SL7207/PCMV-mtHSP70-IRES-EGFP重组沙门菌的构建及在B16细胞中的表达

Construction and expression of attenuated Salmonella typhimurium carrying mycobacterium tuberculosis heat shock protein 70 and Green Fluorescent Protein gene eukaryotic expression vector in B16 Cell

目的构建能够稳定表达结核杆菌热休克蛋白70(mycobacterium tuberculosis heat shock protein 70,mtHSP70)与增强型绿色荧光蛋白(enhanced green fluore scent protein,EGFP)双基因的鼠减毒伤寒沙门菌真核共表达载体,探讨其在小鼠黑色素瘤B16肿瘤细胞中的表达。方法将重组质粒PCMV-mtHSP70-IRES-EGFP电穿孔转入减毒沙门氏菌SL7207中,酶切鉴定,并对构建的重组减毒沙门氏菌体外连续传代20代后再酶切鉴定,对其稳定性进行观察;重组菌体外感染B16,荧光显微镜下观察B16细胞荧光表达及蛋白印迹法(Western blot)检测重组菌在B16细胞内mtHSP70蛋白的表达。结果PCMV-mtHSP70-IRES-EGFP成功导入减毒沙门氏菌SL7207中;该重组菌株在体外能稳定地繁殖、生长和传代;重组菌体外感染B16约36h后,B16细胞胞体变圆并高效表达强荧光,重组菌在B16细胞内表达mtHSP70蛋白。结论成功构建能在真核细胞中稳定表达mtHSP70与EGFP双基因的重组减毒沙门氏菌株,为恶性黑色素瘤靶向基因免疫治疗的后续研究奠定了基础。

Objective To construct recombinant attenuated salmonella typhimurium strains carrying mycobacterium tuberculosis heat shock protein 70 and enhanced green fluorescent protein gene eukaryotic expression vector. Methods Mycobacterium tuberculosis heat shock protein 70 gene was obtained by PCR,then cloned into eukaryotic expression vector PCMV-IRES-EGFP. The recombinants were identified and transformed into attenuated salmonella typhi murium strain SL7207,and the clones were identified by restriction enzyme digestion. The stability of the recombinant attenuated salmonella typhimurium strains was evaluated by reproduced 20 generations and then transfected into mice melanoma B16 cell line. Green fluorescent cells were detected by fluorescence microscopy and for western blot assay to detect the expression of mtHSP70 in B16 cell. Results Confirmed by restriction enzyme digestion,recombinant eukaryoic expression plasmid PCMV-mtHSP70-IRES-EGFP was constructed successfully,and then introduced in to an attenuated salmonella typhimurium SL7207. The recombinant strains stably reproduced,grew and passaged in vitro. When it was transfected into mice melanoma B16 cell line,green fluorescent cells can be detected by fluorescence microscopy and mtHSP70 protein can be detected in B16 cells. Conclusion The recombinant attenuated samonella typhimurium strains carrying plasmid PCMV-mtHSP70-IRES-EGFP have been successfully constructed and identified,and it will provide a base for targeted melanoma genetherapy and immountherapy by recombinant live vaccine carrying mtHSP70 and EGFP.