發(fā)布時(shí)間：2018-04-11 13:25

  本文選題：超抗原 + 慢病毒載體　； 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：目的:1.構(gòu)建攜帶超抗原SEA基因的的低免疫原性慢病毒載體。2.探討攜帶超抗原SEA基因慢病毒載體靶向膀胱腫瘤細(xì)胞BIU-87細(xì)胞表達(dá)能力。3.研究慢病毒載體表達(dá)的SEA蛋白刺激周圍淋巴細(xì)胞的增殖作用及其機(jī)制抗腫瘤作用。方法:應(yīng)用Genebank搜索h TERT-CMV啟動(dòng)子及SEA目的基因序列,設(shè)計(jì)寡聚核苷酸引物,以原有慢病毒載體為模板,應(yīng)用聚合酶鏈?zhǔn)椒磻?yīng)(Polymerase Chain Reaction,PCR)法獲得啟動(dòng)子及目的基因序列。將獲取基因構(gòu)建到慢病毒載體質(zhì)粒中,基因測(cè)序正確后,轉(zhuǎn)入感受態(tài)細(xì)胞中擴(kuò)增、收集。利用包裝系統(tǒng)質(zhì)粒共轉(zhuǎn)染293FT細(xì)胞中包裝病毒,獲取目標(biāo)陽(yáng)性空斑,試劑盒方法提取慢病毒的基因組,進(jìn)行雙酶切鑒定。通過(guò)293FT細(xì)胞感染擴(kuò)增病毒,采用超速離心法獲取濃縮的病毒上清液,病毒滴度通過(guò)熒光滴度法測(cè)定。梯度濃度病毒液轉(zhuǎn)染膀胱腫瘤BIU-87細(xì)胞株后,提取細(xì)胞RNA進(jìn)行RT-PCR反應(yīng),目的基因的PCR產(chǎn)物測(cè)序,Western blot方法檢測(cè)SEA蛋白的表達(dá)。病毒轉(zhuǎn)染后的BIU-87細(xì)胞與人淋巴細(xì)胞共培養(yǎng),不同時(shí)間點(diǎn)后采用倒置顯微鏡觀察淋巴細(xì)胞與腫瘤細(xì)胞生長(zhǎng)情況,收集細(xì)胞培養(yǎng)液,采用酶聯(lián)免疫吸附試驗(yàn)(ELISA)方法檢測(cè)共培養(yǎng)對(duì)細(xì)胞因子分泌的影響。結(jié)果:1.PCR產(chǎn)物基因測(cè)序證實(shí)慢病毒質(zhì)粒中目的基因與啟動(dòng)子連接正確,載體質(zhì)粒大小為10458bp,未發(fā)現(xiàn)明顯堿基突變。熒光滴度法檢測(cè)病毒滴度為(4.30±2)×109TU/m L。構(gòu)建的慢病毒載體雙酶切產(chǎn)物基因測(cè)序與預(yù)期結(jié)果相同。2.Western blot方法結(jié)果顯示BIU-87細(xì)胞中有SEA基因翻譯的超抗原SEA蛋白存在。3.實(shí)驗(yàn)組淋巴細(xì)胞與轉(zhuǎn)染后的BIU-87腫瘤細(xì)胞共培養(yǎng)24、48、96 h后,BIU-87細(xì)胞數(shù)量明顯少于空白對(duì)照組。ELISA結(jié)果顯示實(shí)驗(yàn)組IL-2、IL-4分泌明顯高于空白對(duì)照組。結(jié)論:1.成功構(gòu)建完成表達(dá)SEA基因的重組慢病毒載體,且對(duì)重組慢病毒載體基因組及毒性進(jìn)行檢測(cè),擴(kuò)增、純化一定數(shù)量病毒載體用于體外實(shí)驗(yàn)研究。2.應(yīng)用構(gòu)建的重組慢病毒載體體外作用于膀胱腫瘤BIU-87細(xì)胞株,初步驗(yàn)證載體對(duì)膀胱腫瘤的治療作用及其機(jī)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 鹵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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R737.14

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相關(guān)期刊論文 前3條

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本文編號(hào)：1736185