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:用直接偶联法构建新型蛋白兔抗人IgGCRM9.方法:分别应用异型双功能连接剂(m Maleimido benzoyl N hydoxysuccinimideester,MBS)和N琥珀酰亚胺3(2吡啶二巯基)丙酸盐(SPDP)将兔抗人IgG多抗和白喉毒素突变体(CRM9)进行连接,制备出新...目的:用直接偶联法构建新型蛋白兔抗人IgGCRM9.方法:分别应用异型双功能连接剂(m Maleimido benzoyl N hydoxysuccinimideester,MBS)和N琥珀酰亚胺3(2吡啶二巯基)丙酸盐(SPDP)将兔抗人IgG多抗和白喉毒素突变体(CRM9)进行连接,制备出新的蛋白.结合后的蛋白经SephacrylS300分离纯化,用聚丙烯酰胺凝胶电泳和琼脂糖电泳证明二者的结合情况.采用酶联免疫法检测IgG与CRM9按不同比例反应后所构建的蛋白复合体中抗体部分的活性.结果:用MBS连接剂连接IgG与CRM9后的产物经聚丙烯酰胺凝胶电泳显示出的条带明显滞后于IgG所显示的条带,而在琼脂糖凝胶电泳中IgG与CRM9混合反应的样品所显示的两条条带均与IgG和CRM9单独所在的条带一致.酶联免疫实验测得的各组A450nm值表明,IgG与CRM9按1∶1和1∶3两种比例制备的样品对抗体活性影响最小,而按1∶5和1∶两种比例制备的蛋白,抗体活性受到很大的抑制.结论:兔抗人IgG与CRM9可以通过MBS结合,二者在1∶1～1∶3之间的比例时,可以有较好的连接,而且对抗体活性的影响最小.SP DP则不适用于本实验中的两种蛋白的连接.这将为免疫毒素的新型连接提供有效的方法.展开更多
<strong>Background:</strong> In Nigeria Pentavalent vaccine had replaced Diphtheria-Pertussis- Tetanus [DPT] vaccine in the prevention of pertussis since 2012. <strong>Aims and Objectives:</strong...<strong>Background:</strong> In Nigeria Pentavalent vaccine had replaced Diphtheria-Pertussis- Tetanus [DPT] vaccine in the prevention of pertussis since 2012. <strong>Aims and Objectives:</strong> The aim of this study was to compare the anti-pertussis immunoglobin G (IgG) response of children who received DPT with those who received the pentavalent vaccine. <strong>Subjects and Methods:</strong> This study was carried out in Akpabuyo LGA of Cross River State from April to June 2016. It was a cross-sectional survey of anti-pertussis IgG levels in children aged 6 months to 5 years who received DPT and those who received pentavalent vaccine. IgG antibody levels were determined using enzyme-linked immunosorbent assay. The protective level was set at >11 DU according to manufacturer’s cut off point. <strong>Results:</strong> Seventy eight out of 230 children [33.9%] who had received DPT had protective levels of anti-pertussis IgG compared to 74 out of 192 children [38.5%] who had received pentavalent vaccine. The difference was not statistically significant [<em>p</em> = 0.61]. The median IgG antibody level in those who received DPT was 8.0 DU (interquartile range (IQR) 4.0 - 13.0) compared with 9.0 DU (IQR) 4.0 - 15.0 in those who received pentavalent vaccine [<em>p</em> = 0.18]. No single factor investigated predicted the development of protective levels of antibody in the multivariate analysis. <strong>Conclusion/Recommendation:</strong> There was no difference in the antipertussis antibody response between DPT and pentavalent vaccines recipients. Further study is needed to elucidate factors that could be responsible for low anti-pertussis antibody response in this population.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<strong>Background:</strong> In Nigeria Pentavalent vaccine had replaced Diphtheria-Pertussis- Tetanus [DPT] vaccine in the prevention of pertussis since 2012. <strong>Aims and Objectives:</strong> The aim of this study was to compare the anti-pertussis immunoglobin G (IgG) response of children who received DPT with those who received the pentavalent vaccine. <strong>Subjects and Methods:</strong> This study was carried out in Akpabuyo LGA of Cross River State from April to June 2016. It was a cross-sectional survey of anti-pertussis IgG levels in children aged 6 months to 5 years who received DPT and those who received pentavalent vaccine. IgG antibody levels were determined using enzyme-linked immunosorbent assay. The protective level was set at >11 DU according to manufacturer’s cut off point. <strong>Results:</strong> Seventy eight out of 230 children [33.9%] who had received DPT had protective levels of anti-pertussis IgG compared to 74 out of 192 children [38.5%] who had received pentavalent vaccine. The difference was not statistically significant [<em>p</em> = 0.61]. The median IgG antibody level in those who received DPT was 8.0 DU (interquartile range (IQR) 4.0 - 13.0) compared with 9.0 DU (IQR) 4.0 - 15.0 in those who received pentavalent vaccine [<em>p</em> = 0.18]. No single factor investigated predicted the development of protective levels of antibody in the multivariate analysis. <strong>Conclusion/Recommendation:</strong> There was no difference in the antipertussis antibody response between DPT and pentavalent vaccines recipients. Further study is needed to elucidate factors that could be responsible for low anti-pertussis antibody response in this population.
