人IL-2与EB病毒BZLF1融合基因重组BCG的构建及免疫学研究

Construction and immunological study of recombinant BCG with a fusion gene cosisting of human IL-2 and EB virus BZLF1

目的构建人IL-2与EB病毒(Epstein-Barr virus,EBV)基因BZLF1的融合基因重组BCG(recombinant BCG,rBCG),并分析其免疫效果。方法通过重叠延伸技术,构建融合基因ILBZ,将ILBZ与穿梭表达载体pMV261连接并转化至BCG中进行表达。注射rBCG至C57BL/6J小鼠体内,通过流式细胞术检测和CTL检测分析其免疫反应;注射rBCG至构建的人源性EBV阳性肿瘤的BALB/c-nu裸鼠中,观察rBCG对肿瘤的治疗效果。结果重叠延伸PCR获得的融合基因ILBZ为1248bp;Western blot检测rBCG表达的融合蛋白能被IL-2抗体和ZEBRA抗体识别,大小约55ku;动物免疫试验显示,注射rBCG的小鼠CD4^(+)T细胞和CD8^(+)T细胞百分率分别为45.1%和32.3%,高于BCG(28.5%和24.0%)及PBS的百分率(21.3%和18.1%)(P<0.05);动物治疗实验显示,注射rBCG治疗的裸鼠NK细胞占2.99%,高于BCG(NK细胞为1.89%)和PBS(NK细胞为0.68%)(P<0.05);rBCG注射2周,EBV阳性小鼠肿瘤体积和重量明显小于对照组。结论成功构建rBCG并表达出稳定的融合蛋白,且rBCG能刺激小鼠产生免疫反应,具有抗肿瘤作用。

Objective To construct recombinant BCG(rBCG)with a fusion gene consisting of human IL-2 and BZLF1,agene of the Epstein-Barr virus(EBV),and to analyze its immune action.Methods The single genes IL-2 and BZLF1 were used to construct the fusion gene ILBZ via overlapping extension.The recombinant plasmid ILBZ-pMV261 was constructed via enzyme digestion and ligation of the shuttle expression vector pMV261 to ILBZ.The identified recombinant plasmid ILBZ-pMV261 was transformed into BCG and expressed.After rBCG was injected into C57 BL/6 Jmice,the immune response was analyzed using flow cytometry and CTL.rBCG was injected into C57 BL/6 Jmice,and the immune response was analyzed using flow cytometry and CTL.rBCG was injected into BALB/c-nu nude mice with human EBV-positive tumors.The therapeutic effect of rBCG on tumors was observed using flow cytometry,and changes in tumor size and weight were determined.Results The single gene BZLF1 was 738 bp in length and IL-2 was 465 bp in length.The fusion gene ILBZ obtained via overlapping extension PCR was 1248 bp in length.Western blotting indicated that the fusion protein expressed by rBCG was recognized by IL-2 antibodies and ZEBRA antibodies,and it was about 55 ku in size.An animal immunization test indicated that the percentage of CD4^(+)T cells was 45.1%and the percentage of CD8^(+)T cells was 32.3%in mice injected with rBCG;those percentages were higher than the percentages of CD4^(+)and CD8^(+)T cells in mice injected with BCG(28.5%and 24.0%)or PBS(21.3%and 18.1%)(P<0.05).Animal treatment experiments indicated that the percentage of NK cells in nude mice treated with rBCG was 2.99%,which was higher than the percentage in mice injected with BCG(1.89%)or PBS(0.68%)(P<0.05).After injection of rBCG for 2 weeks,the tumor volume and weight in EBV-positive mice was significantly smaller and lighter than the tumor volume and weight in the control group.Conclusion RBCG was successfully constructed and expressed as a stable fusion protein.rBCG can stimulate an immune response in mice and it has antitumor action.