全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...

结核分枝杆菌Ag85B、TB10.4及Ag85B-TB10.4融合基因 的克隆表达及生物信息学分析

Keywords: 结核分枝杆菌,Ag85B,TB10.4,基因融合,生物信息学

Full-Text   Cite this paper   Add to My Lib

Abstract:

目的构建结核分枝杆菌Ag85B、TB10.4及Ag85B-TB10.4融合表达载体,并进行抗原的生物信息学分析,为候选疫苗筛选奠定基础。方法从结核分枝杆菌H37Rv菌株中分别扩增出Ag85B基因,TB10.4基因,Ag85B-TB10.4;将Ag85B克隆入pET-28a(+)表达载体中,将TB10.4和Ag85B-TB10.4分别克隆入pET-SUMO表达载体中。上述重组质粒转化入大肠埃希杆菌BL21菌中(DE3),经IPTG诱导表达,并对其进行生物信息学分析。结果(1)成功将结核分枝杆菌Ag85B和TB10.4,Ag85B-TB10.4基因分别克隆入pET28a(+)和pET-SUMO表达载体中,经IPTG诱导蛋白表达后,对经超声裂解的菌液上清进行SDS-PAGE电泳,表明获得了与预期蛋白大小一致的表达产物,重组融合蛋白pET-SUMO-Ag85B-TB10.4蛋白分子质量约为54kDa。(2)生物信息学分析显示Ag85B-TB10.4融合蛋白具有多个潜在抗原位点。结论成功构建了重组质粒pET-28a-Ag85B,pET-SUMO-TB10.4和pET-SUMO-Ag85B-TB10.4,并获得了Ag85B,TB10.4和Ag85B-TB10.4的可溶性原核表达产物;生物信息学分析在Ag85B与TB10.4蛋白加入一段蛋白linker后蛋白的亲水性增强,抗原决定簇面积增大,为后续的功能实验研究奠定了基础。

References

[1]  Maher D,Raviglione M.Global epidemiology of tuberculosis[J].Clin Chest Med,2005,26(2):167-182.
[2]  D Young,C Dye.The development and impact of tuberculosis vaccines[J].Cell,2006,124(4):683-6837.
[3]  袁伟,秦川.结核疫苗研究进展[J].中国比较医学杂志,2011,21(2):60-64.
[4]  Brooks JV,Frank.AA,Keen MA,et al.Boosting vaccine for tuberculosis[J].Infect Immun,2001,69(4):2714-2717.
[5]  YA Skeiky,MR AlderSon PJ Ovendale,et al.Differential immuneres responses and protective efficacy induced by components of a tuberculosis polyprotein vaccine,Mtb72F,delivered as naked DNA or recombinant protein[J].J Immunol,2004,172(12):7618-7628.
[6]  Billeskov R,Grandal MV,Poulsen C,et al.Difference in TB10.4 T-cell epitope recognition following immunization with recombinant TB10.4,BCG or infection with Mycobacterium tuberculosis[J].Eur J Immunol,2010,40(5):1342-1354.
[7]  Hervas-Stubbs S.High frequency of CD4+T cells specific for the TB10.4 protein correlates with protection against Mycobacterium tuberculosis infection[J].Infect Immun,2006,74(6):3396-3407.
[8]  Skjot RL.Epitope mapping of the immunodominant antigen TB10.4 and the two homologous proteins TB10.3 and TB12.9,which constitute a subfamily of the esat-6 gene family[J].Infect Immun,2002,70(10):5446-5453.
[9]  Britton WJ,Palengira V.Improving vaccines against tuberculosis[J].Immunal Cell Biol,2003,81(1):34-45.
[10]  Bentley-Hibbert SI,Quan X,NewmanT,et al.Pathophysiology of antigen 85 in patients with active tuberculosis:antigen 85 circulates as complexes with fibronectin and immunoglobulin [J].Infect Immun,1999,67(2):581.
[11]  吴雪琼,张俊仙,郑越.结核分支杆菌Ag85B蛋白在大肠杆菌中的高效表达[J].广东医学,2003,24,(5):487-488.
[12]  Kato-Maeda M,Rhee JT,Gingeras TR et al.Comparing genomes within the species Mycobacterium tuberculosis[J].Genome Res,2001,11(4):547-554.
[13]  王瑞博,井申荣.结核分枝杆菌分泌蛋白TB10.4的功能和应用[J].生命的化学,2011,31(4):598-600.
[14]  李泽鸿,李树民.两种重组融合蛋白的分子生物学特性比较[J].北华大学学报,2007,8(2):497-500.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133