大肠杆菌菌影制备及介导pEGFP-N1在RAW264.7细胞中表达

Preparation of E.coli ghosts and expression of pEGFP-N1 mediated by E.coli ghosts in RAW 264.7 cells

通过PCR扩增噬菌体PhiX174裂解基因E,并将其插入到温控表达载体pBV220中,构建重组温控表达质粒pBV220-E。将重组质粒pBV220-E转化至大肠杆菌DH5α中,升温到42℃诱导E基因的表达。将真核表达质粒pEGFP-N1与大肠杆菌菌影孵育后转染小鼠巨噬细胞RAW264.7,荧光显微镜观察pEGFP-N1的表达情况。结果表明:扩增的E基因全长为276bp,与GenBank公布的基因序列一致。电镜观察结果显示,细菌表面出现跨膜孔道,细菌内容物经孔道排出形成空壳。荧光显微镜观察结果显示,大肠杆菌菌影介导的pEGFP-N1在小鼠巨噬细胞中获得表达。为进一步研究以细菌菌影为基础的新型灭活疫苗和载体疫苗提供参考依据。

The bacteriophage PhiX174 lysis gene E was amplified by PCR, and cloned into the plasmid pBV220 to con- struct recombinant temperature-controlled expression plasmid pBV220-E. The plasmid pBV220 E was subsequently transformed into E. coli DH5a. The recombinant bacteria E. coli （pBV220-E） were cultured at 42 ~C for the generation of ghosts. The mouse RAW264.7 cells were transfected with the bacterial ghosts loading pEGFP-N1, and expression of pEGFP-N1 was detected under fluorescence microscope. The results indicated that E. coli ghosts were prepared success- fully, their contents were released to extracellular region and the lysis efficiency of E. coli （pBV220-E） was 99%. Expression of pEGFP-N1 mediated by E. coli ghosts can be detected in transfected RAW264.7 cells. These results lay foundation for developing the new inactive vaccine or vector vaccine based on bacteria ghosts.