CEA串连表位-HSP70融合基因疫苗诱导小鼠的表位特异性免疫应答

doi:吉林省科技厅重点项目资助课题(20020403，2

CEA串连表位-HSP70融合基因疫苗诱导小鼠的表位特异性免疫应答

安力彬^1,2,刘力华²,方艳秋², 李丹²,段秀梅²,许淑芬²,李树蕾²,谭岩²,宋燕³

1．吉林大学护理学院妇产科护理教研室，吉林长春 130021；2．吉林大学第一医院中心实验室，吉林 长春 130021；3．吉林大学中日联谊医院普通外科，吉林长春 130033

收稿日期:2007-12-21 修回日期:1900-01-01 出版日期:2008-03-28 发布日期:2008-03-28
通讯作者: 谭岩；宋燕

Induction of specific anti- CEA responses by DNA vaccination with fusion sequence of CEA repeat epitopes and heterogenous HSP70 in mice

AN Li-bin^1,2,LIU Li-hua²,FANG Yan-qiu²,LI Dan²,DUAN Xiu-mei²,XU Shu-fen²,LI Shu-lei²,TAN Yan²,SONG Yan³

1. Department of Nursing of Gynecology and Obstetrics,School of Nursing, Jilin University,Changchun 130021,China; 2. Central Laboratary, First Hospital,Jilin University,Changchun 130021,China; 3. Department of General Surgery, China-Japan Union Hospital, Jilin University,Changchun 130033,China

Received:2007-12-21 Revised:1900-01-01 Online:2008-03-28 Published:2008-03-28
Contact: TAN Yan²,SONG Yan³

摘要/Abstract

摘要： 目的：观察癌胚抗原(CEA)串联表位-HSP70融合基因疫苗诱导的免疫应答，为开发新型肿瘤特异性基因疫苗奠定基础。方法：在已经构建含有变异热休克蛋白(HSP)序列的基础上，插入重组CEA串联表位的编码片段获得CEA串联表位-HSP融合基因疫苗。3次肌肉注射免疫Balb/c小鼠，设立注射生理盐水的阴性对照组、注射氢氧化铝佐剂混悬CEA串联表位的阳性对照组及注射pCITriCEA_625-667-mtHSP70的实验组。FCM分析脾脏T细胞亚群；体外培养脾细胞，ELISA检测培养上清中干扰素γ(IFNγ)的相对含量；同时测定小鼠血清CEA特异性抗体的滴度。结果：阴性对照组脾细胞中CD3⁺和CD4⁺T细胞分别为55.1%±6.1%和30.2%±4.1%；实验组脾细胞中CD3⁺和CD4⁺T细胞分别为78.7%±9.2%和48.9%±4.7%，两组比较差异有显著性(P<0.01)。其体外特异性抗原肽诱导的IFNγ分泌处于本底水平，可视为生理性参数，体外非特异性和特异性的IFNγ分泌分别增加3和6倍(P<0.01)。血清中CEA表位特异性抗体滴度阴性对照组为0，阳性对照组＜1∶500,而融合基因疫苗免疫组达到1∶4 000。结论：CEA串联表位-HSP融合基因疫苗在体内诱导以激发辅助性T细胞为特征的免疫应答。

关键词: 热休克蛋白70, T细胞, 抗癌胚抗原抗体

Abstract: Objective To observe the immune responses stimulated by inoculation of DNA vaccine encoding a fusion protein of CEA tandem repeat epitopes and heterogenous HSP70 fragment as a basis for pioneering a novol DNA vaccine specific to carcinomas.Methods The recombinant sequence for two CEA epitopes was tandemly engaged and inserted into-upstream of in-frame-varied HSP70 of Mycobacterium tuberculosis to construct a gene vaccine.Balb/c mice were muscularly injected with recombinant DNA vaccine;negative control (mice were injected with normal saline), positive control (mice were injected with DNA vaccine harboring tandem CEA epitopes plus aluminium hydroxide adjuvant) and experimental group(mice were injected with PCITri CEA_625-667-met HSP70) were set up.After 3 inoculations,the mice were sacrificed and their splenocytes and sera were collected.T cell subsets were analyzed by FCM.IFNγ in their cultural supernatants and seral IgG antibody against CEA were detected with ELISA.Results The amount of CD3⁺and CD4⁺T cells in the mouse spleen in negative control was 55.1%±6.1% and 30.2%±4.1% with no production of IFNγ by in vitro culture of spleenocytes,which could be considered as basal physiological parameters.The fused DNA vaccine induced vigorous T cell responses in mice with nearly 50-fold increases of spleen CD3⁺and CD4⁺T cells and enhanced IFNγ production up to 3 folds for non-specificity and 6 folds 　to specific challenge in cultured splenocytes under stimulation of CEA peptide (P<0.01).The serum titer of IgG antibody against CEA epitope in negative control group was 0 and less than 1∶500 in positive control,whereas that in the mice immunized with the DNA vaccine encoding the fusion protein of the CEA epitopes and HSP70 was up to 1∶4000.Conclusion DNA vaccine for the fusion protein consisted of CEA repeat epitopes and heterogenous HSP70 fragment can induce a strong immune response in mice featured by stimulation of T helper cells.

Key words: heat shock protein-70, T cells, anti-CEA antibody

中图分类号:

安力彬,刘力华,方艳秋, 李丹,段秀梅,许淑芬,李树蕾,谭岩,宋燕. CEA串连表位-HSP70融合基因疫苗诱导小鼠的表位特异性免疫应答[J]. J4, 2008, 34(2): 241-245.

AN Li-bin,LIU Li-hua,FANG Yan-qiu,LI Dan,DUAN Xiu-mei,XU Shu-fen,LI Shu-lei,TAN Yan,SONG Yan. Induction of specific anti- CEA responses by DNA vaccination with fusion sequence of CEA repeat epitopes and heterogenous HSP70 in mice[J]. J4, 2008, 34(2): 241-245.

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