摘要
目的构建结核分枝杆菌Ag85B的真核表达质粒pcD-Ag85B,经肌肉免疫小鼠,观察其对小鼠肺免疫功能及下呼吸道菌群的影响。方法将信号肽基因及编码Ag85B的基因共963 bp核苷酸序列与pcDNA3.1^(+)连接,构建真核表达质粒pcD-Ag85B,经双酶切及测序鉴定后体外转染中国仓鼠卵巢细胞(CHO),并验证其表达;用该质粒经肌肉注射免疫C57BL/6J小鼠,42 d时收集小鼠呼吸道灌洗液,采用间接ELISA法测定SIgA及IgG特异性抗体效价。取小鼠右肺,分离淋巴细胞,采用流式术检测分泌IFN-γ的CD4^(+)T及CD8^(+)T细胞比例;取小鼠左肺,提取核酸进行16S RNA高通量测序及菌群结构分析。结果成功构建真核表达质粒pcD-Ag85B并检测到Ag85B蛋白的表达。ELISA检测pcD-Ag85B免疫C57BL/6J小鼠呼吸道灌洗液SIgA及特异性IgG抗体效价分别为1∶640和1∶400;流式细胞术检测Ag85B能诱导分泌IFN-γ的CD4^(+)T及CD8^(+)T细胞增殖,所占百分比分别为0.72%和0.57%;16S RNA高通量测序分析显示免疫鼠呼吸道菌群多样性增加,厚壁菌门-乳杆菌属丰富度增高(P<0.05)。结论成功构建重组表达载体pcD-Ag85B,该重组载体表达的Ag85B蛋白能诱导小鼠呼吸道粘膜产生适应性免疫效应,增加下呼吸道菌群的多样性并改善益生菌群,为开展Ag85B DNA疫苗抗结核分枝杆菌感染的保护性实验奠定了基础。
Objective To explore the effects of Mycobacterium tuberculosis(MTB)Ag85B eukaryotic expression plasmid pcD-Ag85B on pulmonary immune function and lower respiratory tract microflora in mice after muscular immunization.Methods A total of 963 bp nucleotide sequences of the signal peptide gene and the gene encoding Ag85B were connected to pcDNA3.1^(+)to construct the eukaryotic expression plasmid pcD-Ag85B.After double enzyme digestion and sequencing,the pcD-Ag85B recombinant plasmid was transfected into Chinese hamster ovarian cells(CHO)in vitro,and the correct expression was verified.C57BL/6J mice were immunized by intramuscular injection of pcD-Ag85B.The respiratory tract lavage solution was collected 42 days after immunization.The specific SIgA and IgG antibody titers were determined by indirect ELISA.The lungs were collected,the right lung was used for detect CD4^(+)T and CD8^(+)T cells secreting IFN-γby Flow cytometry,and the left lung was subjected to 16S RNA high-throughput sequencing and microflora structure analysis.Results The eukaryotic expression plasmid pcD-Ag85B was constructed successfully and the specific expression of Ag85B protein was detected in vitro.Compared with the control group,the titers of SIgA and IgG in the immune group were 1∶640,1∶400,respectively.Ag85B could induce CD4^(+)T and CD8^(+)T cells secreting IFN-γ,and the percentages of the two T cells were 0.72%and 0.57%respectively.The diversity of respiratory tract microflora was increased,and the abundance of Firmicutes-Lactobacillus was significantly increased(P<0.05).Conclusion The Ag85B protein induced the adaptive immune effect of mouse respiratory mucosa,increased the diversity of lower respiratory tract microflora and improved the probiotics.This study laid the foundation for the protective experiment of Ag85B DNA vaccine against MTB infection.
作者
张炜
李慧
杨雨欣
姚思
王天松
万巧凤
ZHONG Wei;LI Hui;YANG Yuxin;YAO Si;WANG Tiansong;WAN Qiaofeng(Department of Pathogenic Biology and Immunology,School of Basic Medicine,Ningxia Medical University,Yinchuan 750004,China;School of Clinical Medicine,Ningxia Medical University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2023年第8期905-910,共6页
Journal of Pathogen Biology
基金
宁夏高等学校科学研究项目(No.NYG2022035)
宁夏常见传染病防治重点实验室开放课题(No.XZ2021011)。
