乙肝病毒DNA疫苗pHL-sec-LHBs的构建及小鼠免疫效果检测  

Construction of hepatitis B virus DNA vaccine pHL-sec-LHBs and its immune effect in mice

作　　者：石文刚 徐瑞贤 刘敏 张桃苹[1] Faisal Mahmood 宋玉竹 韩芹芹 夏雪山 张金阳 SHI Wen-gang;XU Rui-xian;LIU Min;ZHANG Tao-ping;FAISAL Mahmood;SONG Yu-zhu;HAN Qin-qin;XIA Xue-shan;ZHANG Jin-yang(Molecular Medicine Research Center of Yunnan Province,Faculty of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500,China;Department of Infectious Diseases and Liver Diseases,First People's Hospital of Yunnan Province,Kunming University of Science and Technology)

机构地区：[1]昆明理工大学生命科学与技术学院,云南省分子医学研究中心,云南昆明650500 [2]昆明理工大学附属云南省第一人民医院感染性疾病及肝病科

出　　处：《中国病原生物学杂志》2023年第4期373-376,384,共5页Journal of Pathogen Biology

基　　金：昆明理工大学分析测试基金项目(No.2021M20202118042);云南省临床病毒学重点实验室开放课题(No.2018DG010);云南(昆明)张文宏专家工作站项目(No.YSZJGZZ-2020051)。

摘　　要：目的构建针对乙肝病毒的DNA疫苗,并检测该DNA疫苗免疫BALB/c小鼠的效果。方法选择乙肝病毒表面蛋白中的LHBs蛋白基因作为抗原基因,以pHL-sec真核外泌型质粒为载体构建pHL-sec-LHBs质粒。使用pHL-sec-LHBs质粒、pHL-sec-LHBs质粒瞬时转染细胞上清和pHL-sec空载质粒在无佐剂或有佐剂条件下分别对BALB/c小鼠进行免疫,共计免疫5次,每次间隔14 d。第5次免疫7 d后对小鼠进行LHBs蛋白特异性抗体检测。结果成功构建pHL-sec-LHBs重组质粒。使用该质粒瞬转293T细胞,Western blot检测到细胞内LHBs蛋白的表达,并有自切外泌信号肽迹象;ELISA检测细胞上清中LHBs蛋白成功外泌。BALB/c小鼠免疫试验显示,pHL-sec-LHBs质粒成功诱发小鼠针对LHBs蛋白特异性抗体产生。结论成功构建pHL-sec-LHBs重组质粒作为乙肝病毒DNA疫苗,该疫苗对BALB/c小鼠中具有良好的免疫原性。Objective To construct a DNA vaccine against hepatitis B virus(HBV)and evaluate its immune effect in BALB/c mice.Methods The LHBs gene of the hepatitis B virus was selected as the antigen gene,and the eukaryotic exosomal plasmid pHL-sec was used as the vector to construct the pHL-sec-LHBs plasmid.BALB/c mice were immunized with pHL-sec-LHBs plasmid,pHL-sec-LHBs plasmid transiently transfected cell supernatant and pHL-sec empty plasmid without or with adjuvant,respectively,for a total of 5 times with an interval of 14 days.Seven days after the fifth immunization,the mice were tested for LHBs protein-specific antibodies.Results The recombinant plasmid pHL-sec-LHBs was successfully constructed.The plasmid was transiently transfected into 293 T cells,and the expression of LHBs protein in the cells was detected by Western blot,and there was a sign of auto-excision of the signal peptide.ELISA detected the successful secretion of LHBs protein in cell supernatant.The immune test of BALB/c mice showed that the pHL-sec-LHBs plasmid successfully induced the production of specific antibodies against LHBs protein in mice.Conclusion The pHL-sec-LHBs recombinant plasmid was successfully constructed as a hepatitis B virus DNA vaccine,which has good immunogenicity in BALB/c mice.

关 键 词：乙肝病毒 DNA疫苗 免疫原性 

分 类 号：R373.21[医药卫生—病原生物学]