口服结核Ag85A-CD226 DNA疫苗在小鼠脾与肠道的表达水平检测

The detection for expression of tuberculosis Ag85A-CD226 DNA in spleen and intestine of mouse after oral inoculation

目的了解结核Ag85A-CD226 DNA疫苗经灌胃方式接种小鼠后在脾淋巴细胞与肠道的表达情况。方法将构建的pcDNA3.1-Ag85A-CD226、pcDNA3.1-Ag85A和pcDNA3.1-CD226真核表达质粒转化DH5α感受态大肠杆菌,扩增并提取纯化质粒,用脂质体包裹制成DNA疫苗。经灌胃方式将制备的DNA疫苗接种C57BL/6小鼠,设置Ag85A-CD226疫苗组、Ag85A疫苗组、CD226疫苗组、pcDNA3.1质粒组和生理盐水对照组。采用间接免疫荧光法检测Ag85A和CD226在肠道的表达。采用流式细胞术检测脾CD4+T细胞、CD8+T细胞和NK细胞的CD226表达。结果 CD226在Ag85A-CD226疫苗组脾脏CD4+T细胞和NK细胞的表达均明显强于Ag85A疫苗组、CD226疫苗组、pcDNA3.1质粒组和生理盐水对照组,差异具有统计学意义(P<0.01);CD226在Ag85A-CD226疫苗组脾脏CD8+T细胞的表达明显强于Ag85A疫苗组、pcDNA3.1质粒组和生理盐水对照组,差异具有统计学意义(P<0.01),与CD226疫苗组相比虽有所增加,但差异无统计学意义(P>0.05)。CD226在Ag85A-CD226疫苗组小肠派氏淋巴结表达明显强于Ag85A疫苗组、CD226疫苗组、pcDNA3.1质粒组和生理盐水对照组,差异具有统计学意义(P<0.01)。Ag85A只在Ag85A-CD226疫苗组和CD226疫苗组小肠固有层表达,且在Ag85A-CD226疫苗组的表达明显强于CD226疫苗组,差异具有统计学意义(P<0.01)。结论 Ag85A-CD226 DNA疫苗接种后,CD226在脾淋巴细胞和肠道表达增强,Ag85A在肠道表达增强,CD226的表达增加会增强Ag85A在肠道的表达水平。

Objective To detect the expression of tuberculosis Ag85A-CD226 DNA vaccine in splenocyte and intestine of mouse by intragastric administration. Methods E. coli DH5α was transformed by pcDNA3. 1-Ag85A-CD226,pcDNA3. 1-Ag85A or pcDNA3. 1-CD226 plasmid. The liposome encapsulated recombinant DNA vaccine was prepared after extraction and amplification of the recombinant plasmid with endotoxin free kit. The C57BL /6 mice were inoculated by intragastric administration. The mice were randomly divided into Ag85A-CD226 vaccine group,Ag85A vaccine group,CD226 vaccine group,pcDNA3. 1 group and normal control group,respectively. Ag85A or CD226 expression in intestine was detected by immunofluorescence and the expressions of CD226 in CD4＋T cells,CD8＋T cells and NK cells of spleen were analyzed by flow cytometry. Results The levels of CD226 expression in splenocyte and the small intestine Peyer＇s patches in Ag85ACD226 vaccine group were higher than those in other groups,P〈0. 01; the expression of Ag85A in the intestinal lamina propria in Ag85A-CD226 vaccine group was higher than that in Ag85A vaccine group,P〈0. 01. Conclusion The expression of CD226 was increased in splenocyte and intestine of mouse,and the increased expression of CD226 could enhance the expression of Ag85A in intestine after inoculation of Ag85A-CD226 DNA vaccination.