含EBV-LMP2基因重组腺病毒疫苗的构建及其诱导CTL应答的初步探讨

The in vitro and in vivo immunogenicity of recombinant adenovirus vaccine containing EBV-latent membrane protein 2

目的 构建包含EBV LMP2基因的重组腺病毒疫苗并探讨它在体内外的免疫性质。方法 采用pAdeasy 1系统构建包含EBV LMP2基因的重组腺病毒疫苗 ,并用IFA、PCR和TCID50 等方法对其特异性进行鉴定。通过重组腺病毒感染人树突状细胞 (DC) ,在体外活化自体T细胞 ;以及通过重组腺病毒感染小鼠淋巴细胞 ,皮下免疫同种小鼠 ,体内活化CTL评价其免疫效果。结果 通过PCR以及间接免疫荧光试验分别证实了病毒目的基因LMP2的存在以及蛋白在 2 93细胞中的表达。采用TCID50 方法 ,测定第 6代的病毒滴度为 2× 10 8。体内外的免疫实验结果显示通过这两种方式均可以有效地引发针对EBV LMP2的CTL反应。结论 包含EBV LMP2基因的重组腺病毒疫苗可以在体内外有效地引发CTL应答 ,这些资料为下一步临床应用含LMP2的重组腺病毒作为疫苗治疗和预防EBV相关肿瘤奠定了基础。

Objective To construct the recombinant adenovirus vaccine containing LMP2 gene and assess its immunogenicity in vitro and in vivo. Methods Using pAdeasy-1 system, we constructed the recombinant adenovirus vaccine containing LMP2 gene. To estimate its immunogenicity, we used the DC infected by Adeasy-LMP2 to stimulate autologous T cells in vitro and the murine lymphocyte infected by Adeasy-LMP2 to immunize mice through hypodermic injection. Results With PCR and immunofluorescence assay, we identified the existence of target gene LMP2 in the Adeasy-LMP2 and the expression of LMP2 protein in the 293 cells infected by Adeasy-LMP2. Using TCID 50 method, we determined the titer of the sixth generation Adeasy-LMP2 was 2×108. The immunology assay in vitro and in vivo showed that these two methods both can effectively elicit anti-LMP2 CTL reaction. Conclusions The recombinant adenovirus vaccine containing EBV-LMP2 gene effectively elicits cell immunity. These data are the base of our clinical test of LMP2 recombinant adenovirus vaccine in prevention and treatment of EBV-associated tumors.