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 DT...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.展开更多
目的 :将rBCG IL 2应用于动物实验 ,观察其对肿瘤生长和免疫功能的影响。方法 :采用基因工程技术构建rBCG IL 2 ,用黑色素瘤细胞株B16接种于小鼠小腿外侧皮下 ,再用rBCG IL 2局部治疗 ,2周后处死小鼠 ,用ELISA法检测脾淋巴细胞培养上清...目的 :将rBCG IL 2应用于动物实验 ,观察其对肿瘤生长和免疫功能的影响。方法 :采用基因工程技术构建rBCG IL 2 ,用黑色素瘤细胞株B16接种于小鼠小腿外侧皮下 ,再用rBCG IL 2局部治疗 ,2周后处死小鼠 ,用ELISA法检测脾淋巴细胞培养上清中IL 2 ,IFN γ和GM CSF的含量和肉眼及镜下观察肿瘤生长和局部免疫效应细胞浸润情况。结果 :rBCG IL 2对肿瘤生长的抑制作用明显优于对照组 ,肿瘤细胞周围有较多的巨噬细胞和淋巴细胞浸润 ,肿瘤细胞减少。同时rBCG IL 2还能诱导机体产生IL 2和IFN γ。结论 :rBCG IL 2能通过诱导局部免疫效应细胞浸润和机体细胞因子的产生 ,抑制肿瘤生长 。展开更多
Objective.To construct recombinant BCG again st leptospirosis.Methods.We amplified the entire open readin g frame of the OmpL1gene from the genome of the leptospire serovar Lai strain 017.Two recombin ant plasmids pBQ...Objective.To construct recombinant BCG again st leptospirosis.Methods.We amplified the entire open readin g frame of the OmpL1gene from the genome of the leptospire serovar Lai strain 017.Two recombin ant plasmids pBQ1and pBQ2were constructed by oriented ligation based on the E.coli-BCG shuttle plasmids pMV261and pMV361respectively.The recombinant plasmids were transformed into BCG by electroporation.The rBCGs bearing pBQ1and pBQ2were induced by high temperature of 45℃.Results.The expressed product,a 35kD prote in was detected by SDS-PAGE.The resu lt indicates that pBQ1and pBQ2can express OmpL1in rBCG.Conclusion.The 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.展开更多
基金Research supported by Bio-Manguinhos/FIOCRUZ,PAPES II/FIOCRUZ,FAPERJ,CNPq,CAPES,Programa de Núcleo de Excelencia(PRONEX/MCT/CNPq).
文摘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.
文摘Objective.To construct recombinant BCG again st leptospirosis.Methods.We amplified the entire open readin g frame of the OmpL1gene from the genome of the leptospire serovar Lai strain 017.Two recombin ant plasmids pBQ1and pBQ2were constructed by oriented ligation based on the E.coli-BCG shuttle plasmids pMV261and pMV361respectively.The recombinant plasmids were transformed into BCG by electroporation.The rBCGs bearing pBQ1and pBQ2were induced by high temperature of 45℃.Results.The expressed product,a 35kD prote in was detected by SDS-PAGE.The resu lt indicates that pBQ1and pBQ2can express OmpL1in rBCG.Conclusion.The 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.
