结核分枝杆菌ESAT-6蛋白免疫优势肽段的重组表达、纯化与鉴定  

Expression,purification and characterization of recombinant multiepitope peptide of ESAT-6 of Mycobacterium tuberculosis

作　　者：王海波[1] 朱中元[1] 刘贤杰[2] 

机构地区：[1]海南医学院附属新华医院检验科,海口570311 [2]海南大学植物保护学院,海南儋州571737

出　　处：《临床检验杂志》2009年第3期170-173,共4页Chinese Journal of Clinical Laboratory Science

基　　金：海南省卫生厅科研基金(编号:琼卫2006-61号)

摘　　要：目的构建结核分枝杆菌(MTB)ESAT-6免疫优势肽段(ESAT-6P)的基因表达工程菌,以获得大量重组肽段。方法根据编码ESAT-6P的MTB基因序列,设计1对特异引物,通过PCR技术从H37Rv基因组DNA扩增出ESAT-6P编码基因,目的片段克隆到原核表达载体pET-30 a中,构建N端带有6H is-tag的表达载体。将重组表达载体转化E.coliBL21(DE3)后,经IPTG诱导和SDS-PAGE分析表达产物,斑点免疫结合法(D IBA)鉴定重组肽段的免疫反应性。结果成功诱导表达ESAT-6蛋白的免疫优势肽段,与表达载体氨基酸序列融合后,表达产物相对分子质量约为16 500,该重组肽段以包涵体形式存在。D IBA结果表明,该重组肽段可被结核患者血清特异识别。结论获得MTB ESAT-6免疫优势肽段的基因表达工程菌,为将此重组肽段应用于结核诊断试剂和新型疫苗的研制奠定了基础。Objective To obtain sufficient recombinant multiepitope peptide of ESAT-6. Methods DNA encoding ESAT-6 peptide was amplified by PCR using a pair of primers which were designed in accordance with the reported ESAT-6 gene sequence. The ESAT-6P gene was inserted into the prokaryotic expression vector pET-30a with the N-terminal 6His-tag. The recombinant peptide vector pET30a- ESAT-6P was transformed into E. coli BL21 （ DE3 ） via chemical transformation. The positive clone was screened by means of PCR. The target peptide was expressed in E. coli after induction with IPTG and analyzed by SDS-PAGE. The immunogenicity of the peptide was evaluated by dot immunobinding assay. Results The target peptide was successfully expressed and purified. The solubility analysis showed that the recombinant peptide existed as inclusion body in the E. coli. DIBA indicated that the ESAT-6P was specifically reactive to sera from TB patients. Conclusions The recombinant muhiepitope peptide of.ESAT-6 engineering bacteria has been obtained, which will be quite helpful to develop novel specific diagnostic reagents and effective vaccine.

关 键 词：结核分枝杆菌 ESAT-6蛋白 抗原表位 表达 

分 类 号：R446.5[医药卫生—诊断学]