结核分枝杆菌分泌蛋白EspB 在RAW264.7细胞中的稳定表达

Stable expression of Mycobacterium tuberculosis protein EspB in RAW264. 7

摘要:

摘要:【目的】建立能够稳定表达结核分枝杆菌(Mycobacterium tuberculosis，Mtb)分泌蛋白EspB(Rv3881c)的RAW264.7细胞系，为研究EspB 蛋白在调控巨噬细胞功能中所起的作用提供科学依据。【方法】首先成功构建的重组质粒pEGFP-C1-EspB，然后将重组载体pEGFP-C1-EspB和空载体pEGFP-C1以脂质体介导的方法转染至小鼠巨噬细胞RAW264. 7 中，经过G418 筛选后建立稳定表达EGFP-EspB 融合蛋白以及EGFP的细胞系，并通过RT-PC

Abstract:

Abstract:［Objective］We established a cell line stably expressing Mycobacterium tuberculosis secretory protein EspB in order to provide evidences for studying EspB in modulating the functions of macrophage．［Methods］ The recombinant plasmid pEGFP-C1-EspB was first constructed，then RAW264. 7 cell was transfected with pEGFP-C1-EspB and pEGFPC1 by liposome respectively． After screening with a high level of G418，the macrophage cell lines that stably expressed EGFP-EspB fusion protein or EGFP were established． The gene and protein expression levels were further analyzed by RTPCR，fluorescence microscopy and western blot．［Results］ The EGFP-EspB fusion gene was integrated into the chromosome and the protein was stably expressed in the selected macrophage cell line．The macrophage cell lines that stably expressed EGFP-EspB fusion protein or EGFP were established．［Conclusion］ These results gave us a tool for the future study in the effects of EspB protein in modulating the functions of macrophage and its interaction with other molecules of macrophage．