EB病毒LMP2-LMP1Δ融合基因免疫效果的初步研究

目的构建EBV LMP2-LMP1Δ融合基因,初步观察融合基因体内诱导EBV特异性细胞免疫应答的效果。方法 （1）应用PCR方法构建包含EBV-LMP2全长和去除致癌基因的EBV-LMP1Δ融合基因,并将融合基因插入到pcDNA3.1（＋）-his真核表达载体中,构建重组表达质粒pcDNA-LMP2-LMP1Δ,Western blot和免疫荧光方法检测融合蛋白的表达。（2）使用pcDNA-LMP2-LMP1Δ及携带LMP1Δ和LMP2基因的重组腺病毒单独或联合免疫Balb/c小鼠,末次免疫1周后应用IFN-γELISPOT方法检测小鼠脾淋巴细胞中EBV特异性CTL水平。结果 （1）真核表达质粒pcDNA-LMP2-LMP1Δ能够在293细胞中表达LMP2-LMP1Δ融合蛋白,融合蛋白分子量大小正确,具有免疫原性。（2）重组质粒pcDNA-LMP2-LMP1Δ免疫小鼠能够诱导出EBV特异性的CTL,但诱导的CTL水平很低,1×106小鼠脾淋巴细胞中平均斑点数只有12个,远远低于LMP1Δ、LMP2重组腺病毒平均495个斑点数的免疫结果。但使用pcDNA-LMP2-LMP1Δ和重组腺病毒联合免疫,与只使用重组腺病毒相比,诱导的EBV特异性CTL水平能够显著提高,1×106小鼠脾淋巴细胞中平均斑点数可达1 001个（P〈0.01）。结论构建的EBV LMP2-LMP1Δ融合基因能够有效表达LMP2-LMP1Δ融合蛋白,并能够在小鼠体内诱导出EBV特异性的CTL反应,与重组腺病毒联合使用,可以提高特异性CTL应答水平。

Construction and preliminary immunological study of EB virus LMP2-LMP1Δ fusion gene

Objective To construct EBV LMP2-LMP1Δ fusion gene and preliminarily investigate an induced EBV-specific cellular immunological response in vivo.Methods A eukaryotic expression vector was constructed by inserting pcDNA3.1（＋）-his plasmid of the fusion gene of full-length EBV-LMP2Δ and partial sequence of EBV-LMP1Δ,later of which had been removed of oncogene.The expression of fusion protein was determined by Western blot analysis and immunofluorescence essay in 293 cell line.Balb/c mice were divided into 4 groups and immunized with PBS,pcDNA3.1-LMP2-LMP1Δ,adenovirus carrying LMP1Δ /LMP2 genes and the combination of pcDNA3.1-LMP2-LMP1Δ（wk 0,2） and the recombinant adenovirus（wk 4）,respectively.The EBV-specific CTL levels of the mouse spleen lymphocytes were analyzed with IFN-γ ELISPOT essay in one week after the final immunization.Results The reconstructed pcDNA3.1-LMP2-LMP1Δ-his expressed the LMP2-LMP1Δ fusion protein in 293 cells correctly per MW and immunogenicity.The induced EBV-specific CTL response was lower in the mice immunized with the recombinant plasmid pcDNA3.1-LMP2-LMP1Δ-his alone（12 spots/1×106 mouse spleen lymphocytes on average） than that in the mice immunized with the recombinant adenovirus vector vaccine（495 spots/1×106 mouse spleen lymphocytes spots on average）.However,the induced EBV-specific CTL response was greatly improved（1 001 spots/ 1×106 mouse spleen lymphocytes on average） when mice were immunized with the combination of pcDNA3.1-LMP2-LMP1Δ-his and the recombinant adenovirus（P0.001）.Conclusions The new reconstructed EBV LMP2-LMP1Δ fusion gene eukaryotic expression vector improved the EBV-specific cellular CTL response when used together with recombinant adenovirus vaccine.