|本期目录/Table of Contents|

[1]王 博,丁 晓,颜 贝,等.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[J].宁夏医科大学学报,2013,(07):738-742,747.
 WANG Bo,DING Xiao,YAN Bei,et al.Cloning, Expression and Bioinformatics of the Ag85B,TB10.4 and Ag85B-TB10.4 Fusion Gene in Mycobacterium Tuberculosis[J].Ningxia Medical University,2013,(07):738-742,747.
点击复制

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(PDF)
分享到:

《宁夏医科大学学报》[ISSN:1005-8486/CN:64-1029/R]

卷:
期数:
2013年07期
页码:
738-742,747
栏目:
论著
出版日期:
2013-07-20

文章信息/Info

Title:
Cloning, Expression and Bioinformatics of the Ag85B,TB10.4 and Ag85B-TB10.4 Fusion Gene in Mycobacterium Tuberculosis
作者:
王 博 丁 晓 颜 贝 高玉婧 蒲 静 裴秀英
宁夏医科大学基础医学院生化与分子生物学系,宁夏生殖与遗传重点实验室, 医学科学技术研究中心,银川 750004
Author(s):
WANG Bo DING Xiao YAN Bei GAO YU-jing PU Jing PEI Xiu-ying
Biochemistry and Molecular Biology department of school of basic medical sciences, Key Laboratory of Reproduction and Heredity of Ningxia Hui Autonomous Region, Medical Sci-Tech Research Center, Ningxia Medical University,Yinchuan 750004
关键词:
结核分枝杆菌 Ag85B TB10.4 基因融合 生物信息学
Keywords:
Mycobacterium tuberculosis Ag85B TB10.4 gene fusion bioinformatics
分类号:
R378.91+1
DOI:
-
文献标志码:
A
摘要:
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。
Abstract:
Objective To clone the Ag85B,TB10.4,Ag85B-TB10.4 fusion gene of Mycobacterium tuberculosis and to analyze the bioinformatics of the antigen, in order to lay the foundation for the candidate vaccine screening. Methods Ag85B, TB10.4and Ag85B-TB10.4 were amplified from the M. tuberculosis H37Rv genome by polymerase chain reaction(PCR). Ag85B was cloned into pET-28a(+)expression vector, TB10.4 and Ag85B-TB10.4 were cloned into pET-SUMO expression vector. All recombinant plasmids were transformed into Ecoli BL21(DE3),and the expression products induced by IPTG were analyzed through bioinformatics. Results The recombinant plasmids TB10.4, Ag85B and Ag85B-TB10.4 were constructed successfully. The SDS-PAGE results showed that the expression products were consistent with the expected results. The molecular weight of the fusion protein pET-SUMUO-Ag85B-TB10.4 was 54 KDa. Bioinformatics analysis showed that there was a plurality of potential antigenic sites in Ag85B-TB10.4 fusion protein. Conclusion The recombinant plasmids pET-28a-Ag85B, pET-SUMO-TB10.4 and pET-SUMO-Ag85B-TB10.4 were constructed successfully, and the soluble prokaryotic expression products of TB10.4, Ag85B and Ag85B-TB10.4 were obtained. The hydrophilicity and antigenic determinant cluster area of Ag85B-TB10.4 fussion protein increased after added linker between Ag85B and TB10.4 by the bioinformatics analysis, which laid the foundation for subsequent functional experimental study.

参考文献/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.

相似文献/References:

[1]杨桂茂,朱明星,张爱君,等.结核分枝杆菌宁夏分离株pst S1基因的扩增及生物信息学分析[J].宁夏医科大学学报,2010,(09):960.
 YANG Gui-mao,ZHU Ming-xing,ZHANG Ai-jun,et al.Amplification and Bioinformatic Analysis of the pst S1 Gene from Ningxia Isolated Mycobacterium Tuberculosis[J].Ningxia Medical University,2010,(07):960.
[2]丁淑琴,王 洁,张 焱,等.结核分枝杆菌phoS2基因的克隆及序列分析[J].宁夏医科大学学报,2010,(06):669.
 DING Shu-qin,WANG Jie,ZHANG Yan,et al.Cloning and Sequence Analysis of the PhoS2 Gene from Mycobacterium Tuberculosis [J].Ningxia Medical University,2010,(07):669.
[3]朱佳佳,刘宏鹏,张爱君,等.结核分枝杆菌ESAT-6诊断抗原基因的克隆及同源性分析[J].宁夏医科大学学报,2010,(03):339.
 ZHU Jia-jia,LIU Hong-peng,ZHANG Ai-jun,et al.Cloning and Homologous Analysis of the ESAT-6 Gene from Mycobacterium Tuberculosis [J].Ningxia Medical University,2010,(07):339.
[4]雄 英,王娅娜,李宗吉,等.细粒棘球蚴中国大陆株铁蛋白生物信息学的初步分析 及其结构和功能预测[J].宁夏医科大学学报,2010,(03):327.
 XIONG Ying,WANG Ya-na,LI Zong-ji,et al.Bioinformatics Analysis for the Structure and Function of Ferritin Gene from Echinococcus Granulosus (Chinese Mainland Strain)[J].Ningxia Medical University,2010,(07):327.
[5]孙俊锋,棘怀庆,雄 英,等.生物信息学方法初步筛选细粒棘球蚴防治靶位蛋白[J].宁夏医科大学学报,2010,(02):172.
 SUN Jun-feng,JI Huai-qing,XIONG Ying,et al.High-throughput Screening Valuable Target Protein of Echinococcusgranulosus via Bioinformatics Methods[J].Ningxia Medical University,2010,(07):172.
[6]丁淑琴,杨园园,杨凤琴,等.结核分支杆菌重组蛋白rps12的结构与功能预测[J].宁夏医科大学学报,2012,(11):1130.
 DING Shu-qin,YANG Yuan-yuan,YANG Feng-qin,et al.Bioinformatics Analysis of the Structure and Fuction in Mycobacterium Tuberculosis Recombinant Protein rps12[J].Ningxia Medical University,2012,(07):1130.
[7]王 元,王娅娜,师志云,等.细粒棘球绦虫(中国大陆株)特异性抗原P-29 重组蛋白的生物信息学分析[J].宁夏医科大学学报,2012,(07):673.
 WANG Yuan,WANG Ya-na,SHI Zhi-yun,et al.Bioinformatics Analysis of the Echinococcus Granulosus Recombinant Ferritin Antigen[J].Ningxia Medical University,2012,(07):673.
[8]张 婵,朱明星,杨风琴,等.结核分枝杆菌宁夏分离株pstS1基因在毕赤酵母中的表达[J].宁夏医科大学学报,2013,(08):860.
 ZHANG Chan,ZHU Mingxing,YANG Fengqin,et al.Expression of Mycobacterium Tuberculosis pstS1Gene Isolated from Ningxia in Pichia Pastoris[J].Ningxia Medical University,2013,(07):860.
[9]朱明星,杨桂茂,孙惠敏,等.结核分枝杆菌宁夏分离株MPT64基因的生物信息学分析[J].宁夏医科大学学报,2013,(07):778.
 ZHU Ming-xing,YANG Gui-mao,SUN Hui-min,et al.The Bioinformatic Analysis of the MPT64 Gene of Mycobacterium Tuberculosis Isolated in Ningxia[J].Ningxia Medical University,2013,(07):778.
[10]张瑞芹,汤建中,贾 伟,等.结核分枝杆菌优势蛋白PPE37重组载体的构建及原核表达[J].宁夏医科大学学报,2013,(03):240.
 ZHANG Rui-qin,TANG Jian-zhong,JIA Wei,et al.The Construction and Prokaryotic Expression of Mycobacterium Tuberculosis Dominant Protein PPE37 Recombinant Vector[J].Ningxia Medical University,2013,(07):240.

备注/Memo

备注/Memo:
收稿日期:2013-03-22 基金项目:国家自然基金(30660185); 宁夏医科大学博士学位建设学科开放课题(宁医校发[2010]195号KF2010-21) 作者简介:王博(1985-),男,陕西人,在读硕士研究生,主要从事结核疫苗研究。 通信作者:裴秀英,教授,主要从事疾病相关基因的研究 E-mail:peixy@nxmu.edu.cn
更新日期/Last Update: 2013-07-20