钙网蛋白基因120片段和人乳头瘤病毒6bE7基因诱导的小鼠细胞免疫反应

The Specific Immune Response Elicited by Recombinant CRT120/HPV6bE7 DNA Vaccine in Mice

目的研究嵌合DNA疫苗钙网蛋白基因120片段(CRT120)和人乳头瘤病毒6b型E7基因(HPV6bE7)的免疫学特性。方法克隆、构建重组质粒即嵌合DNA疫苗pcDNA3．1-GFP-CRT120／ HPV6bE7、pcDNA3．1-GFP-HPV6bE7、pcDNA3．1-GFP-CRT120;经脂质体转染获得HPV6bE7转染阳性 B16细胞株;用肌内注射法对C57BL/6小鼠进行重组质粒DNA疫苗免疫;检测免疫小鼠的外周血T淋巴细胞亚群变化、小鼠脾细胞毒性T淋巴细胞杀伤活性以及与靶细胞共孵育后白介素2和干扰素γ的分泌水平。结果获得构建正确的嵌合DNA疫苗pcDNA3．1-GFP-CRT120／HPV6bE7以及稳定表达HPV6bE7 的阳性细胞株。CRT120／HPV6bE7较之HPV6bE7能引起免疫小鼠外周血中CD8+T淋巴细胞亚群分化和 TCRγδ T细胞上调(P<0．05),脾淋巴细胞与靶细胞共孵育上清液中白介素2和干扰素γ的浓度也明显高于HPV6bE7和CRT120组(P<0．05);CRT120／HPV6bE7免疫的小鼠淋巴细胞对靶细胞B16／HPV6bE7 有明显的杀伤活性。结论 CRT120／HPV6bE7嵌合DNA疫苗能够诱导免疫小鼠的病毒抗原特异性细胞免疫。

Objective To investigate the cellular immune response to the recombinant DNA vaccine CRT120/HPV6bE7 in mice. Methods The recombinant encoding HPV6bE7, linked with CRT120, was constructed in pcDNA3.1 eukaryotic vector with the report gene GFP. The pcDNA3,1-GFP -HPV6bE7 was transfected into B16 cells by a lipofectamine kit. The C57BL/6 mice were vaccinated with recombinant DNA plamids, The T-cell phenotype in the peripheral blood lymphoeytes of the immunized mice was measured by flow cytometry, The CTL activity of the lymphocytes from the spleens and lymph nodes, and the levels of IL-2 and IFN-γ were analyzed. Results The constructed recombinant plasmid CRT120/HPV6bE7 was analyzed. Positive transfected cell clones were established and could stably express the target gene HPV6bE7. Compared with HPV6bE7, CRT120/HPV6bE7 plasmid enhanced to a greater extent CD8＊ T-lymphocyte differentiation, the number of TCR＇y8 T-lymphocytes and the levels of IL-2 and IFN-＇y （all P 〈 0,05）. The lymphocytes of CRT120/HPV6bE7 vaccinated mice exhibited significant CTL activity against B16/HPV6bE7 cells. Conclusions The recombinant CRT120/HPV6bE7 DNA vaccine can effectively elicits specific cellular immunity in mice.