摘要
目的:构建Cpn0308重组质粒pcDNA3.1/His A-Cpn0308,将重组质粒腹腔注射小鼠后观察小鼠免疫反应变化,以期为进一步研究Cpn0308免疫保护性奠定基础。方法:构建pcDNA3.1/His A-Cpn0308,并用菌落PCR、双酶切、序列测定等多种技术确定其正确性;将重组质粒转染HeLa细胞,间接免疫荧光法检测细胞内蛋白表达情况;重组质粒免疫BALB/c小鼠,一定时间后Western blot检测血清Cpn0308抗体特异性,间接ELISA法检测小鼠血清中Cpn0308 IgG抗体水平、ELISA试剂盒检测血清中细胞因子。结果:pcDNA3.1/His A-Cpn0308重组质粒构建成功且序列正确;重组质粒转染的HeLa细胞胞浆观察到黄绿色荧光,对照组无荧光;重组质粒组血清抗体A450x±s为0.343±0.024,pcDNA3.1/His A质粒组为0.174±0.018,PBS组为0.156±0.023,WB结果显示重组质粒组小鼠血清稀释800倍后仍有特异性目的条带出现,对照组不出现;重组质粒组小鼠IFN-γ浓度均值为264 ng/L,IL-4浓度均值为22 ng/L,pcDNA3.1/His A质粒组为:IFN-γ120 ng/L,IL-4 10 ng/L,PBS组为:IFN-γ99 ng/L,IL-4 9 ng/L。重组质粒组IgG抗体水平和细胞因子水平明显升高,与对照组相比有统计学差异。结论:pcDNA3.1/His A-Cpn0308真核表达重组质粒构建成功,且能够在真核细胞中表达目的蛋白;重组质粒对小鼠进行免疫后,提高了小鼠血清IgG抗体水平和细胞因子水平,为进一步研究Cpn DNA疫苗以及研究该蛋白的生物学功能提供实验基础。
Objective：To construct a recombinant plasmid containing Cpn0308 gene,then transfect it into HeLa cells and detect its expression;and detect the mice immunity to pcDNA3.1/His A-cpn0308.Methods：The pcDNA3.1/His A-Cpn0308 was constnected by using molecular cloning technology,then transfected into HeLa cells.The expressed protein was identified by indirect immunofluorescence assay.After this,pcDNA3.1/His A-Cpn0308 were injected intraperitoneally into normal BALB/c mice,pcDNA3.1/His A and PBS were used as control groups.The cytokines and anti-Cpn0308 antibodies in the sera were detected by ELISA,and the specificity of serum antibody to Cpn0308 were detected by Western blot.Results：Eukaryotic expression recombinant pcDNA3.1/His A-Cpn0308 had been successfully constructed;Cpn0308 protein expressed in the HeLa cells;in the pcDNA3.1/His A-Cpn0308 group,the average level of IFN-γ was 264 ng/L,and IL-4 was 22 ng/L;pcDNA3.1/His A group the average level of IFN-γ was 120 ng/L,and IL-4 was 10 ng/L,and PBS group was 99 ng/L and 9 ng/L;in the pcDNA3.1/His A-Cpn0308 group,the A450 x±s of IgG was 0.343±0.024,pcDNA3.1/His A group was 0.174±0.018,and PBS group was 0.156±0.023.WB results showed that mice serums of recombinant plasmid group still had specific purpose bands at dilution of 1∶ 800,but the control groups didn＇t appear.The antibody and cytokines level were higher in experimental group than control groups.There was statistical significance between experimental group and control groups.Conclusion：Recombinant plasmid can express Cpn0308 protein in eukaryotic system.The mice immune fuctions were enhanced obviously after intraperitoneal immunization with Cpn0308,which lay the foundation for studying the biological activities and the development of the Chlamydia pneumoniae vaccine.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2012年第9期782-786,共5页
Chinese Journal of Immunology
