结核杆菌热休克蛋白70刺激表位在融合基因疫苗中的佐剂作用

J4 ›› 2012, Vol. 38 ›› Issue (3): 506-511.

结核杆菌热休克蛋白70刺激表位在融合基因疫苗中的佐剂作用

段秀梅|邹亚斌|马洪喜|李树蕾|王银萍|王超|李玉琴

吉林大学第一医院病理诊断中心|吉林长春 130021

收稿日期:2012-01-06 出版日期:2012-05-28 发布日期:2012-05-28
通讯作者: 段秀梅（Tel:0431-88782143，E-mail:xiumeiduan428@yahoo.com.cn）;李玉琴（Tel:0431-88782740,E-mail:liyuqin-401@163.com） E-mail:xiumeiduan428@yahoo.com.cn；liyuqin-401@163.com
作者简介:段秀梅（1967-）|女|吉林省长春市人|教授|医学博士|主要从事肿瘤免疫学及分子生物学方面的研究。
基金资助:
吉林省长春市科技计划项目资助课题（2008093）

Adjuvant effect of mycobacterium tuberculosis heat shock protein 70 stimulating epitope on fusion gene vaccine

DUAN Xiu-mei,ZOU Ya-bin,MA Hong-xi,LI Shu-lei,WANG Yin-ping,WANG Chao,LI Yu-qin

Pathological Diagnosis Center, First Hospital,Jilin University,Changchun 130021,China

Received:2012-01-06 Online:2012-05-28 Published:2012-05-28

摘要/Abstract

摘要：

目的：探讨结核杆菌热休克蛋白70（mtHSP70）的刺激表位在肝细胞癌相关抗原661（HCA661）融合基因疫苗pCI-HCA661-mtHSP70中的佐剂作用，为提高基因疫苗的免疫效果提供依据。方法：18只BALB/c小鼠随机分为pCI-neo组、pCI-HCA661-mtHSP70C免疫组和pCI-HCA661-mtHSP70E免疫组，每组6只，分别肌肉注射100μg空质粒pCI-neo、重组质粒pCI-HCA661-mtHSP70C和pCI-HCA661-mtHSP70E，流式细胞术分析小鼠脾T细胞亚群变化；ELISA法检测体外培养脾细胞上清中干扰素γ(IFNγ)的含量及免疫小鼠血清中HCA661特异性抗体的滴度；体外特异性细胞毒T淋巴细胞(CTL)杀伤试验观察融合基因疫苗对肿瘤细胞的抑制作用。结果：pCI-HCA661-mtHSP70C免疫组和pCI-HCA661-mtHSP70E免疫组的CD3+、CD4+T淋巴细胞的百分比和CD4+/CD8+比值与pCI-neo组比较差异有统计学意义（P<0.05）；pCI-HCA661-mtHSP70E免疫组的CD3+、CD4+百分比和CD4+/CD8+比值均高于pCI-HCA661-mtHSP70C免疫组，差异有统计学意义（P<0.05）；在抗原肽刺激下，pCI-HCA661-HSP70E免疫组小鼠脾细胞培养上清中IFNγ含量较pCI-mtHCA661-HSP70C免疫组明显提高（P<0.05）；pCI-HCA661-mtHSP70C免疫组和pCI-HCA661-mtHSP70E免疫组IFNγ含量均高于pCI-neo组(P<0.05)。pCI-HCA661-mtHSP70E免疫组诱导的HCA661特异性抗体水平较pCI-HCA661-mtHSP70C免疫组显著增高（P<0.05）；pCI-HCA661-mtHSP70E免疫组可以产生更强的CTL抗肿瘤效应。结论：mtHSP70刺激表位可以诱导机体对HCA661融合基因疫苗的特异性免疫应答，发挥更强的佐剂作用。

Abstract:

Abstract:Objective To explore the adjuvant effect of stimulating epitope of mycobacterium tuberculosis heat shock protein 70(mtHSP70) on fusion gene vaccine of hepatocellular carcinoma associated antigens 661(HCA661), and to provide basis for improving immune effect of gene vaccine. Methods Eighteen BALB/c mice were randomly divided into three groups:pCI-neo group(n=6),pCI-HCA661-HSP70C group(n=6) and pCI-HCA661-HSP70E group(n=6).The BALB/c mice were immunized intramuscularly with empty plasmid pCI-neo,recombinant plasmid pCI-HCA661-mtHSP70C and pCI-HCA661-mtHSP70E.The changes of T cell subsets were analyzed by FCM. The antigen-stimulated production of IFNγ in the supernatants of splenocytes in vitro and the specific IgG antibody against HCA661 were detect by ELISA. The specific CTL cytotoxicity against tumor cells was used to detect the inhibitory effect on tumor cells. Results The percentagse of CD3+,CD4+T lymphocytes and the ratio of CD4+/CD8+ were significantly increased in pCI-HCA661-mtHSP70C group and pCI-HCA661-mtHSP70E group compared with pCI-neo group（P<0.05）. The percentages of CD3+,CD4+ T lymphocytes and the ratio of CD4+/CD8+ were significantly increased in pCI-HCA661-mtHSP70E group compared with pCI-HCA661-mtHSP70C group(P<0.05). The concentration of IFNγ in pCI-HCA661-mtHSP70E group was higher than that in pCI-HCA661-mtHSP70C group in supernatants from cultural splenocytes under HCA661 peptide stimulation（P<0.05）. The IFNγ levels in pCI-HCA661-mtHSP70C group and pCI-HCA661-mtHSP70E group were higher than that in pCI-neo group(P<0.05).The levels of specific antibodies induced by fusion gene in pCI-HCA661-mtHSP70E group were increased significantly compared with pCI-HCA661-mtHSP70C group（P<0.05）.The anti-tumor effect of CTL was enhanced strongly in pCI-HCA661-mtHSP70E group. Conclusion Stimulating epitope of mtHSP70 can induce a strong specific immune response to HCA661 fusion gene vaccine in mice by its adjuvant effect.

Key words: fusion gene vaccine;hepatocellular carcinoma-associated antigens 661;mycobacterium tuberculosis heat protein 70;genetic adjuvant

中图分类号:

段秀梅|邹亚斌|马洪喜|李树蕾|王银萍|王超|李玉琴. 结核杆菌热休克蛋白70刺激表位在融合基因疫苗中的佐剂作用[J]. J4, 2012, 38(3): 506-511.

DUAN Xiu-mei,ZOU Ya-bin,MA Hong-xi,LI Shu-lei,WANG Yin-ping,WANG Chao,LI Yu-qin. Adjuvant effect of mycobacterium tuberculosis heat shock protein 70 stimulating epitope on fusion gene vaccine[J]. J4, 2012, 38(3): 506-511.

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