结核杆菌Ag85A和mGM-CSF共同表达载体的构建与CTL活性的诱导

构建、鉴定结核杆菌Ag85A和细胞因子GM CSF共同表达质粒 ,为研究新型抗结核杆菌DNA疫苗提供新的策略。先从质粒pBSby5中扩增出结核杆菌Ag85A基因序列并插入到质粒pIRES上成为pI85A ,用PCR方法从pc mGM CSF质粒中扩增出mGM CSF ,构建于pI85A质粒上 ,成为共同表达质粒pI85AGM。然后转染 772 1细胞 ,进行蛋白的表达和鉴定。结果经酶切鉴定和DNA测序证实重组质粒构建正确。通过RT PCR、SDS PAGE、Westernblot检测证实Ag85A与mGM CSF基因的转录和蛋白表达正确。Ag85A与mGM CSF免疫组小鼠的CTL活性明显增强。表明细胞因子GM CSF和结核杆菌Ag85A共同表达载体构建成功 ,并能诱导小鼠的CTL活性 ,为研制新型结核病疫苗奠定了基础

Construction of the Eukaryotic Coexpression Vector for Mycobacterium Tuberculosis Antigen Ag85A and Murine GM-CSF with a Preliminary Study on the Immune Functions

To construct the eukaryotic coexpression vector for Mycobacterium tuberculosis antigen Ag85A and murine G-CSF, th e gene encoding Ag85A was amplified from plasmid pBSby5 by PCR, and then directly cloned into plasmid pIRES to form pI85A. Also by PCR, the gene encoding mGM-CSF was amplified from pc-mGM-CSF and cloned directly into plasmid pI85A to form coexpression vector pI65A-GM. This eukaryotic expression recombinant plasmid con taining genes for both Ag85A and mGM-CSF, was then transfected to 7721 cells and th e expressed proteins were deterrmined. When mice were immunized with the recombinant plasmid, the immune effects of Ag85A and mGM-CSF to mice were determined by CTL function testings. Experimental results showed that the recombinant plasmid was constructed correctly as monstrated by restriction enzyme digestion and DNA sequencing. The genes encoding Ag85A and mGM-CSF transcrited and expressed correctly as shown by RT-PCR, SDS-PAGE and mGM-CSF transcrited and expressed correctly as shown by RT-PCR, SDS-PAGE and Western blot analysis. The CTL activities significantly increased when mice were immunized with coexpression recombinant plasmid pI85AGM. It is concluded that an eukaryotic coexpression plasmid pI8 5AGM has been constructed successfully, and this plasmid can induce CTL activities in mice. These results might provide basis for the further development of new v accine against tuberculosis.