结核分枝杆菌Ag85B DNA疫苗免疫效果的初步评价  

Preliminary evaluation of immune effect of DNA vaccine against Mycobacterium tuberculosis Ag85B

作　　者：王娜[1] 刘琼 杨雨欣 李慧[1] 张炜[1] 马国荣 陈耀庚 万巧凤[1] WANG Na;LIU Qiong;YANG Yu-xin;LI Hui;ZHANG Wei;MA Guo-rong;CHEN Yao-geng;WAN Qiao-feng(不详;Department of Pathogen Biology and Immunology,School of Basic Medicine,Ningxia Medical University,Yinchuan 750004,Ningxia Hui Autonomous Region,China)

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,宁夏银川750004 [3]宁夏医科大学理学院数学系,宁夏银川750004

出　　处：《中国生物制品学杂志》2022年第12期1427-1431,共5页Chinese Journal of Biologicals

基　　金：国家自然科学基金(82160027);宁夏自然科学基金(2020A0459);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(XZ2021010)。

摘　　要：目的初步评价结核分枝杆菌(Mycobacterium tuberculosis,M.tb)Ag85B DNA疫苗的免疫效果。方法对含HSV2-gD信号肽基因和Rv1886c基因(共963 bp核苷酸序列)的重组质粒pcD-sRv1886c进行双酶切和测序鉴定。将鉴定正确的重组质粒pcD-s Rv1886c转染CHO细胞,Western blot法检测CHO细胞培养液中Ag85B蛋白的表达情况。将重组质粒pcD-sRv1886c经肌肉注射免疫C57BL/6J小鼠,共免疫2次,间隔14 d,首次免疫后14、28及42 d经小鼠眼眶后静脉丛采血,分离血清,间接ELISA法检测小鼠血清中IgG及IgG1抗体效价;首次免疫后42 d无菌取小鼠脾脏,制成单细胞悬液,流式细胞术检测分泌IFNγ^(+)的CD4^(+)T及CD8+T细胞比例,CCK8法检测脾淋巴细胞增殖情况。结果双酶切和测序鉴定证明重组质粒pcD-sRv1886c构建正确,转染CHO细胞后表达的蛋白可与鼠抗Ag85B单克隆抗体发生特异性结合。首次免疫后14 d,重组质粒免疫组小鼠血清中IgG及IgG1特异性抗体效价分别为1∶1720和1∶460,28 d后为1∶5760和1∶2560,42 d后为1∶6080和1∶2720。首次免疫后42 d,重组质粒免疫组和PBS阴性对照组小鼠脾细胞中CD3^(+)CD4^(+)IFNγ^(+)细胞比例分别为(2.03±0.23)%和(0.14±0.02)%,CD3^(+)CD8+IFNγ^(+)细胞比例分别为(1.05±0.08)%和(0.13±0.01)%,差异均有统计学意义(t分别为18.38和11.58,P均<0.001)。重组质粒免疫组小鼠脾淋巴细胞SI为5.02,明显高于PBS对照组(1.06),且差异有统计学意义(t=15.66,P<0.001)。结论M.tb Ag85B DNA疫苗可有效诱导机体产生体液免疫及细胞免疫效应。Objective To preliminarily evaluate the immune effect of DNA vaccine against Mycobacterium tuberculosis(M.tb)Ag85B.Methods Recombinant plasmid pcD-sRv1886c containing HSV2-gD signal peptide gene and Rv1886c gene(963 bp nucleotides in total)was identified by restriction analysis and sequencing,and the correct one was transfected to CHO cells.The expression of Ag85B protein in cell culture was determined by Western blot.C57BL/6J mice were immunized i.m.with recombinant plasmid pcD-sRv1886c for 2 times at an interval of 14 d,of which the serrm samples were collected 14,28 and 42 d after the first immunization and determined for IgG and IgG1 antibody titers by indirect ELISA.The spleens of mice were taken aseptically 42 d after the first immunization,prepared into single-cell suspension and determined for proportion of CD4^(+)T and CD8+T cells secreting IFNγby flow cytometry,and for lymphocytes proliferation by CCK8 assay.Results Recombinant plasmid pcD-sRv1886c was constructed correctly as proved by restriction analysis and sequencing,and the protein expressed in CHO cells after transfection with the plasmid showed specific binding to mouse monoclonal antibody(McAb)against Ag85B.The titers of IgG and IgG1 in sera of mice were 1∶1720 and 1∶46014 d,1∶5760 and 1∶256028 d,and 1∶6080 and 1∶272042 d after the first immunization,respectively.The proportions of CD3^(+)CD4^(+)cells secreting IFNγ^(+)in spleen of mice 42 d after the first immunization with the recombinant plasmid and PBS as negative control were(2.03±0.23)%and(0.14±0.02)%,while those of CD3^(+)CD8+cells secreting IFNγ^(+)were(1.05±0.08)%and(0.13±0.01)%,respectively,both of which showed significant differences(t=18.38 and 11.58 respectively,each P<0.001).Splenic lymphocyte SI of mice immunized with the recombinant plasmid was 5.02,which was significantly higher than that in PBS control group(1.06)(t=15.66,P<0.001).Conclusion DNA vaccine against M.tb Ag85B effectively induced humoral and cellular immunities.

关 键 词：结核分枝杆菌 DNA疫苗 Ag85B蛋白 

分 类 号：R183.3[医药卫生—流行病学]