减毒鼠伤寒沙门菌体内定位分析…

Localization Analysis of attenuated Salmonella typhimurium with oral immunization

摘要:

目的: 分析减毒鼠伤寒沙门菌口服感染后在小鼠体内定位的情况。方法: 将构建的红色荧光蛋白(RFP)原核质粒pYA33-DsRed, 以电穿孔法转化减毒鼠伤寒沙门菌X4550, 重组菌命名为X4550(33-DsRed)。重组菌分别感染巨噬细胞RAW264.7和骨髓源树突状细胞(BMDC), 并用流式细胞术检测红色荧光细胞荧光强度。此外, 以不同剂量重组菌口服免疫BALB/c小鼠, 并于免疫后1d、2d、3d、5d、7d取小鼠脾、肝、肠系膜淋巴结(MLN)、派伊尔氏结(PP)、腹股沟淋巴结(ILN)细胞, 检测各组织器官中的红色荧光阳性细胞百分率。结果: 重组菌对RAW264.7细胞和BMDC均具有良好的侵袭力。口服小鼠后, 第1d, 仅在MLN及PP中检测到RFP阳性细胞, 其中PP中阳性细胞达到1.4%; 第2 d, 在ILN中达到0.4%; 第3 d, 各个组织器官中RFP阳性细胞均有上升趋势, 此时在脾、肝中也检测到RFP阳性细胞。第5 d, RFP阳性细胞均减少, 第7 d则未检测到任何RFP阳性细胞。减毒鼠伤寒沙门菌具有良好的侵袭力, 其黏膜移行方式以及对免疫组织器官靶向定位性, 在优化黏膜疫苗以及提高疫苗免疫效力等方面都具有重要作用。

Abstract:

AIM: To analyze the localization of attenuated Salmonella typhimurium after oral immunization. METHODS: Prokaryotic expression plasmid pYA33-DsRed, carrying the RFP gene, was constructed and electro-transformed into an attenuated strain X4550 of Salmonella typhimurium, the recombinant bacteria were named as X4550(33-DsRed). The macrophage cell line RAW264.7 and bone marrow dendritic cell (BMDC) were invaded by X4550(33-DsRed) in vitro. Furthermore, BALB/c mice were immunized with recombinant bacteria orally. RFP positive cells (RFP+ cells) were detected by Flow Cytometry (FACS) from spleen, liver, Mesenteric lymp node (MLN), Peyer’s patch (PP), Inguinal lymph node (ILN). RESULTS: The invasion rate increased when the multiplicity of infection(MOI) were improved in this two kinds of cells respectively. After oral immunization with X4550(33-DsRed), RFP+ cells were detected by FACS on 1d, 2d, 3d, 5d, 7d in spleen, liver, MLN, PP, ILN cells. The first day, RFP+ cells were detected in MLN and PP, and in PP at a higher rate of 1.4% than that of MLN. 0.4% RFP+ cells were detected the next day in ILN. On 3th day, the rates of RFP+ cells were increased in all of above tissues or organs and decreased on the 5th day. At the 7th day, RFP+ cells couldn’t be detected in all tissues or organs tested. CONCLUSIONS: It is suggested that the invasion ability and the transfer througy mucosal pathway and targeting to recognize immune tissue or organs are favor of the research in mucosal vaccine and the vaccine efficiency.