【摘要】：目的: 構建免疫毒素IL-18-PE38融合基因真核表達重組載體,并研究該重組質粒對IL-18受體超表達的白血病及類風濕性關節(jié)炎是否有治療作用。方法與結果: (1) 通過限制性內切酶雙酶切從質粒PRKL459K-IL18-PE38中獲取IL18-PE38融合基因,將其與分泌性真核表達載體pSecTag2B連接,轉化感受態(tài)菌,挑取單克隆培養(yǎng)并提取質粒,EcoRI單酶切后電泳鑒定顯示所構建真核表達載體pSecTag2B-IL-18—PE38片段長度約為6.8kb。DNA測序結果顯示IL-18和PE38序列與基因文庫中報道序列相符。 (2) 脂質體轉染法將構建的真核表達質粒轉染入3T3細胞,熒光免疫細胞化學法熒光顯微鏡照片顯示轉染融合基因組熒光表達強,空載體對照組熒光表達微弱。轉染之3T3細胞用Zeocin篩選純系轉染陽性細胞,篩選50天后,空白組細胞死亡,對照組與轉染組存活細胞成克隆性生長。采用斑點ELLSA和Western-blot法鑒定細胞培養(yǎng)上清,證實細胞培養(yǎng)上清中有融合蛋白表達。將上清作用于白血病L615細胞株,MTT法測定細胞生長情況,結果顯示轉染重組基因組細胞死亡率高于轉染空載體對照組。流式細胞術檢測結果示轉染重組基因組可見明顯凋亡峰。AO/EB熒光雙染色熒光顯微鏡照片顯示轉染組較對照組呈現(xiàn)明顯凋亡細胞染色特征。
[Abstract]:Aim: to construct the eukaryotic expression recombinant vector of immunotoxin IL-18-PE38 fusion gene and to study the therapeutic effect of the recombinant plasmid on leukemia and rheumatoid arthritis with IL-18 receptor overexpression. Methods and results: (1) IL18-PE38 fusion gene was obtained from plasmid PRKL459K-IL18-PE38 by restriction endonuclease digestion, then ligated with secretory eukaryotic expression vector pSecTag2B and transformed into competent bacteria. The pSecTag2B-IL-18-PE38 fragment length of the constructed eukaryotic expression vector was about 6.8kb.DNA sequencing results showed that the IL-18 and PE38 sequences were consistent with the reported sequences in the gene library. The results of EcoRI single enzyme digestion and electrophoresis showed that the constructed eukaryotic expression vector pSecTag2B-IL-18-PE38 fragment length was about the same as that reported in the gene library. (2) Eukaryotic expression plasmid was transfected into 3T3 cells by lipofectamine transfection. Fluorescence immunocytochemical fluorescence microscopy showed that the fluorescent expression of fusion genome was strong, while that of empty vector control group was weak. The transfected 3T3 cells were screened by Zeocin. After 50 days of screening, the cells in the blank group died, and the surviving cells in the control group and the transfected group grew into clones. Dot ELLSA and Western-blot methods were used to identify the expression of fusion protein in the supernatant of cell culture. The cell growth of L615 cells was measured by MTT assay. The results showed that the death rate of transfected recombinant genomic cells was higher than that of blank vector control group. The results of flow cytometry showed that apoptosis peaks could be seen in the transfected recombinant genome, and AO / EB fluorescence double staining fluorescence microscope photographs showed that the transfected group showed obvious apoptotic cells staining characteristics compared with the control group.
【學位授予單位】：四川大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：Q789;R346

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1 胡洪慧,王鳳山,凌沛學;白細胞介素-4的研究進展[J];中國藥學雜志;2005年10期

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