HPV16 CTL表位E749-57以HSP110为分子伴侣的免疫原性研究

Immunogenicity of the immunodominant cytotoxic T lymphocyte epitope E749-57 in HPV16 oncoprotein E7 chaperoned by HSP110

目的 探讨以mHSP110为分子伴侣的HPV16 CTL表位E749-57的免疫原性.方法 将mHSP110基因克隆、原核表达和纯化,SDS-PAGE和Western blot鉴定.在热休克状态与E749-57结合形成复合物,高压液相色谱（HPLC）鉴定其结合程度.用mHSP1 10-E749-57复合物免疫小鼠,IFN-γ胞内染色、MTT法检测小鼠脾细胞中特异CTL.结果 克隆mHSP110片段经DNA序列测定与基因库中其CDS一致,长度为2577 bp 经SDS-PAGE和Western印迹证实mHSP110表达、纯化成功,HPLC分析E749-57能够与mHSP110形成复合物.复合物免疫小鼠的脾淋巴细胞中CD8＋IFN-γ＋T细胞的频率、脾淋巴细胞增殖活性明显高于E749-57组、HSP110组和PBS组.复合物免疫小鼠可明显抑制TC-1肿瘤的生长.结论 mHSP110-E749-57复合物能诱导产生特异性CTL并产生抗肿瘤效应.

Objective To investigate the immunogenicity of immunodominant cytotoxic T lymphocyte epitope E749-57 of human papilloma virus （HPV） 16 oncoprotein E7 chaperoned by heat shock protein （HSP）110. Methods Mouse HSP110 gene was cloned into prokaryotic expression vector pQE-80L for the expression of HSP110 protein, which was purified using Ni-NTA column. SDS-PAGE and Western-blot were conducted to confirm the purified mHSP110 protein, which was subsequently incubated with E749-57 peptide under heat shock condition, and high-performance liquid chromatography （HPLC） was used to evaluate the binding efficiency of the recombinant protein and E749-57 peptide. Twenty mice were divided into 4 groups to be immunized with mHSP110 protein, E749-57 peptide, mHSP110-E749-57 complex and phosphate buffered saline （PBS）,respectively. Two weeks after the last immunization, spleen cells were collected from the immunized mice and divided into 2 parts： one were stimulated by E749-57 peptide followed by the detection of CD8＋ INF-γ＋ T cells with flow cytometry the other one were subjected to MTT analysis for the estimation of cell proliferation. The mHSP110-E749-57 complex was also used to immunize TC-1 tumor bearing mice to observe its anti-tumor effect.Results The full-length 2577 bp-sized mHSP110 gene was amplified from mouse liver cDNA and cloned into pQE-80L vector. Direct sequencing confirmed the correctness of the cloning. SDS-PAGE and Western-blot demonstrated the successful purification of mHSP110. HPLC assay showed that the purified mHSP110 protein could bind with E749-57 to form a relatively stable protein complex. The percentage of IFN-γ＋ CD8＋ T cells in and proliferation index of spleen cells from the complex-immunized mice were statistically higher than those from the other 3 groups of mice. Moreover, the complex could obviously inhibit the growth of TC-1 tumor in mice. Conclusion The mHSP110-E749-57 complex could enhance the generation of specific cytotoxic T lymphocytes and exert anti-tumor effects in mice.