【作者】 谢宇锋；

【导师】 杨吉成；

【作者基本信息】 苏州大学 ， 免疫学， 2005， 硕士

【摘要】 目的:克隆人工IL-17F基因,在大肠杆菌中重组表达并构建人IL-17F转基因细胞和真核稳定表达,研究其生物学活性。 方法:分离人外周血单个核细胞,经PHA刺激后,提取细胞总RNA,根据GenBank公布的人IL-17F cDNA序列设计并合成一对引物,采用RT-PCR技术克隆人IL-17F基因,将其亚克隆到pUCm-T载体并鉴定。以测序正确的pUCm-T/hIL-17F为模板,PCR扩增出目的基因片段,并分别正向插入原核表达载体pGEX-5X-3和逆转录病毒载体pSIV-1,构建重组表达载体pGEX-5X-3/hIL-17F和pSIV-1/hIL-17F。重组原核表达载体pGEX-5X-3/hIL-17F导入大肠杆菌BL21并经IPTG诱导表达包涵体融合蛋白,纯化后进行Western印迹鉴定。MTT法分析GST-hIL-17F纯品蛋白对人脐静脉内皮细胞ECV304的生长抑制作用和ELISA法检测其对ECV304内皮细胞表达IL-6、IFN_γ和TNF-α的生物学作用,并采用鸡胚绒毛尿囊膜(CAM)法分析其对血管形成的影响。重组逆转录病毒载体pSIV-1/hIL-17F与辅助病毒载体pHIT456和pHIT60混合后,脂质体法共转染包装细胞293T细胞。获得的具有感染能力的成熟重组逆转录病毒感染SMMC-7721肝癌细胞,经G418加压筛选获得抗性细胞株,并RT-PCR、Western blot检测目的基因的转录和表达。更多还原

【Abstract】 Objective: To clone and express ML-17F gene in E.coli and construct hIL-17F-transgenetic cell stably expressing hIL-17F protein and to study the biological activities of recombinant hIL-17F protein.Methods: To design and synthesize one pair of primers according to declared hIL-17F cDNA sequence from GenBank. The cDNA fragment encoding hIL-17F was amplified by RT-PCR from the total RNA extracted from PHA-activated peripheral blood mononuclear cells. It was cloned into pUCm-T vector and sequenced. Then recombinant prokaryotic expression vector pGEX-5X-3/hIL-17F and recombinant eukaryotic expression vector pSIV-1/hIL-17F were constructed and identified. The pGEX-5X-3/hIL-17F was transfected into E.coli BL21 and the inclusion bodies(IB) fusion protein was induced by IPTG The purified GST-hIL-17F fusion protein was identified by Western blot. The proliferation of ECV304 cells was observed by incubating them with soluble GST-hIL-17F fusion protein by MTT assay. The concentrations of IL-6, IFN-γ and TNF- α in the supernatants of ECV304 cells were determined by ELISA. The effect of GST-hIL-17F on the angiogenesis ofthe chick chorioallantoic membrane was assessed by CAM assay. The recombinant retrovirus vector pSIV-l/hIL-17F together with its two helper virus vectors pHIT456 and pHIT60 cotransfected into the package cell 293T by Liposome to produce mature recombinant retrovirus, then which was used to infect SMMC-7721 cell. The SMMC-7721 cell line stably expressing ML-17F protein was selected in the presence of G418 and identified by RT-PCR and Western blot.Results: A 41kD fusion protein was effciently induced in E.coli BL21 by IPTG, accounting for about 55% of the total bacterial protein. The purified GST-hIL-17F fusion protein was identified by Western blot. GST-hIL-17F fusion protein had obvious biological activities to inhibit the proliferation of ECV304 cells and enhance IL-6 secretion. GST-hIL-17F had a marked antiangiogenic activity. Results of RT-PCR and Western blot indicated that hIL-17F-transgenetic SMMC-7721 cell could stably express ML-17F target protein.Conclusion: ML-17F was effciently expressed in E.coli and the recombinant ML-17F protein had a marked antiangiogenic activity and had obvious biological activities to inhibit the proliferation of ECV304 cells and enhance IL-6 secretion, and the successful construction of hIL-17F-transgenetic SMMC-7721 cell stably expressing ML-17F protein by infection of retrovirus vector, which lays a foundation forfuture research on the mechanism of antiangiogenesis and tumor therapy by antiangiogenesis of recombinant hIL-17F protein.更多还原