CAJ | 학술논문

目的构建表达结核分枝杆菌(Mycobacterium tuberculosis,Mtb)免疫优势抗原Ag85A的DNA疫苗,分析其加强免疫后提高卡介苗(BCG)初免小鼠的抗结核T细胞免疫应答.方法以Mtb毒株H37Rv基因组DNA为模板,PCR扩增Ag85A抗原编码的结构基因并克隆至真核表达载体pVAX1中构建其DNA疫苗；接着,将纯化后的该DNA疫苗加强免疫BCG初免小鼠2针,以BCG和DNA单独免疫小鼠为对照,免疫8周后无菌分离脾淋巴细胞,分别应用IFN-γ ELISPOT和多因子胞内流式细胞术(intracellular staining)分析免疫小鼠的Mtb抗原特异性效应细胞免疫水平与分泌IFN-γ/TNF-α/IL-2的多功能CD4+T细胞频率及其强度以及CD8+T细胞免疫应答.结果与BCG免疫及DNA单独免疫组相比,Ag85A DNA加强免疫不仅能显著提高小鼠IFN-γ+TNF-α+IL-2+多功能T细胞,IFN-γ+IL-2+、IL-2+TNF-α+双功能T细胞与IL-2+单功能T细胞的频率以及IL-2的分泌能力,还能显著诱导小鼠产生更多分泌IFN-γ和IL-2的CD8+T细胞.结论本研究成功构建了表达Mtb免疫优势抗原Ag85A的DNA疫苗并分析了其免疫原性,证实了BCG初免-DNA加强的免疫策略可同时显著增强实验小鼠的Mtb抗原特异性CD4+T和CD8+T细胞应答水平,有利于提高BCG的免疫原性,为增强BCG逐渐下降的抗结核保护效果提供新思路.
목적 구건표체결핵분지간균(Mycobacterium tuberculosis,Mtb)면역우세항원Ag85A적DNA역묘,분석기가강면역후제고잡개묘(BCG)초면소서적항결핵T세포면역응답.방법 이Mtb독주H37Rv기인조DNA위모판,PCR확증Ag85A항원편마적결구기인병극륭지진핵표체재체pVAX1중구건기DNA역묘；접착,장순화후적해DNA역묘가강면역BCG초면소서2침,이BCG화DNA단독면역소서위대조,면역8주후무균분리비림파세포,분별응용IFN-γ ELISPOT화다인자포내류식세포술(intracellular staining)분석면역소서적Mtb항원특이성효응세포면역수평여분비IFN-γ/TNF-α/IL-2적다공능CD4+T세포빈솔급기강도이급CD8+T세포면역응답.결과 여BCG면역급DNA단독면역조상비,Ag85A DNA가강면역불부능현저제고소서IFN-γ+TNF-α+IL-2+다공능T세포,IFN-γ+IL-2+、IL-2+TNF-α+쌍공능T세포여IL-2+단공능T세포적빈솔이급IL-2적분비능력,환능현저유도소서산생경다분비IFN-γ화IL-2적CD8+T세포.결론 본연구성공구건료표체Mtb면역우세항원Ag85A적DNA역묘병분석료기면역원성,증실료BCG초면-DNA가강적면역책략가동시현저증강실험소서적Mtb항원특이성CD4+T화CD8+T세포응답수평,유리우제고BCG적면역원성,위증강BCG축점하강적항결핵보호효과제공신사로.
Objective To construct DNA vaccine expressing Mycobacterium tuberculosis(Mtb) immunodominant antigen Ag85A and analyze its anti-tuberculosis T cell responses in BCG primed-mice after DNA vaccination boosting.Methods The coding gene of Ag85A mature fragment was amplified by PCR with H37Rv genomic DNA as template,and then cloned into the eukaryotic expression vector pVAX1 to construct Ag85A DNA vaccine.After purification,Ag85A DNA vaccine was injected intramuscularly twice in BCG primed-mice with BCG vaccination and DNA vaccination alone as control.Eight weeks post-vaccination,spleen lymphocytes were separated and were then used to analyze Mtb antigen specific effector T cell response and polyfuntional IFN-γ/TNF-α/IL-2 secreting CD4+ T cell frequencies and intensities,and CD8+T cell responses by IFN-γ ELISPOT assay and intracellular staining,respectively.Results Compared to BCG vaccinated-and DNA vaccinated-mice,Ag85A DNA boosting not only enhanced significantly BCG primed-mice IFN-γ+TNF-α+IL-2+,IFN-γ+ IL-2+,TNF-α+IL-2+ and IL-2+ CD4+ T cell frequencies and IL-2 secretion,but also improved significantly IFN-γ-secreting and IL-2-secreting CD8+ T cell frequencies.Condusion Ag85A DNA vaccine was constructed successfully and was demonstrated to enhance significantly BCG primed-mice Mtb antigen specific CD4+ and CD8+ T cell responses when boosting,which is beneficial to improve BCG immunogenicity and its waning immune protection against Mtb.