Cpn0308真核表达载体的构建及对小鼠免疫功能的影响

发布时间：2018-04-22 03:01

本文选题：肺炎衣原体 + Cpn　；参考：《中国免疫学杂志》2012年09期

【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。
[Abstract]:Aim: to construct Cpn0308 recombinant plasmid pcDNA3.1/His A-Cpn0308, and observe the changes of immune response in mice after intraperitoneal injection of the recombinant plasmid, so as to lay a foundation for further study on the protection of Cpn0308. Methods: pcDNA3.1/His A-Cpn0308 was constructed, and its correctness was confirmed by colony PCR, double enzyme digestion, sequencing, etc. The recombinant plasmid was transfected into HeLa cells, and the expression of intracellular protein was detected by indirect immunofluorescence. BALB/c mice were immunized with recombinant plasmid. After a certain period of time, Western blot was used to detect the specificity of serum Cpn0308 antibody, indirect ELISA method was used to detect the level of Cpn0308 IgG antibody in mouse serum and Elisa kit was used to detect cytokines in serum. Results the recombinant plasmid of 1% pcDNA3.1% his A-Cpn0308 was successfully constructed and the sequence was correct. The cytoplasm of HeLa cells transfected with the recombinant plasmid showed yellowish green fluorescence, while the control group had no fluorescence. The serum antibody A450x 卤s of recombinant plasmid group was 0.343 卤0.024% pcDNA3.1% of his A plasmid group was 0.174 卤0.018 + PBS group: 0.156 卤0.023 group. The results showed that there were still specific bands of purpose after the serum dilution of the recombinant plasmid group, but not in the control group. The average concentration of IFN- 纬 in recombinant plasmid group was 264 ng / L ~ (-1) IL-4 = 22 ng / L ~ (-1 / L) pcDNA3.1 / his A plasmid group: 1% IFN- 纬 ~ (120) ng / L ~ (-1) IL-4 / L ~ (10) ng / L ~ (-1) PBS group:: IFN- 纬 ~ (99) ng / L ~ (99) ng / L ~ (-9) ng 路L ~ (-1). The levels of IgG antibody and cytokines in the recombinant plasmid group were significantly higher than those in the control group. Conclusion the recombinant plasmid of pcDNA3.1 / his A-Cpn0308 was successfully constructed and could express the target protein in eukaryotic cells, and the level of serum IgG antibody and cytokine were increased after immunizing mice with the recombinant plasmid. It provides experimental basis for further study of Cpn DNA vaccine and the biological function of the protein.
【作者单位】：河北北方学院医学检验学院;
【分类号】：R392.1

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