Tumstatin-EGFP在CHO细胞中的表达和活性分析  

Expression and biological activity of tumstatin-EGFP in CHO cells

作　　者：姚丽娟[1] 罗以勤[1] 孔建新[1] 赵亮[1] 濮跃晨[1] 马筱玲[1] 

机构地区：[1]安徽医科大学附属省立医院检验科,合肥230001

出　　处：《安徽医科大学学报》2008年第6期603-607,共5页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:30672437)

摘　　要：目的分析融合蛋白tumstatin-EGFP在中国仓鼠卵巢细胞(CHO)中的表达及生物学活性。方法经过酶切和测序正确的真核表达载体PIRESneo3/STL-EGFP、PIRES-neo3/sig-EGFP和空载体稳定转染CHO细胞后,用Western blot从细胞上清液中检测融合蛋白的表达、倒置荧光显微镜观察绿色荧光蛋白的表达。通过生长曲线分析外源基因的导入对CHO细胞生长的影响,MTT法和管状形成抑制试验评估tumstatin-EGFP融合蛋白的tumstatin样活性,体外靶向试验验证tumstatin能否携带融合蛋白特异性结合内皮细胞。结果获得了稳定转染的CHO细胞系。生长曲线表明转染后的较未转染的CHO细胞生长缓慢,MTT试验和管状形成抑制试验分别证实了融合蛋白tumstatin-EGFP具有抑制内皮细胞增殖,明显抑制血管管状结构的形成的作用;体外靶向试验也验证了tumstatin-EGFP能够特异性结合内皮细胞。结论重组真核表达载体在CHO细胞获得稳定表达,融合蛋白tumstatin-EGFP的表达不影响tumstatin和EGFP各自的生物学活性,并且在体外tumstatin能特异性携带EGFP靶向内皮细胞,为进一步tumstatin及其融合蛋白的研究奠定基础。Objective To analyze the expression of tumstatin-EGFP in a Chinese hamster ovary cell line （CHO） and examine biologic activity of the fusion proteins. Methods Correct expression vector of PIRESneo3/STL -EG- FP,PIRESneo3/sig-EGFP and PIRESneo3 were stably tansfected into CHO-K1 cells. Tumstatin was identified by Western blotting, EGFP was determined by fluorescent microscope. Experiments with growth curve test, MTT assay, tubular inhibition test and cell binding assay in vitro were used to assess the biological activity of tumstation-EGFP. Results The stably aboundant expression was obtained in transfected CHO-K1 cells. Growth curve test showed that transfected ceils grew lower than that of untransfected, MTT test and inhibition test of tube formation had strongerly demonstrated that tumstatin-EGFP not only suppressed endothelial cell proliferation but also displayed the property of inhibiting vascular tube information. Furthermore, it had been provided unambiguous evidence that the protein of tumstatin-EGFP couId specifically target to endithlial cells in vitro test. Conclusion We successfully obtain stably expressions in CHO-K1. Tumstatin and EGFP biologic activity are contained in the recombinant and the fusion protein could specially bind to endothelial ceils in vitro study. This work provides foundations for further study on the founction of tumstatin or its fusion protein.

关 键 词：绿色荧光蛋白质类 

分 类 号：R341[医药卫生—基础医学] R394