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Plasmid instability when the hsp60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant bcg

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American Journal of Molecular Biology 2013年第2期3卷 81-86页

作者： Dilzamar V.Nascimento Odir A.Dellagostin Raphael Hirata Jr Geraldo M.B.Pereira Ana Luiza Mattos-Guaraldi Geraldo R.Garcia Armoa Faculdade de Ciencias Médicas Universidade do Estado do Rio de JaneiroRio de JaneiroBrazil Centro de Biotecnologia Universidade Federal de PelotasCampus UniversitárioPelotasBrazil Instituto Oswaldo Cruz Fundacao Oswaldo CruzRio de JaneiroBrazil

The genetic modification of the live attenuated Mycobacterium bovis bcg to deliver a protective Corynebacterium diphtheriae antigen in vivo could be a safer and less costly alternative to the new and more expensive DTP vaccines available today, in particular to third world-countries. The stability of expression of heterologous antigens in bcg, however, is a major challenge to the use of live recombinant bacteria in vaccine development and appears to be dependent to a certain extent, on a genetic compatibility between the expression cassette within the plasmid construct and the mycobacterium host. In the quest for the best recombinant bcg transformant to express the dtb gene of C. diphtheriae we generated two new rbcg strains by transforming the Moreau substrain of bcg with the mycobacterial expression vectors pUS973 and pUS977, each one carrying a different promoter to drive the expression of the target antigen. After transformation recombinant bcg clones were selected on Middlebrook 7H10 kanamycin Agar plates, expanded in Middlebrook 7H9 kanamycin Broth and analyzed by agarose gel electrophoresis and immunoblotting. rbcgs transformed with the construct carrying the weak PAN promoter from M. paratuberculosis stably expressed the dtb gene. Conversely, rbcgs transformed with the construct carrying the strong mycobacterium hsp60 promoter were unstable and consequently unfit for the expression of the C. diphtheriae gene.

关键词： bcg Vaccine recombinant bcg Fragment B of Diphtheria Toxin Anti-Diphtheria Vaccine

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The Progress of bcg and recombinant bcg in Development of Vaccines against Infectious Diseases and Tumor Immunotherapy

The Progress of BCG and Recombinant BCG in Development of Va...

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第十届全国免疫学学术大会

作者： Yuanqiang Zheng Yumei Zhou Lu Lv Zhenli Chen Caixia Wang Zhe Chen Sen Xu Lan Wu Xinrong Han Zhengrong Cui Yanchun Shi Inner Mongolia Key Laboratory of Molecular Biology Inner Mongolia Medical University School of Basic Medical Science Beijing University of Chinese Medicine School of Nursing Inner Mongolia Medical University Pharmaceutics Division College of Pharmacy the University of Texas at Austin

Bacillus Calmette-Guerin(bcg) vaccines are attenuated live strains of Mycobacterium bovis and are among the most widely used vaccines in the world. bcg is proven effective in preventing severe infant meningitis and miliary tuberculosis. Intravesical instillation of bcg is also a standard treatment for non-muscle invasive bladder cancer. bcg has numerous favorable properties such as its extensive safety record in humans(i.e. bcg has been used in humans for about 100 years), thermal stability, low production costs, adjuvant activity(i.e. a strong inducer of long-lasting CD4+ type 1 helper T cell(Th1) and humoral immunity), and ability to induce mucosal immunity when administered by a proper mucosal route(e.g. oral, intrarectal, or intranasal). bcg is also an attractive bacterial vector in developing vaccine candidates using recombinant bcg(r bcg) technology, which is based on the use of bcg bacteria as a vector to express foreign genes. In the past few decades, r bcg technology had been extensively applied to develop vaccine candidates for a variety of infectious diseases, including bacterial, viral, and parasite infections, and to improve the efficacy of bcg in bladder cancer therapy. This review is intended to show the vast applications of bcg and r bcg in prevention of infectious diseases and in cancer immunotherapy, with a special emphasis on the recent approaches and trends on both preclinical and clinical levels.

关键词： bcg recombinant bcg vaccine development infectious disease tumor

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THE CONSTRUCTION AND EXPRESSION OF recombinant SHUTTLE PLASMID WITH OMPL1 GENE FROM LEPTOSPIRA INTERROGANS SEROVAR LAI STRAIN 017 IN BACILLE CALMETTE-GUERIN

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Chinese Medical Sciences Journal 2002年第2期17卷 81-84页

作者：鲍朗邱洪宇晏菊芳谢勇恩陈玮 Res. U. Leptospirosis/Infection West China Univ. of Med. Sci. Chengdu 610041 China

*** construct recombinant bcg again st *** amplified the entire open readin g frame of the OmpL1gene from the genome of the leptospire serovar Lai strain *** recombin ant plasmids pBQ1and pBQ2were constructed by oriented ligation based on the ***-bcg shuttle plasmids pMV261and *** recombinant plasmids were transformed into bcg by *** rbcgs bearing pBQ1and pBQ2were induced by high temperature of 45℃.*** expressed product,a 35kD prote in was detected by *** resu lt indicates that pBQ1and pBQ2can express OmpL1in *** technical methods in this study may help detect the immunogenicity a nd immunoprotection of OmpL1and develop more safe,highl y effective rbcg bearing leptospira l antigen with long-lasting protection.

关键词： Leptospira interrogans serovar Lai recombinant bcg OmpL1gene

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Mycobacterium bovis bcg as a Delivery System for the dtb Gene Antigen from Diphtheria Toxin

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American Journal of Molecular Biology 2017年第4期7卷 176-189页

作者： Dilzamar V.Nascimento Odir A.Dellagostin Denise C.S.Matos Douglas McIntosh Raphael Hirata Jr. Geraldo M.B.Pereira Ana Luíza Mattos-Guaraldi Geraldo R.G.Armoa Faculdade de Ciências Médicas Universidade do Estado do Rio de JaneiroRio de JaneiroBrazil Núcleo de Biotecnologia Universidade Federal de PelotasCampus UniversitárioPelotasBrazil Instituto de Tecnologia em Imunobiológicos Fundacao Oswaldo CruzRio de JaneiroBrazil Instituto Oswaldo Cruz Fundacao Oswaldo CruzRio de JaneiroBrazil Instituto de Veterinária Universidade Federal Rural do Rio de JaneiroSeropedicaRio de JaneiroBrazil

Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of bcg which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into bcg cells by electroporation. Mice immunized with recombinant bcg expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rbcgs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rbcgpUS977dtbPW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay.

关键词： recombinant bcg Diphtheria Toxin dtb Gene Park Williams 8(PW8) Corynebacterium diphtheriae rDTBPW8 pUS977 Vector

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Evaluation of a recombinant bacillus calmette-guérin vaccine expressing P39-L7/L12 of Brucella melitensis:An immunization strategy against brucellosis in BALB/c mice

Evaluation of a recombinant bacillus calmette-guérin vaccin...

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第十四届全国免疫学学术大会

作者： Yanchun Shi Yuanqiang Zheng Yajing Chen Inner Mongolia Key Laboratory of Molecular Biology Inner Mongolia Medical University

Brucellosis is a chronic infectious disease caused by bacteria from the genus *** has a serious global economic impact,and poses a threat to public ***,vaccines should exhibit a safe profile as well as enhanced protective ***,there are no licensed vaccines available for humans against *** this study,for the first time,a recombinant bcg(rbcg) vaccine expressing the P39-L7/1 2 fusion protein of Brucella melitensis against brucellosis was *** rbcg vaccine elicited strong antigen-specific humoral and T cell-mediated immunity in *** rbcg vaccine also induced a significant level of protection following challenge with a virulent strain of *** M28 in BALB/c ***,the protection level induced by the rbcg vaccine was sig nificantly higher than that induced by ***,these results suggested that rbcg vaccine acted as a suitable vaccine candidate and a new strategy against human brucellosis.d as a suitable vaccine candidate and a new strategy against human brucellosis.

关键词： recombinant bcg Vaccine P39-L7/12 Brucellosis

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结核杆菌潜伏抗原—建立重组卡介苗的新策略

结核杆菌潜伏抗原—建立重组卡介苗的新策略

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中华医学会结核病学分会2010年学术年会

作者：范雄林华中科技大学同济医学院病原生物学系

目的评价以结核杆菌潜伏抗原为基础的重组卡介苗的抗结核保护效果。方法基因工程技术分别建立过表达结核杆菌潜伏抗原HspX,结核杆菌活跃生长分泌抗原Ag85B的重组卡介苗。分别免疫BALB/C和C57BL/6小鼠,ELISA检测抗原特异性抗体;Elispot... 详细信息

目的评价以结核杆菌潜伏抗原为基础的重组卡介苗的抗结核保护效果。方法基因工程技术分别建立过表达结核杆菌潜伏抗原HspX,结核杆菌活跃生长分泌抗原Ag85B的重组卡介苗。分别免疫BALB/C和C57BL/6小鼠,ELISA检测抗原特异性抗体;Elispot检测抗原特异的IFN-γ;并用结核杆菌H37Rv毒株攻击感染免疫小鼠,分析感染小鼠肺脏、脾脏含细菌数量,肺脏病理改变和病理评分。结果分别成功构建过表达结核杆菌潜伏抗原HspX的重组卡介苗rbcg::X和过表达结核杆菌活跃生长分泌抗原Ag85B的重组卡介苗rbcg::85B。rbcg::X可诱导免疫小鼠产生高水平的抗原特异性的抗体产生,以及高水平的IFN-γ。rbcg::X可提供显著抗结核杆菌的攻击感染。结论结核杆菌的潜伏抗原用于发展重组卡介苗预防结核具有重要的前景。

关键词： recombinant bcg Tuberculosis Latency antigen Vaccine HspX

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