融合基因EBNA1-LMP1重组腺病毒的构建及其免疫学研究  

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

作　　者：于美美 邵为林 崔晏硕 陈廷 薛庆节 YU Mei-mei;SHAO Wei-lin;CUI Yan-shuo;CHEN Ting;XUE Qing-jie(School of Basic Medical,Jining Medical University,Ji'ning,Shandong 272067,China;Department of Medical Laboratory,Weifang Medical University,Weifang,Shandong 261053,China)

机构地区：[1]济宁医学院基础医学院,山东济宁272067 [2]潍坊医学院医学检验学院,山东潍坊261053

出　　处：《中国热带医学》2022年第1期14-19,共6页China Tropical Medicine

基　　金：山东省重点研发计划项目(No.2018GSF118137);山东省医药卫生科技发展计划项目(No.2017WS339);山东省高等学校科技计划项目(No.J17KB085);济宁市重点研发计划项目(No.2019SMNS020);济宁医学院科研项目(No.JYFC2018KJ043,No.16008);济宁医学院省级大创项目(No.S202010443044);贺林院士工作站重点项目(No.JYHL2019ZD03)。

摘　　要：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(1161 bp),通过重叠延伸PCR获得EBNA1-LMP1(1923 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70000。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。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(1161 bp)were obtained by PCR.EBNA1-LMP1(1923 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 EBNA1and LMP1 in 293T cells,with a size of about 70000.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-αand IFN-γof recombinant adenovirus immunized

关 键 词：重组腺病毒 EB病毒阳性肿瘤 基因重组 

分 类 号：R392[医药卫生—免疫学]