and immunogenic during C. trachomatis infection in humans and that the cytoplasmic exposure may enhance the immunogenicity. Copyright © 2008, American Society for Microbiology. All Rights Reserved.
语种:
英文
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肺炎嗜衣原体蛋白酶样活性因子重组蛋白的血清学诊断价值初探
作者:
郑江花;吴移谋;丁滔;陈丽丽;莫小辉;...
期刊:
国际检验医学杂志 ,2008年29(11):970-972+975 ISSN:1673-4130
作者机构:
南华大学附属第二医院检验科,湖南衡阳,421000;南华大学医学院病原生物学研究所,湖南衡阳,421001;南华大学附属第一医院泌尿外科,湖南衡阳,421001;[陈丽丽; 李忠玉; 吴移谋; 莫小辉] 南华大学;[丁滔] 南华大学第一附属医院
关键词:
衣原体;肺炎;重组蛋白质类;免疫活性;临床实验室技术
摘要:
目的 克隆、表达肺炎嗜衣原体(Cpn)蛋白酶样活性因子(CPAF)的免疫优势区基因(CPAFm),初步评价其在血清学诊断中的应用价值.方法 构建pGEX6p-2/CPAFm重组质粒,诱导表达重组蛋白,十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定结果;将纯化蛋白免疫新西兰兔,间接酶联免疫吸附试验(ELISA)检测其免疫原性;与人抗Cpn抗血清反应分析其免疫反应性.间接ELISA法检测Cpn IgG和沙眼衣原体(Ct)阳性血清.结果 高效表达和纯化出一相对分子质量约为51.3×103的重组蛋白,蛋白质印迹法(Western blot)证明其能与人抗Cpn IgG抗体发生反应;在被免疫的新西兰兔体内,特异性IgG抗体的滴度为1∶16 000以上,间接ELISA法检测40例Cpn IgG参考血清,阴性和阳性结果的符合率均为100.0%(20/20);与微量免疫荧光法(MIF)对照,检测300例临床血清标本中的IgG抗体,符合率为98.3%;检测Ct阳性血清未见交叉反应.结论 表达的Cpn CPAF免疫优势区重组蛋白具有良好的免疫活性,在Cpn的血清学诊断中具有较高的应用价值.
语种:
中文
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Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice
作者:
Li ZhongYu;Wang ShiPing* ;Wu YiMou;Zhong GuangMing;Chen Ding
期刊:
中国科学:生命科学(英文版) ,2008年51(11):973-980 ISSN:1674-7305
通讯作者:
Wang ShiPing
作者机构:
[Wang ShiPing; Li ZhongYu] Cent S Univ, Xiangya Sch Med, Changsha 410078, Hunan, Peoples R China.;Univ Texas Hlth Sci Ctr, San Antonio, TX 78229 USA.;[Wu YiMou; Li ZhongYu] Univ S China, Dept Microbiol & Immunol, Hengyang 421001, Peoples R China.;[Zhong GuangMing; Chen Ding] Univ Texas Hlth Sci Ctr San Antonio, San Antonio, TX 78229 USA.
通讯机构:
[Wang ShiPing] C;Cent S Univ, Xiangya Sch Med, Changsha 410078, Hunan, Peoples R China.
关键词:
Chlamydia trachomatis;pORF5;DNA vaccine;immune protection;Th1;immune response
摘要:
To validate the immune protective efficacy of pORF5 DNA vaccine and to analyze potential mechanisms related to this protection. In this study, pORF5 DNA vaccine was constructed and evaluated for its protective immunity in a mouse model of genital chlamydial infection. Groups of BALB/c mice were immunized intranasally with pORF5 DNA vaccine. Humoral and cell mediated immune responses were evaluated. The clearance ability of chlamydial challenge from the genital tract and the chlamy- dia-induced upper genital tract gross pathology and histopathological characterization were also de- tected. The results showed that the total and the IgG2a anti-pORF5 antibody levels in serum were sig- nificantly elevated after pcDNA3.1-pORF5 vaccination, as were the total antibody and IgA levels in vaginal fluids. pcDNA3.1-pORF5 induced a significantly high level of Th1 response as measured by robust gamma interferon (IFN-γ). Minimal IL-4 was produced by immune T cells in response to the re-stimulation with pORF5 protein or the inactive elementary body in vitro. pcDNA3.1-pORF5-vacci- nated mice displayed significantly reduced bacterial shedding upon a chlamydial challenge and an accelerated resolution of infection. 100% of pcDNA3.1-pORF5 vaccinated mice successfully resolved the infection by day 24. pcDNA3.1-pORF5-immunized mice also exhibited protection against patho- logical consequences of chlamydial infection. The stimulated index was significantly higher than that of mice immunized with pcDNA3.1 and PBS (P<0.05). Together, these results demonstrated that immu- nization with pORF5 DNA vaccine is a promising approach for eliciting a protective immunity against a genital chlamydial challenge.
语种:
英文
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穿透支原体脂质相关膜蛋白诱导人单核细胞产生炎症性细胞因子
作者:
吴移谋;曾焱华;邓仲良;詹利生;李忠玉;...
期刊:
中华传染病杂志 ,2006年24(4):251-253 ISSN:1000-6680
作者机构:
[吴移谋; 曾焱华; 邓仲良; 詹利生; 李忠玉; 陈超群] 南华大学医学院病原生物学研究所
关键词:
细胞;艾滋病;病毒
摘要:
穿透支原体(M.penetrans,Mpe)是从感染人类免疫缺陷病毒的患者尿液中分离出来的,被称为艾滋病相关支原体.
语种:
中文
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pEGFP/MOMP真核表达重组体的构建及表达
作者:
粟盛梅;李忠玉;余敏君;占利生;唐双阳
期刊:
中国麻风皮肤病杂志 ,2005年21(7):509-511 ISSN:1009-1157
作者机构:
南华大学医学院病原生物学研究所,湖南衡阳,421001;[唐双阳; 占利生; 李忠玉; 余敏君; 粟盛梅] 南华大学
关键词:
真核表达重组体;真核表达重组质粒;HeLa细胞;沙眼衣原体;PCR扩增;主要外膜蛋白;真核表达载体;全基因片段;PCR方法;阳性重组子;脂质体介导;核酸疫苗;表达情况;序列测定;基因组;克隆人;特异性;D型;酶切;鉴定
摘要:
目的:克隆沙眼衣原体主要外膜蛋白(MOMP)基因,构建pEGFP/MOMP真核表达重组质粒,为沙眼衣原体核酸疫苗的研制提供依据.方法:用PGR方法从D型Ct基因组中扩增MOMP全基因片段,克隆入真核表达载体pEGFP相应的酶切位点中,阳性重组子经BamH I、Kpn I双酶切、PCR扩增及测序鉴定;并经脂质体介导转染HeLa细胞,36 h后观察瞬时表达情况.结果:PCR扩增得到约1.2kb的特异性MOMP基因片段;序列测定证实与GenBank登录的D型沙眼衣原体一致;筛选鉴定出真核表达重组体pEGFP/MOMP.结论:成功地构建了pEGFP/MOMP真核表达重组体,并在HeLa细胞中表达了MOMP.
语种:
中文
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沙眼衣原体外膜蛋白2重组蛋白的表达纯化和免疫性鉴定
作者:
陈超群;吴移谋;李忠玉;尹卫国;谭立志
期刊:
中华检验医学杂志 ,2005年28(7):697-699 ISSN:1009-9158
作者机构:
421001,衡阳,南华大学医学院微生物学教研室
关键词:
衣原体;沙眼;细菌外膜蛋白质类;免疫性
摘要:
目的 表达沙眼衣原体外膜蛋白2,纯化表达产物,并对其免疫性进行鉴定.方法应用聚合酶链反应(PCR)技术获得沙眼衣原体D型外膜蛋白2第167~434位氨基酸的编码基因片段,将此片段克隆于表达载体pET28b(+),转化大肠埃希菌BL21(DE3),IPTG诱导表达,SDS-PAGE、蛋白印迹试验分析表达产物,亲和层析法纯化重组蛋白;重组蛋白免疫家兔以检测其免疫原性.结果重组质粒酶切分析及DNA测序证实成功构建pET28b(+)/Omp2aa167~aa434表达质粒;通过优化表达和纯化条件,获得了相对分子质量约为35.0×103纯化蛋白产物,蛋白印迹试验证实该重组蛋白能与Ct 感染者阳性血清反应;ELISA法测定免疫血清特异性抗体效价在1:1 280以上.结论表达的重组外膜蛋白2aa167~aa434具有良好的免疫性.
语种:
中文
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鹦鹉热衣原体rrn操纵元序列双重PCR检测方法的建立
作者:
唐国芳;陈丽丽;刘良专;李忠玉;王绍胜;...
期刊:
中国农业大学学报 ,2005年10(5):65-68 ISSN:1007-4333
作者机构:
[唐国芳; 陈丽丽; 刘良专; 李忠玉; 王绍胜; 徐磊; 吴移谋] 湖南省南华大学病原生物学研究所
关键词:
鹦鹉热衣原体;rrn操纵元序列;双重PCR;检测
摘要:
为快速检测鹦鹉热衣原体,基于鹦鹉热衣原体rrn操纵元序列(即16S-23S rRNA间隔区基因和23S rRNA基因序列)建立了双重PCR法,对100份临床标本进行检测,并与单一PCR法、免疫荧光法进行了比较.结果显示:双重PCR法特异性与敏感性均高于单一PCR法、免疫荧光法.双重PCR平均阳性检出率为83.4%~97.2%,敏感性为500 fg/μL.结果显示鹦鹉热衣原体rrn操纵元序列双重PCR法不仅能够检测衣原体和非衣原体感染,而且能够同时实现鹦鹉热衣原体检测和鉴定,从而克服了单一引物应用时的不足,为未来鹦鹉热衣原体病分子流行病学调查及临床早期诊断提供了有效的检测手段.
语种:
中文
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Humoral and cellular immune responses induced by DNA vaccine based on major outer membrane protein of chlamydia trachomatis
作者:
李忠玉;吴移谋;余敏君;陈超群
期刊:
免疫学杂志 ,2005年21(6):457-459 ISSN:1000-8861
作者机构:
[李忠玉; 吴移谋; 余敏君; 陈超群] Department of Microbiology, School of Medicine, Nanhua University
关键词:
沙眼衣原体;DNA疫苗;体液免疫应答;细胞免疫应答
摘要:
目的构建沙眼衣原体主要外膜蛋白核酸疫苗,并观察其诱导小鼠产生的体液免疫和细胞免疫.方法将核酸疫苗(pcDNA3.1-MOMP)或对照空质粒(pcDNA3.1)注射于4~6周龄小鼠后腿股四头肌,每次剂量为100mg.间隔2周加强免疫2次.末次免疫后,ELISA法测定脾淋巴细胞培养上清液中IFN-γ及小鼠血清中抗MOMP水平;MTT法测定脾淋巴细胞特异性增殖反应.结果 小鼠接种核酸疫苗后,能产生特异性抗体,第3次免疫后抗体最高滴度达1:1024,培养上清液中IFN-γ达(532.0±45.4)pg/mL;实验组小鼠脾淋巴细胞刺激指数为3.94±0.25,其抗原特异性反应明显高于对照组.结论 沙眼衣原体主要外膜蛋白核酸疫苗能刺激机体产生特异的细胞免疫和体液免疫.
语种:
中文
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沙眼衣原体Omp2的原核表达与鉴定(英文)
作者:
陈超群;吴移谋;李忠玉;朱翠明;尹卫国
期刊:
中华性传播感染杂志:英文版 ,2004年(2):67-71+132 ISSN:1608-6260
作者机构:
DepartmentofMicrobiology,CollegeofMedicalScience,NanhuaUniversity,Hengyang,Hunan421001,China
关键词:
原核表达;外膜蛋白2;衣原体;沙眼;重组器官;质粒
摘要:
Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 × His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coil M15, providing an efficient and simple system for assaying the immunological properties of Omp2.
语种:
英文
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沙眼衣原体60 kDa外膜蛋白基因的克隆与表达
作者:
陈超群;吴移谋;李忠玉;朱翠明;余敏君
期刊:
实用预防医学 ,2004年11(1):7-10 ISSN:1006-3110
作者机构:
南华大学医学院微生物学教研室,中国湖南,衡阳,421001
关键词:
沙眼衣原体;omp2基因;基因克隆;表达
摘要:
目的克隆和表达沙眼衣原体60 kDa外膜蛋白(Omp2)基因. 方法 D型沙眼衣原体感染McCoy细胞后,抽提基因组DNA,经PCR扩增出omp2基因片段,与pUCm-T克隆载体连接,酶切纯化后克隆到原核表达载体pQE30,构建重组表达载体pQE30/omp2,PCR、酶切及测序鉴定.IPTG诱导表达,SDS-PAGE检测有无蛋白的表达. 结果 (1)获得长约1 650 bp的PCR产物, 酶切结果显示所构建的重组质粒已成功地克隆了omp2 基因,序列分析结果与已知omp2序列相同;(2)SDS-PAGE检测表达产物,在相对分子量60 kDa处有表达条带;(3)诱导表达之菌体超声破碎后,目的蛋白主要以包涵体形式存在. 结论获得了Ct omp2基因片段,并在E.coli M15中成功表达.
语种:
中文
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沙眼衣原体ompA基因真核表达载体的构建及在HeLa细胞中的表达
作者:
李忠玉;吴移谋;陈超群;尹卫国
期刊:
实用预防医学 ,2004年11(2):212-214 ISSN:1006-3110
作者机构:
南华大学医学院病原生物学研究所,中国湖南衡阳,421001;[尹卫国; 李忠玉; 吴移谋; 陈超群] 南华大学
关键词:
ompA基因;转染;真核表达
摘要:
目的构建沙眼衣原体(Ct)ompA基因真核表达重组质粒,利用脂质体体外转染HeLa细胞,为核酸疫苗的研制作准备.方法应用PCR技术从D型Ct基因组中扩增ompA全长基因,重组入pUCm-T克隆载体.将pUCm-T/ompA中的ompA外源基因片段经酶切、连接等反应,亚克隆人pcDNA3.1真核表达载体的相应位点,进行序列分析和酶切鉴定后,运用脂质体将重组体pcDNA3.1/ompA转染HeLa细胞,免疫组化法观察目的基因的表达.结果PCR扩增得到约1.2kb的特异性ompA基因片段;序列测定证实与发布序列一致;构建得到真核表达重组体;重组质粒pcDNA3.1/ompA在HeLa表达MOMP.结论Ct ompA基因能够在体外真核细胞中表达,为进一步研究CtDNA疫苗以及研究该蛋白的生物学功能提供实验基础.
语种:
中文
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沙眼衣原体主要外膜蛋白在大肠杆菌中的表达与鉴定
作者:
李忠玉;吴移谋;陈超群;余敏君
期刊:
中华皮肤科杂志 ,2004年37(12):709-711 ISSN:0412-4030
作者机构:
[李忠玉; 吴移谋; 陈超群; 余敏君] 南华大学医学院微生物学教研室;南华大学医学院微生物学教研室 421001湖南省衡阳市
关键词:
衣原体;沙眼;细菌外膜蛋白质类;基因表达
摘要:
目的克隆沙眼衣原体主要外膜蛋白(MOMP)基因,构建原核表达重组载体,并通过E.coliBL21实现MOMP的融合表达.方法用PCR法扩增MOMP基因,克隆插入到pUCm-T载体中,序列测定正确后,将其亚克隆到原核表达载体pET-22b(+)中,采用Ni-NTA亲和层析法纯化表达产物,对表达产物进行SDS-PAGE和蛋白质印迹鉴定.结果PCR扩增出约1 200bp DNA片段,序列测定证实与基因库登陆的D型沙眼衣原体一致;表达产物的相对分子质量为47000,与预期分子质量相符,蛋白印迹证实表达产物为特异性蛋白.结论沙眼衣原体MOMP基因可以在大肠杆菌中得到表达,其表达产物能与相应的抗体结合,为沙眼衣原体核酸疫苗的研究奠定了基础.
语种:
中文
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