HPV58型E7基因重组痘苗疫苗免疫保护作用的实验研究

目的制备含人乳头瘤病毒(humanpapillomavirus,HPV)58型E7基因的重组痘苗病毒疫苗,并利用动物肿瘤模型观察疫苗接种效果。方法利用PCR方法扩增HPV58E7DNA片段,在消除其转化活性的基础上,构建表达HPV58E7的重组痘苗病毒疫苗并免疫小鼠,观察该疫苗对E7阳性肿瘤细胞生长的抑制作用,体外诱导、测定细胞毒性T淋巴细胞(cytotoxicTlymphocytes,CTL)活性。结果经定点突变的HPV58E7基因转化活性显著降低,用突变的E7基因构建的重组痘苗病毒免疫小鼠后,能抑制E7阳性肿瘤细胞在小鼠体内的生长并延长小鼠的生存期;免疫小鼠脾淋巴细胞体外可诱导产生针对E7阳性肿瘤的CTL细胞。结论在消除转化活性的基础上,HPV58E7可用于治疗与HPV58感染有关的肿瘤。

Experimental study of the immuno-protective activity of recombinant vaccinia virus expressing HPV58 E7

To develop a recombinant vaccinia virus vaccine expressing HPV58E7and to determine its immuno-protective activity in mice bearing HPV58E7 + tumor.Methods E7DNA was amplified and cloned from a plasmid containing HPV58E7genome by PCR.To abolish its transforming activity,the nucleotides coding for amino acid residues at positions24and92were modified by site-directed mutagenesis so that cysteine was substituted by glycine.Balb/c3T3cells were transfected with mE7.The expression of E7protein by the mE7-transfected Balb/c cells was confirmed by immunofluorescence staining.The transfected cells were observed in vitro for anchorage-independent growth and tumorigenesis in nude mice.Recombinant E7vaccinia virus vaccine was constructed by homologous recombination of HPV58E7vaccinia expression plasmid and vaccinia virus(Tiantan stain).The immuno-protective activity of the vaccines was determined by tumor growth inhibition and cytotoxic T lymphocytes(CTL)induction in vaccine-immunized syngeneic mice.Results Substitution of cysteine by glycine at both positions24and92of HPV58E7abolished its transforming activity.Growth of HPV E7 + tumor in mice immunized with the recombinant vaccinia virus expressing HPV58E7was inhibited,and the surviving time of the immunized mice was prolonged.CTL activity was induced as revealed by in vitro cytotoxicity assay using E7 + tumor cells as target cells.Conclusions HPV58E7,with its transforming potential abolished,may be used as vaccine for immunotherapy of patients with HPV58related cancers.