【作者】 任杰；

【导师】 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， 2010， 硕士

【摘要】 背景与目的：人前列腺干细胞抗原(prostate stem cell antigen, PSCA)高表达于进展期前列腺癌组织及前列腺癌转移灶,与前列腺癌发生、发展密切相关,在前列腺癌诊断和治疗研究中备受重视。本研究选择人PSCA作为免疫治疗的靶点,以塞姆利基森林病毒(Semliki Forest Virus, SFV)复制子载体pSCK为基础,构建用于治疗前列腺癌的可复制型DNA疫苗pSCK-PSCA3,同时为评价疫苗的免疫功能,在大肠杆菌BL21系统中表达和纯化了人PSCA的融合蛋白,构建了可以高效表达人PSCA的小鼠B16细胞系。本研究为pSCK-PSCA3疫苗的免疫功能评价奠定了坚实的实验基础,也为前列腺癌的免疫治疗提供了一种全新策略。方法：(1)利用DNA重组技术将PSCA片段克隆入含有GST标签的原核表达载体pET42a,双酶切鉴定后得到正确的重组质粒pET42a-PSCA,转化大肠杆菌BL21后,IPTG诱导表达,用Glutathione Sepharose 4B柱对诱导蛋白进行纯化,纯化产物进行SDS-PAGE及Western Blot鉴定；(2)利用PCR扩增出人PSCA全长基因的cDNA编码区序列,插入到真核表达载体pcDNA3.1中,双酶切及测序鉴定后得到正确的重组质粒pcDNA3.1-PSCA。利用脂质体法将其转染入小鼠黑色素瘤B16细胞,G418加压筛选后得到阳性克隆。通过流式细胞术、免疫荧光及Western Blot检测PSCA在细胞系中的表达情况；(3)将sig-PSCA3-Fc-GPI-GM/B7基因片段自pVAX1-PSCA3-FcGB质粒上切下,插入可复制型载体pSCK中,构建可复制型DNA疫苗pSCK-PSCA3,并应用流式细胞术及免疫组化鉴定其在真核细胞中的表达情况。结果：(1)pET42a-PSCA原核表达质粒构建正确,SDS-PAGE及Western Blot检测显示GST-PSCA融合蛋白诱导表达成功,分子量大小约为43KD。(2) pcDNA3.1-PSCA真核表达质粒构建正确,建立了稳定转染人PSCA的B16细胞系,其表达率接近100%。(3)成功构建了可复制型DNA疫苗pSCK-PSCA3,流式细胞术及免疫组化检测证明该疫苗可以在真核细胞中有效表达。结论：在大肠杆菌BL21菌株中成功表达了PSCA融合蛋白,筛选出的B16细胞系可以高效表达PSCA基因,成功构建了可复制型PSCA基因疫苗pSCK-PSCA3,为后续肿瘤疫苗的体内外免疫功能研究奠定了基础。更多还原

【Abstract】 Background and Objective:Human prostate stem cell antigen(PSCA) is highly expressed in advanced prostate cancer and metastasis, and is closely related to the genesis and progression of prostate cancer, thus has caught much attention in the diagnosis and treatment of the disease. In this research, we intend to choose PSCA as a potential target for the immunotherapy of prostate cancer and construct a replication-competent DNA vaccine pSCK-PSCA3 based on Semliki Forest Virus(SFV) replicon vector pSCK. To evaluate the function of the vaccine, we expressed a PSCA fusion protein in E.coli BL21 and established a stably transfected B16 cell line which can highly express PSCA. This research has provided a solid experimental foundation for the function evaluation of the vaccine pSCK-PSCA3, and has offered a novel strategy for the immunotherapy of prostate cancer.Methods:(1)The fragment of PSCA was cloned into prokaryotic expression vector pET42a which contains a glutathione s-transterase(GST) tag. Following the double restriction enzyme digestion analysis, the recombinant plasmid pET42a-PSCA was transformed into E.coli BL21(DE3). GST-PSCA fusion protein was expressed under IPTG, and purified by passing over Glutathione Sepharose 4B column. The purified fusion protein was identified by SDS-PAGE and Western Blot.(2)The full length of PSCA cDNA fragment was amplified by PCR and inserted into eukaryotic expression vector pcDNA3.1. The recombinant plasmid pcDNA3.1-PSCA was identified by double restriction enzyme digestion and DNA sequencing. Then the plasmid pcDNA3.1-PSCA was transfected into B16 cells by lipofection. After screening culture by G418, a stably transfected B16 cell line was established. The expression of PSCA gene was identified by Flow Cytometry, Immunofluorescence and Western Blot. (3)The genetic fragment sig-PSCA3-Fc-GPI-GM/B7 was acquired from pVAXl-PSCA3-FcGB plasmid, then the fragment was cloned into the SFV replicon vector pSCK to construct the final DNA vaccine pSCK-PSCA3. The expression of the vaccine in eukaryote cells was identified by Flow Cytometry and Immunol Histochemistry.Results:(1) The prokaryotic expression plasmid pET42a-PSCA was successfully constructed. The GST-PSCA fusion protein was expressed effectively and its relative molecular weight was 43kD analyzed by SDS-PAGE and Western Blot.(2) The eukaryotic expression plasmid pcDNA3.1-PSCA was successfully constructed. A stably transfected B16 cell line was established and the expression rate of PSCA gene was virtually 100 percent.(3) The replication-competent DNA vaccine pSCK-PSCA3 was successfully constructed. The DNA vaccine can effectively express in eukaryotic cells analyzed by Flow Cytometry and Immunol Histochemistry.Conclusion:The PSCA fusion protein is expressed effectively in the BL21 strains. The established B16 cell line can highly express PSCA gene. A new type replication-competent DNA vaccine pSCK-PSCA3 was successfully constructed. The research has provided a solid experimental foundation for further studies on the function of the vaccine.更多还原