结核分枝杆菌Hsp65-Ag85B、Hsp65-ESAT6融合基因DNA疫苗株的构建及免疫原性研究  

Construction and immunogenicity investigation of Hsp65-Ag85B/Hsp65-ESAT6 fusion gene DNA vaccine strain of Mycobacterium Tuberculosis

作　　者：张真 赵玲娜 申梦 王希良 杨鹏辉 包金风 ZHANG Zhen;ZHAO Lingna;SHEN Meng;WANG Xiliang;YANG Penghui;BAO Jinfeng(School of Basic Medicine,Inner Mongolia Medical University,Hohhot 010059,China;Fifth Medical Center of Chinese PLA General Hospital,Beijing 100039,China;Beijing Institute of Microbiology and Epidemiology/State Key Laboratory of Pathogenic Microbiology and Biological Safety,Beijing 100071,China)

机构地区：[1]内蒙古医科大学基础医学院,呼和浩特010059 [2]中国人民解放军总医院第五医学中心,北京100039 [3]军事科学院军事医学研究院微生物流行病研究所/病原微生物与生物安全国家重点实验室,北京100071

出　　处：《免疫学杂志》2020年第2期109-115,共7页Immunological Journal

摘　　要：目的构建结核分枝杆菌Hsp65-Ag85B、Hsp65-ESAT6 DNA候选疫苗株并鉴定,初步评价其免疫原性。方法从GenBank中获取M.tuberculosis H37Rv的Ag85B、ESAT6和Hsp65基因序列优化后合成,经PCR、酶切、连接与真核表达载体PVAX1重组,构建重组质粒PVAX1-Hsp65-Ag85B和PVAX1-Hsp65-ESAT6。重组质粒体外转染验证其表达后,免疫C57BL/6小鼠,检测特异性细胞免疫应答反应。结果Hsp65-Ag85B、Hsp65-ESAT6 DNA疫苗株在体内外均能表达,Western blot结果显示,DNA疫苗株体外转染293T细胞,24 h可检测到特异性蛋白条带,48 h蛋白表达量达最高。小鼠体内实验表明,Hsp65-Ag85B、Hsp65-ESAT6 DNA疫苗株均可激发较强的免疫反应,可诱导机体产生分泌高水平TNF-α的Th型免疫应答,且Hsp65-Ag85B优于Hsp65-ESAT6。结论Hsp65-Ag85B、Hsp65-ESAT6 DNA疫苗株均具有较强的免疫原性,呈Th型细胞介导的免疫应答,推测Hsp65-Ag85B DNA疫苗的保护效力优于Hsp65-ESAT6,为新型结核DNA疫苗研制提供了实验依据。Tuberculosis(TB),a chronic respiratory infectious disease caused by Mycobacterium tuberculosis(Mtb)infection,is listed as one of the major infectious diseases in China and seriously endangers the health of people in developing countries.The purpose of this study is to construct and identify candidate vaccine strains of Mycobacterium tuberculosis Hsp65-Ag85B and Hsp65-ESAT6 DNA,and to initially evaluate their immunogenicity.The PVAX1-Hsp65-Ag85B and PVAX1-Hsp65-ESAT6 plasmids were constructed by gene synthesis,doubleenzyme digestion and ligation,and the correctness of the sequences were identified by PCR,agarose gel electrophoresis and sequencing.Western blot showed after recombinant plasmid transfection into 293 T cells for 24 and 48 hours,there were clear and specific target protein bands;the results of mouse immunoassay showed that DNA vaccine could induce high level of TNF-α,which was responsible for Th-type immune response.In conclusion,the synthesized stable DNA vaccine in vitro has good immunogenicity,which lays a foundation for the improvement of DNA vaccine research and provides new ideas for the development of tuberculosis vaccine.

关 键 词：DNA疫苗 AG85B ESAT6 HSP65 分枝杆菌 

分 类 号：R392.5[医药卫生—免疫学]