【作者】 韩刚；

【导师】 王晓雄； 高江平； 于继云；

【作者基本信息】 中国人民解放军军医进修学院 ， 外科学， 2009， 博士

【摘要】 [目的]选择人前列腺干细胞抗原（prostate stem cell antigen,PSCA）作为治疗靶点,以本实验室自主研发的pVAX1-neo-Fc-GPI-IRES-GM/B7 DNA疫苗载体为基础,构建用于治疗前列腺癌的异种化PSCA融合抗原免疫增效DNA疫苗,评价以PSCA作为前列腺癌治疗靶点的可行性,并为前列腺癌,特别是激素非依赖性前列腺癌的治疗提供新策略。[方法]（1）检索GenBank,选择包含人主要T细胞抗原表位序列的人PSCA基因片段,应用异种化抗原设计技术,保留人T细胞抗原表位,设计异种化PSCA融合抗原片段;（2）根据核酸序列按中心模板法设计引物,应用重叠延伸PCR技术拼接合成异种化PSCA融合抗原片段基因,以PCR、限制性酶切和DNA序列测定法进行鉴定:（3）利用DNA限制性内切酶BssHⅡ和MluⅠ酶切后粘端互补的特点,采用同尾酶法构建1—4拷贝异种化PSCA融合抗原片段（PNCA₁-PSCA₄）,并将上述片段分别插入真核表达载体pCI-neo-Fc-GPI中,转染293T细胞,借助免疫荧光+流式细胞术考察插入片段表达效率,最终选定PSCA₃片段进行下一步研究;（4）将sig-PSCA₃-Fc-GPI基因片段自pCI-PSCA₃-Fc-GPI质粒上切下,插入pVAX1-neo-IRES—GM/B7载体中,构建免疫增效DNA疫苗pVAX1-PSCA₃-FcGB,并应用转染Cos7细胞+免疫荧光/流式细胞术方法鉴定其在真核细胞中的表达情况;（5）给8周龄雄性C57BL/6小鼠皮下种植RM-1细胞,制备小鼠前列腺癌模型,并采用股四头肌肌肉注射+电脉冲法（Electroporation,EP）接种DNA疫苗质粒pVAX1-PSCA₃-FcGB,同时接种pVAX1空载体质粒和pVAX1-PSCA₁-FcGB质粒作为对照,通过观察计算免疫动物的成瘤时间、肿瘤体积和抑瘤率,来评价该DNA疫苗在小鼠体内的抑瘤效果。[结果]（1）拼接合成了含有人主要T细胞表位的异种化PSCA融合抗原片段基因,经DNA测序证实与设计序列完全一致;（2）将异种化PSCA融合抗原片段进行1-4拷贝串联后,转染293T细胞+免疫荧光/流式细胞术结果证实PSCA₃表达效率较高;（3）成功构建了免疫增效DNA疫苗pVAX1-PSCA₃-FcGB,并通过转染Cos7细胞+免疫荧光/流式细胞术检测证明该疫苗可以在真核细胞中有效表达;（4）动物实验证明抗前列腺癌免疫增效DNA疫苗pVAX1-PSCA₃-FcGB能够推迟RM-1细胞肿瘤的形成,但在抑制肿瘤生长方面效果欠佳。[结论]1.成功拼接合成了异种化PSCA融合抗原片段基因;2.构建了以PSCA为靶点的抗前列腺癌免疫增效DNA疫苗pVAX1-PSCA₃-FcGB:3.该疫苗能够推迟RM-1细胞肿瘤的形成。更多还原

【Abstract】 Objective:To construct a DNA vaccine mainly containing heterological genetic fragment encoding most cytotoxic lymphocyte epitopes of PSCA -pVAX1-PSCA₃-FcGB,and to investigate the feasibility of using PSCA as a potential target for prostate cancer therapy,and to seek for a new strategy of immutherapy of advanced and androgen-indepent prostate cancer.Methods:The heterological PSCA fusion antigen genetic fragment was synthesized by overlapping-extending-PCR.Then the former fragment was linked one by one by co-adhesive end restriction and ligation strategy until the 4 copies fragment was accomplished.The 1 to 4 copies fragments（PSCA₁-PSCA₄） were inserted into eukaryotic expression vector pCI-Fc-GPI respectively.Four kinds of corresponding plasmids were transfected into 293T cell respectively and the expression of four kinds of fusion genetic fragments were detected by immunofluorescence and flow cytometry.The PSCA₃ fragment was selected for its superior expression level in eukaryotic cells.Then the sig-PSCA₃-Fc-GPI genetic fragment was cloned into pVAX1-neo-IRES-GM/B7 vector to construct the final immunological inhanced DNA vaccine pVAX1-PSCA₃-FcGB. Immunofluorescence and flow cytometry were used to confirm the expression of PSCA₃ fragment by transfected into Cos7 cell.Finally,the anti-tumor effect of pVAX1-PSCA₃-FcGB was tested in murine prostate cancer model generated by RM-1 cell line.The animal was immunized with pVAX1-PSCA₃-FcGB DNA vaccine by intramuscular injection plus electroporation,pVAX1 and pVAX1-PSCA₁-FcGB plasmid were used as control.The inhibitory effect of tumor was investigated by observion of forming time,volume and inhibition ratio of tumor.Results:DNA sequencing conformed that the heterological PSCA fusion antigen fragment which was synchronized by overlapping-extending-PCR,was consistent to design.Enzyme digestion analysis showed that the 1 to 4 copies heterological PSCA fusion antigen fragments were constructed successfully.And corresponding eukaryotic plasmids pCI-PSCA_1/2/3/4-Fc-GPI expressed well in 293-T cells.Enzyme digestion analysis showed that the recombinant plasmid-pVAX1-PSCA₃-FcGB DNA vaccine was constructed.The expression of the DNA vaccine in Cos7 cells was demonstrated by immunofluorescence and flow cytometry.A significant delay of tumor forming time was observed in murine model with prostate cancer which immunized with pVAX1-PSCA₃-FcGB （u=-2.104,P=0.038）;But inhibitory effect of tumor development was far from satisfactory.Conclusion:（1）The heterological PSCA fusion antigen genetic fragment encoding most cytotoxic lymphocyte epitopes of PSCA was synchronized by overlapping-extending-PCR.（2）A new type immunologic inhanced DNA vaccine -pVAX1-PSCA₃-FcGB,was constructed successfully.（3）This DNA vaccine can delay tumor of RM-1 cell line in vitro.更多还原