结核分枝菌Ag85B DNA疫苗免疫对小鼠结肠SIgA及菌群的影响  

Effect of DNA vaccine immunization of Mycobacterium tuberculosis Ag85B on colon SIgA and microflora in mice

作　　者：李慧[1] 杨雨欣 姚思 张炜[1] 马国荣 万巧凤[1] LI Hui;YANG Yu-xin;YAO Si;ZHANG Wei;MA Guo-rong;WAN Qiao-feng(School of Clinical Medicine,Department of Pathogen Biology and Immunology,Ningxia Medical University,Yinchuan 750004,China;School of Clinical Medicine, Ningxia Medical University)

机构地区：[1]宁夏医科大学基础医学院病原生物学与免疫学系,宁夏银川750004 [2]宁夏医科大学临床医学院

出　　处：《中国病原生物学杂志》2022年第12期1404-1409,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.82160027);2022年宁夏高等学校科学研究项目(NYG2022035);宁夏常见传染病防治重点实验室开放课题(No.XZ2021011)。

摘　　要：目的 探究结核分枝杆菌Ag85B的真核表达质粒pcD-Ag85B经肌肉免疫后对小鼠结肠黏膜SIgA及菌群的影响。方法 以pcDNA3.1+为真核表达载体,将信号肽基因和编码Ag85B的Rv1886c基因共963 bp核苷酸序列连接到pcDNA3.1+,构建真核表达质粒pcD-Ag85B。通过双酶切和测序鉴定无误后,将pcD-Ag85B重组质粒转染中国仓鼠卵巢癌细胞(CHO)并验证其表达。采用微针注射法将pcD-Ag85B重组质粒经大腿肌肉免疫C57BL/6J小鼠;初免后42 d收集小鼠粪便及结肠黏液进行16S RNA高通量测序和菌群结构分析,并测定SIgA特异性抗体效价。结果 成功构建真核表达质粒pcD-Ag85B并检测到Ag85B蛋白的特异性表达,蛋白相对分子质量约为35×10~3,与预期相符。一免疫后42 d,Ag85B组小鼠结肠厚壁菌门-乳杆菌属的罗伊氏乳杆菌(Lactobacillus reuteri)和约氏乳杆菌(Lactobacillus johnsonii)丰度与对照组相比显著增高(均P<0.05);肠粘膜SlgA效价为1∶4500。结论 成功获得表达Ag85B蛋白的真核表达质粒,用该质粒免疫能诱导小鼠结肠粘膜产生体液免疫效应,改善结肠益生菌-乳酸杆菌群丰度。Objective To investigate the effects of Mycobacterium tuberculosis Ag85 B eukaryotic expression plasmid PCD-Ag85 bB on SIgA and bacterial community of colonic mucosa in mice after muscular immunization.Methods Using pcDNA3.1+ as the eukaryotic expression vector,the signal peptide gene and the Rv1886 c gene encoding Ag85 B were connected to pcDNA3.1+ with a total of 963 bp nucleotide sequence to construct the eukaryotic expression plasmid PGD-AG85 B.After double enzyme digestion and sequencing,the recombinant plasmid pcD-Ag85 B was transfected into Chinese hamster ovarian cancer cells(CHO) and its expression was verified.C57 BL/6 J mice were immunized with pcD-Ag85 B recombinant plasmid via thigh muscle by micronal injection.Mice feces and colonic mucus were collected 42 days after primary immunization for high-throughput sequencing of 16 S RNA and bacterial structure analysis,and SIgA specific antibody titer was determined.Results The eukaryotic expression plasmid pcD-Ag85 B was successfully constructed and the specific expression of Ag85 B protein was detected.The molecular weight of the protein was about 35×10~3,which was consistent with the expectation.At 42 days after immunization,the abundance of Lactobacillus reuteri and Lactobacillus johnsonii in the colon of Ag85 B group was significantly higher than that of the control group(all P< 0.05);The SlgA titer of intestinal mucosa was 1∶45000.Conclusion The Ag85 B protein expression plasmid was successfully obtained,which can effectively induce the humoral immune effect of colonic mucosa in mice,and improve the colonic probiotic-lactobacillus community.

关 键 词：结核分枝杆菌 AG85B 真核表达质粒 抗体效价 益生菌 

分 类 号：R378.911[医药卫生—病原生物学]