Hsp70-NP融合蛋白增强诱导HTNV NP特异性CTL的实验研究

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,Westernblots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。

Hsp70-NP Recombinant Protein Enhances HTNV NP Specific Cellular Immune Response in vivo

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.