结核杆菌Hsp65和hGM-CSF双顺反子表达质粒的构建与表达

Construction and expression of bicistronic expression plasmid containing Hsp65 of Mycobacterium tuberculosis and hGM-CSF

目的:应用分子生物学技术,构建pIHsp65GM双顺反子真核表达质粒,并在真核细胞中表达.方法:以结核分枝杆菌H37Rv株基因组为模板经聚合酶链反应(PCR)扩增出Hsp65基因,同时从质粒pORF-hGM-CSF中扩增出基因佐剂hGM-CSF,分别克隆到真核表达载体pIRES的多克隆位点A(MCSA)和B(MCSB)中,构建pIHsp65GM真核表达质粒,并转染HepG-2细胞中表达和检测.结果:酶切鉴定提示插入的基因片段大小分别为1.64kb和0.47kb,测序分析表明克隆的Hsp65和hGM-CSF序列与GenBank上公布序列完全一致;免疫组织化学方法检测到表达Hsp65的阳性细胞;ELISA方法检测到pIHsp65GM转染组细胞培养上清中hGM-CSF的表达,与空载体对照组比较差异有统计学意义(P<0.01).结论:成功构建和表达了pIHsp65GM质粒,为研制优于卡介苗的新型抗结核病DNA疫苗奠定基础.

AIM： To construct and express the bicistronic eukaryotic expression plasmid pIHsp65GM by molecular-biological methods. METHODS： We used Mycobacterium tuberculosis H37Rv genome as template to amplify Hsp65 gene and used plasmid pORF-hGM-CSF as template to amplify gene adjuvant hGM-CSF gene （ granulocyte-macrophage colony-stimulating factor） by PCR, and then recombined them with the two multiple cloning sites （ MCSA and MCSB） of pIRES vector to construct a bicistronic eukaryotic expression plasmid pIHsp65GM. The plasmid was finally transfected into HepG-2 cells to detect the expression of Hsp65 and hGM-CSF proteins by immunohistochemistry and ELISA respectively. RESULTS： Restriction enzyme digestion demonstrated that the inserted gene fragments were 1.64 kb and 0. 47 kb. DNA sequencing revealed that the cloned Hsp65 and hGM-CSF sequences were consistent with the GeneBank reported. The Hsp65-positive cells were found by immunohistochemistry. hGM-CSF expression from pIHsp65GM transfected cells was detected by ELISA, and the difference between pIHsp65GM transfected cells and pIRES transfected cells was statistically significant. CONCLUSION： The successful construction and expression of a bicistronic eukaryotic expression vector containing Hsp65 gene and gene adjuvant hGM-CSF are fulfilled, which lays a foundation for the further research on a new DNA vaccine that is better than BCG vaccine in the prevention of tuberculosis.