融合基因EBNA1-LMP1重组腺病毒的构建及其免疫学研究

目的 构建EB病毒（Epstein-Barr virus, EBV）EBNA1和LMP1融合基因的重组腺病毒,探究重组腺病毒的免疫学作用。方法 以质粒pCXWB-EBNA1和pMV261-LMP1为模板,通过PCR扩增EBNA1和LMP1基因,并通过linker以重叠延伸PCR的方式构建融合基因EBNA1-LMP1,将其与腺病毒穿梭质粒pDC316-mCMV-EGFP通过双酶切、连接构建重组质粒EBNA1-LMP1-pDC316-mCMV-EGFP,并通过PCR、酶切和基因测序进行鉴定。将重组质粒EBNA1-LMP1-pDC316-mCMV-EGFP与包装质粒pBHGlox(delta)E1,3Cre转染293T细胞以获得重组腺病毒;通过Western blot检测融合基因EBNA1-LMP1在293T细胞中的表达;用重组腺病毒免疫C57BL/6J小鼠,测定小鼠体内CD4⁺T细胞、CD8⁺T细胞和细胞因子TNF-α、IFN-γ的比例,观察重组腺病毒的免疫效果。结果 通过PCR获得基因EBNA1（717 bp）和LMP1（1 161 bp）,通过重叠延伸PCR获得EBNA1-LMP1 （1 923 bp）融合基因,经PCR、双酶切以及基因测序证实融合基因EBNA1-LMP1已成功插入腺病毒穿梭载体pDC316-mCMV-EGFP;Western blot表明融合基因在293T细胞中表达蛋白EBNA1和LMP1,大小约70 000。动物实验显示,重组腺病毒免疫组的脾CD4⁺T细胞、CD8⁺T细胞占总细胞的54.2%和39.2%;小鼠眼球血中重组腺病毒免疫组CD4⁺T淋巴细胞和CD8⁺T淋巴细胞占总细胞的54.2%和39.2%;而重组腺病毒免疫组的脾TNF-α和IFN-γ占总细胞因子的0.72%和7.63%,均高于对照组,差异均具有统计学意义（P<0.05）。结论 本实验成功获得了能稳定表达融合蛋白的重组腺病毒,动物实验显示重组腺病毒能有效刺激小鼠体内T淋巴细胞、B淋巴细胞的增殖和细胞因子TNF-α、IFN-γ的分泌。

Construction of recombinant adenovirus with fusion gene EBNA1-LMP1 and its immunogenicity

Objective To construct recombinant adenovirus with Epstein-Barr virus (EBV) fusion gene expression vector containing EBV gene EBNA1 and LMP1, and to explore the immunological effect of recombinant adenovirus, so as to obtain a vaccine with certain preventive and therapeutic effects on EBV positive tumor. Methods Using plasmids pcxwb-EBNA1 and pMV261-LMP1 as templates, EBNA1 and LMP1 genes were amplified by PCR, and the fusion gene EBNA1-LMP1 was constructed by overlapping extension PCR through linker sequence. The recombinant plasmid EBNA1-LMP1-pDC316-mCMV-EGFP was constructed by double enzyme digestion and connection with adenovirus shuttle vector pDC316-mCMV-EGFP, and identified by PCR, enzyme digestion and gene sequencing. The recombinant plasmid EBNA1-LMP1-pDC316-mCMV-EGFP and the packaging plasmid pBHGlox(delta)E1,3Cre were transfected into 293T cells. The expression of fusion gene EBNA1-LMP1 in 293T cells was detected by Western blot; C57BL/6J mice immunized with recombinant adenovirus. After immunization, the eye blood and spleen of mice were taken to determine the proportion of CD4⁺T cells, CD8⁺T cells and cytokine TNF-α, IFN-γ, so as to obtain the immune effect of recombinant adenovirus. Results EBNA1 (717 bp) and LMP1 (1 161 bp) were obtained by PCR. EBNA1-LMP1 (1 923 bp) fusion gene was obtained by overlapping extension PCR. PCR, double enzyme digestion and gene sequencing confirmed that the fusion gene EBNA1-LMP1 had been successfully inserted into adenovirus shuttle vector pDC316-mCMV-EGFP; Western blot showed that the fusion gene expressed proteins EBNA1 and LMP1 in 293T cells, with a size of about 70 000. Animal experiments showed that the spleen CD4⁺T cells and CD8⁺T cells in the recombinant adenovirus immunized group accounted for 54.2% and 39.2% of the total cells, which was higher than that in the control group (P<0.05). CD4⁺T lymphocytes and CD8⁺T lymphocytes in mouse eye blood immunized with recombinant adenovirus accounted for 54.2% and 39.2% of the total cells (P<0.05). The spleen TNF-α and IFN-γ of recombinant adenovirus immunized group accounted for 0.72% and 7.63% of the total cytokines respectively, with statistical difference (P<0.05). Conclusions The recombinant plasmid EBNA1-LMP1-pDC316-mCMV-EGFP is successfully constructed and transfected into 293T cells, and the recombinant adenovirus that can stably express the fusion protein. Animal experiments show that the recombinant adenovirus can effectively stimulate the proliferation of T and B lymphocytes and the cytokine TNF-α and IFN-γ in mice. It lays a foundation for the research of EBV positive tumor vaccine.