携带超抗原SEA基因低免疫原性病毒的构建及潜在抗肿瘤作用

发布时间：2018-04-11 13:25

本文选题：超抗原 + 慢病毒载体　；参考：《江苏大学》2017年硕士论文

【摘要】：目的:1.构建携带超抗原SEA基因的的低免疫原性慢病毒载体。2.探讨携带超抗原SEA基因慢病毒载体靶向膀胱肿瘤细胞BIU-87细胞表达能力。3.研究慢病毒载体表达的SEA蛋白刺激周围淋巴细胞的增殖作用及其机制抗肿瘤作用。方法:应用Genebank搜索h TERT-CMV启动子及SEA目的基因序列,设计寡聚核苷酸引物,以原有慢病毒载体为模板,应用聚合酶链式反应(Polymerase Chain Reaction,PCR)法获得启动子及目的基因序列。将获取基因构建到慢病毒载体质粒中,基因测序正确后,转入感受态细胞中扩增、收集。利用包装系统质粒共转染293FT细胞中包装病毒,获取目标阳性空斑,试剂盒方法提取慢病毒的基因组,进行双酶切鉴定。通过293FT细胞感染扩增病毒,采用超速离心法获取浓缩的病毒上清液,病毒滴度通过荧光滴度法测定。梯度浓度病毒液转染膀胱肿瘤BIU-87细胞株后,提取细胞RNA进行RT-PCR反应,目的基因的PCR产物测序,Western blot方法检测SEA蛋白的表达。病毒转染后的BIU-87细胞与人淋巴细胞共培养,不同时间点后采用倒置显微镜观察淋巴细胞与肿瘤细胞生长情况,收集细胞培养液,采用酶联免疫吸附试验(ELISA)方法检测共培养对细胞因子分泌的影响。结果:1.PCR产物基因测序证实慢病毒质粒中目的基因与启动子连接正确,载体质粒大小为10458bp,未发现明显碱基突变。荧光滴度法检测病毒滴度为(4.30±2)×109TU/m L。构建的慢病毒载体双酶切产物基因测序与预期结果相同。2.Western blot方法结果显示BIU-87细胞中有SEA基因翻译的超抗原SEA蛋白存在。3.实验组淋巴细胞与转染后的BIU-87肿瘤细胞共培养24、48、96 h后,BIU-87细胞数量明显少于空白对照组。ELISA结果显示实验组IL-2、IL-4分泌明显高于空白对照组。结论:1.成功构建完成表达SEA基因的重组慢病毒载体,且对重组慢病毒载体基因组及毒性进行检测,扩增、纯化一定数量病毒载体用于体外实验研究。2.应用构建的重组慢病毒载体体外作用于膀胱肿瘤BIU-87细胞株,初步验证载体对膀胱肿瘤的治疗作用及其机制。
[Abstract]:Purpose 1.A low immunogenicity lentivirus vector containing superantigen SEA gene was constructed.Objective: to investigate the expression ability of lentivirus vector carrying superantigen SEA gene targeting bladder tumor cells BIU-87 cells. 3.To study the proliferation of peripheral lymphocytes stimulated by SEA protein expressed by lentivirus vector and its mechanism of anti-tumor effect.Methods: h TERT-CMV promoter and SEA target gene sequence were searched by Genebank, and oligonucleotide primers were designed. Using the original lentivirus vector as template, the promoter and target gene sequence were obtained by polymerase chain reaction (PCR) method.The obtained gene was constructed into the vector plasmid of lentivirus. After the gene was sequenced correctly, the gene was transferred to the receptive cells for amplification and collection.Packaging system plasmids were co-transfected into 293FT cells to obtain the target positive plaque. The genome of lentivirus was extracted by kit method and identified by double enzyme digestion.The supernatant of the virus was obtained by ultracentrifugation and the virus titer was determined by fluorescence titer method.After transfection of gradient concentration virus into BIU-87 cell line of bladder tumor, RNA was extracted for RT-PCR reaction. The expression of SEA protein was detected by PCR product sequencing and Western blot method.The BIU-87 cells were co-cultured with human lymphocytes after transfection. The growth of lymphocytes and tumor cells was observed by inverted microscope at different time points, and cell culture medium was collected.Enzyme linked immunosorbent assay (Elisa) was used to detect the effect of co culture on cytokine secretion.Results 1. PCR product gene sequencing confirmed that the target gene and promoter of lentivirus plasmid were connected correctly. The plasmid size was 10458bp. no significant base mutation was found.The titer of virus detected by fluorescence titer assay was 4.30 卤2 脳 109TU/m L.The sequence of the double enzyme digested product gene of the constructed lentivirus vector was the same as the expected result. 2. Western blot assay showed that there was a superantigen SEA protein translated by SEA gene in BIU-87 cells.The number of BIU-87 cells in the experimental group was significantly lower than that in the blank control group. Elisa showed that the IL-2 IL-4 secretion in the experimental group was significantly higher than that in the blank control group.Conclusion 1.The recombinant lentivirus vector expressing SEA gene was successfully constructed, and the genome and toxicity of recombinant lentivirus vector were detected, amplified and purified.The recombinant lentivirus vector was applied to bladder tumor BIU-87 cell line in vitro. The therapeutic effect of the vector on bladder tumor and its mechanism were preliminarily verified.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14

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