AR-3可增强内含肽介导的全长凝血因子Ⅷ的基因表达  

Enhancing effect of AR-3 on protein splicing-based eukaryotic cell gene transfer of full-length coagulation factor Ⅷ

作　　者：朱甫祥[1] 刘泽隆[1] 缪静[1] 屈慧鸽[1] 迟晓艳[1] 

机构地区：[1]鲁东大学生命科学学院生物医学研究所,山东烟台264025

出　　处：《中华医学杂志》2010年第48期3435-3439,共5页National Medical Journal of China

基　　金：山东省自然科学基金（ZR2010CM061）;烟台市科技发展计划（2008152）

摘　　要：目的 观察具有促进凝血因子Ⅷ（fⅧ）重链分泌的酸性区3（AR-3）对蛋白质剪接作用连接的全长fⅧ分泌的影响.方法 以双载体转融合内含肽的全长fⅧ重链和轻链基因,并将AR-3融合于重链基因,瞬时共转培养的293细胞,用Western印迹观察转基因细胞内的蛋白质剪接;用酶联免疫吸附试验（ELISA）和Coatest法定量分析分泌至培养上清中的剪接的全长fⅧ蛋白和由其产生的生物活性.结果 共转基因细胞内可见明显的剪接fⅧ蛋白形成,培养上清中剪接的fⅧ蛋白量和活性分别为（1 12±18）ng/ml和（0.76±0.13）U/ml,明显高于共转未融合AR-3的重链与轻链基因细胞[（64±11）ng/ml和（0.37±0.05）U/ml],而且,混合培养的分别单独转AR-3融合重链和轻链基因细胞的上清中亦检测到剪接的fⅧ蛋白及活性[（27±7）ng/ml和（0.16±0.05）U/ml].结论 AR-3可通过改善内含肽剪接的全长fⅧ的分泌增强转fⅧ基因的效果.Objective To study the effect of an acidic region-3 （ AR-3）, capable of improving the secretion of heavy chain of coagulation factor Ⅷ （ fⅧ ）, on the secretion of protein spicing ligated full-length fⅧ. Methods A pair of vectors was used to deliver intein fused heavy and light chain genes of a full-length tⅧ gene with an additional AR-3 incorporated on the end of heavy chain into cultured 293 cells. The intracellular protein splicing was observed by Western blot. And the secretion of spliced fⅧ and activity in culture supernatant were quantified by enzyme-linked immunosorbent assay （ELISA） and Coatest assay respectively. Results A noticeable spliced fⅧ protein band was observed from the gene co-transfected cells. The culture supernatant displayed a spliced fⅧ of （ 112 ± 18） ng/ml with an activity of （0. 76 ±0. 13） U/ml greater than that of cells co-transfected with AR-3-free heavy chain and light chain genes [（64±11） ng/ml and （0.37±0.05） U/ml]. And a spliced fⅧ of （27±7） ng/ml with an activity of （0. 16 ± 0. 05） U/ml was detected in the culture supernatant from combined cells separately transfected with AR-3-fused heavy chain gene and light gene. Conclusion AR-3 can enhance the fⅧ gene transfer by improving the secretion of intein spliced full-length fⅧ.

关 键 词：因子Ⅷ 蛋白质剪接 转基因 酸性区3 

分 类 号：R686[医药卫生—骨科学]