PDF《第21 卷4期》

第21 卷4期中国病毒学21(4) :339-342

2006 年7月VIROLOGICA SINICA July

2006 收稿日期:2006-01-11,修回日期:2006-04-20 * 基金项目:国家自然科学基金(30271186) 作者简介:李静(1975-) ,女,四川省籍,博士研究生,主要从事传染病的免疫治疗研究.

** 通讯作者:杨守京(1965-) ,男,山东省籍,教授,第四军医大学病理学教研室.Corresponding author. Tel: 029-84773527 Email: [email protected] Hsp70-NP 融合蛋白增强诱导 HTNV NP 特异性 CTL 的实验研究* 李静1,叶正旭

2 ,杨守京 1** ,李铠男

1 ,高娟3,刘彦仿

1 (1.第四军医大学病理学教研室;

2.第四军医大学西京医院骨科;

3.第四军医大学西京医院消化内科 陕西西安 710032) Hsp70-NP Recombinant Protein Enhances HTNV NP Specific Cellular Immune Response in vivo LI Jing1 ,YE Zheng-xu2 ,YANG Shou-jing1** ,LI Kai-nan1 ,GAO Juan3 ,LIU Yan-fang1 (1.Department of Pathology;

2. Department of Orthopaedics;

3. Department of Gastroenterology, Xi Jing hospital, the Fourth Military Medical University, Xi'

an 710032, China) Abstract:To examine whether the recombinant protein Hsp70-NP could induce the N-specific CTLs, we immunized C57/BL6 mice with purified protein Hsp70-NP, NP or Hsp70. The lymphoproliferative responses and CTL activities of spleen cells from vaccinated mice were determined. Accordingly, in order to get the target cells for cytotoxic assay, we tranfected pcDNA3.1/S into mice melanoma cells B16 with Lipofectamine2000, and selected stable transfectants with G418. RT-PCR, Western blots and immunofluorescence were used to identify the B16-N. The lymphoproliferative responses of spleen cells of mice vaccinated with Hsp70-NP, NP clearly showed proliferation in the presence of the N protein. But the stimulation indexes of splenocytes to NP of Hsp70-NP group were significantly higher than that of NP group. The CTL results showed that the cytotoxic effect of lymphocyte of the Hsp70-NP group was higher than that of the NP gorup. The results showed that Hsp70 could enhance the potency of NP to elicit HTNV NP specific cellular immune responses in vivo. Key words:Heat shock protein (HSP);

Hantaan virus Nucleocapsid protein;

Transfection;

Cytotoxic T lymphocyte 摘要:本文采用纯化蛋白 Hsp70-NP,NP,Hsp70 分别免疫 C57/BL6 小鼠,取各组小鼠脾淋巴细胞进行淋巴细胞 增殖试验和细胞毒试验.此外,为了获得细胞毒实验的靶细胞,本文还采用脂质体介导质粒 pcDNA3.1/S 转染黑 色素瘤细胞 B16,通过 G418 筛选稳定克隆,并用 RT-PCR,Western blots 以及免疫荧光染色证实 N 蛋白在胞浆 中表达.淋巴细胞增殖实验表明,Hsp70-NP,NP 组小鼠脾淋巴细胞均能够对体外抗原刺激产生增殖反应,而Hsp70-NP 组的增殖指数明显高于 NP 免疫组. 细胞毒实验结果表明, LDH 的释放具有效应细胞依赖性, Hsp70-NP, NP 免疫组脾淋巴细胞均可以特异性杀伤靶细胞 B16-N,而Hsp70-NP 免疫组的杀伤率显著高于 NP 免疫组.实验 结果显示,Hsp70 可以增强 NP 诱导产生特异性 CTL 的能力.本研究结果为进一步设计基于 NP 的合成肽疫苗或 基因疫苗提供了重要实验依据. 关键词:热休克蛋白(HSP);

汉滩病毒核蛋白;

转染;

细胞毒性 T 淋巴细胞 中图分类号:R373 文献标识码:A 文章编号: 1003-5125(2006)04-0339-04 由汉坦病毒(Hantaviruses, HV)引起的肾综合 征出血热严重危害着我国人民的身体健康.现有 的用于出血热防治的灭活疫苗主要诱导机体产生 体液免疫,难以获得持久的免疫力.而一般说来, 抗病毒免疫需要有效的由 CTL 和Th1 介导的细胞 免疫.寻求更加安全、有效的疫苗一直是国内外 学者多年研究的目标.目前,热休克蛋白(HSPs) 因其独特的分子伴侣和抗原提呈功能在肿瘤和传 染病的免疫治疗研究中备受关注.研究者已证实 从感染或转染细胞中提取的热休克蛋白抗原肽复