人OSM基因的克

发布时间：2018-06-03 20:29

本文选题：人抑瘤素M + 真核表达　；参考：《苏州大学》2005年硕士论文

【摘要】：目的:克隆人抑瘤素(hOSM)基因,在COS7细胞和WI-38细胞中进行重组表达并检测表达蛋白的生物学活性,初步阐明OSM细胞因子的抑瘤作用以及在造血中的作用,为OSM的基础和应用性研究打下基础。方法:用PMA刺激U937细胞,抽提总RNA,经RT-PCR获得cDNA,与pUCm-T载体连接后,经PCR获得全长hOSM基因,再与pcDNA3.1/myc-his(-)A真核表达载体连接,构建重组体pcDNA3.1A-hOSM,采用PCR、限制性酶双酶切法鉴定和经DNA测序正确后在COS-7细胞中进行瞬时表达,采用RT-PCR鉴定hOSM基因的转录,MTT法研究表达产物对A375细胞的生长抑制活性。并将pcDNA3.1A-hOSM真核表达载体转染WI-38细胞,经G418筛选获得稳定表达的转基因细胞;采用RT-PCR鉴定hOSM基因在WI-38细胞中的转录,MTT法研究表达产物抑制A375细胞和A549细胞生长的生物学特性,流式细胞仪法测定A375细胞的细胞周期,利用原代培养的第4代骨髓基质细胞研究hOSM对造血的支持作用。结果:重组入载体中的基因片段不仅方向正确,而且所克隆的片段确为hOSM基因,与GeneBank报导序列完全一致,并成功的在COS-7细胞中瞬时表达了具有抑制A375细胞生长rhOSM蛋白。获得稳定表达的转基因WI-38细胞,rhOSM表达产物能引起A375细胞的细胞周期G1→S
[Abstract]:Objective: to clone the hOSM gene, express it in COS7 cells and WI-38 cells, detect the biological activity of the expressed protein, and elucidate the anti-tumor effect of OSM cytokines and its role in hematopoiesis. It lays a foundation for the basic and applied research of OSM. Methods: U937 cells were stimulated with PMA, total RNAs were extracted, cDNAs were obtained by RT-PCR, ligated with pUCm-T vector, then the full-length hOSM gene was obtained by PCR, and then connected with pcDNA3.1/myc-his(-)A eukaryotic expression vector. The recombinant pcDNA3.1A-hOSMwas constructed and expressed in COS-7 cells by PCR, restriction enzyme digestion and DNA sequencing. RT-PCR was used to identify the growth inhibitory activity of hOSM gene on A375 cells. The pcDNA3.1A-hOSM eukaryotic expression vector was transfected into WI-38 cells, and the stable expression transgenic cells were obtained by G418 screening. RT-PCR was used to identify the expression of hOSM gene in WI-38 cells to study the biological characteristics of the expression products inhibiting the growth of A375 cells and A549 cells. The cell cycle of A375 cells was measured by flow cytometry, and the supporting effect of hOSM on hematopoiesis was studied by primary cultured bone marrow stromal cells (BMSCs). Results: the recombinant gene fragment was not only in the right direction, but also a hOSM gene cloned, which was completely consistent with the GeneBank report sequence. The recombinant gene was successfully expressed in COS-7 cells by transient expression of rhOSM protein which could inhibit the growth of A375 cells. Stable expression of rhOSM in transgenic WI-38 cells can induce cell cycle G1 / S of A375 cells
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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