幽门螺杆菌Lpp20-IL2核酸疫苗免疫活性  

Construction of Helicobacter pylori Lpp20-IL2 DNA vaccine and evaluation of its immunocompetence in C57BL/6 mice

作　　者：于文[1] 张艳[1] 靖吉芳[1] 刘志杰[1] 

机构地区：[1]湖南省南华大学病原生物研究所,衡阳421001

出　　处：《微生物学报》2010年第4期554-559,共6页Acta Microbiologica Sinica

基　　金：湖南省自然科学基金优秀面上项目(06JJ2093);湖南省教育厅青年基金(08B067)~~

摘　　要：【目的】观察pcDNA3.1(+)/Lpp20-IL2免疫C57BL/6小鼠后所产生的体液免疫和细胞免疫应答水平,为研制高效、新型的幽门螺杆菌核酸疫苗提供实验依据。【方法】构建pcDNA3.1(+)/Lpp20-IL2重组载体,并转染HeLa细胞,用Western-blot观察鉴定其在真核细胞得到表达后免疫C57BL/6小鼠,ELISA间接法测定小鼠血清中抗Lpp20IgG抗体水平,ELISA双抗体夹心法检测脾淋巴细胞培养上清中IFN-γ、IL4水平,MTT比色法检测脾淋巴细胞增殖反应,免疫荧光组化法检测Lpp20蛋白在小鼠肌肉组织中的表达情况。【结果】成功构建了pcDNA3.1(+)/Lpp20-IL2真核表达载体,且重组质粒能在HeLa细胞内有效表达目的蛋白;小鼠接种pcDNA3.1(+)/Lpp20-IL2核酸疫苗后能产生特异性IgG抗体,8w后ELISA测定血清抗体A450值明显升高。核酸疫苗pcDNA3.1(+)/Lpp20-IL2免疫组小鼠脾淋巴细胞经特异性抗原刺激后,培养上清中IFN-γ、IL4含量明显升高。pcDNA3.1(+)/Lpp20-IL2和pcDNA3.1(+)/Lpp20核酸疫苗组小鼠脾淋巴细胞经特异性抗原刺激后,刺激指数明显高于空质粒组和PBS组。Lpp20蛋白在小鼠肌肉组织中能够有效表达。【结论】幽门螺杆菌Lpp20-IL2融合基因核酸疫苗和Lpp20单基因核酸疫苗均能刺激机体产生较强细胞免疫应答和体液免疫应答,且前者能诱导更强的细胞免疫应答。ObjectivesTo evaluate the humoral and cellular immune responses in C57BL /6 mice induced by pcDNA3.1（＋ ） /Lpp20-IL2 for further development of DNA vaccine against H.pylori infection.Methods The eukaryotic expression vector pcDNA3.1（＋ ） /Lpp20-IL2 was constructed and then transfected into HeLa cells using liposome.Western blot was used to verify the expression of Lpp20 antigen gene in HeLa cells.Then five groups of 6-week-old C57BL /6 mice were immunized intramuscularly with either of the following vaccines：pcDNA3.1（＋ ） /Lpp20-IL2, pcDNA3.1（＋ ） /Lpp20,pcDNA3.1（＋ ） /IL2,mock plasmid pcDNA3.1（＋ ）,or PBS four times at 1-week interval.Six weeks after the last injection,the specific IgG in the sera of C57BL /6 mice and the cytokine content of IFN-γ and IL4 in mice spleen lymphocyte culture medium after stimulation by recombinant Lpp20（rLpp20 ） was tested quantitatively by ELISA,respectively.The proliferation of spleen cells was measured by MTT assay.Lpp20-IL2 fusion protein in mouse muscular tissue was detected by immunofluorescence histochemistry.ResultsThe eukaryotic expression recombinant pcDNA3.1（＋ ） /Lpp20-IL2 was successfully constructed and could be expressed in HeLa cells .The significant specific antibody titers were detected by ELISA in pcDNA3.1（＋ ） /Lpp20-IL2 DNA vaccine groups and the highest titer was 1∶ 4096 after eight weeks.The cytokines content of IFN-γ and IL4 in cultural supernatant of spleen lymphocytes from mice immunized with pcDNA3.1（＋ ） /Lpp20-IL2 increased significantly.After stimulated by corresponding antigen,the stimulation index of pcDNA3.1（＋ ） /Lpp20-IL2 and pcDNA3.1（＋ ） /Lpp20 group were higher than that of control groups（P 0.01）.Lpp20-IL2 fusion protein could be expressed in mouse muscular tissue.ConclusionsWe have successfully constructed the recombinant eukaryotic expression plasmid pcDNA3.1（＋ ） /Lpp20-IL2 and the Lpp20-IL2 fusion protein could be effectively expressed in HeLa cells.Both pcDNA3.1（�

关 键 词：幽门螺杆菌 脂蛋白Lpp20 白细胞介素2 核酸疫苗 免疫活性 

分 类 号：R392[医药卫生—免疫学]