中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2010年
48期
3435-3439
,共5页
朱甫祥%刘泽隆%缪静%屈慧鸽%迟晓艳
硃甫祥%劉澤隆%繆靜%屈慧鴿%遲曉豔
주보상%류택륭%무정%굴혜합%지효염
因子Ⅷ%蛋白质剪接%转基因%酸性区3
因子Ⅷ%蛋白質剪接%轉基因%痠性區3
인자Ⅷ%단백질전접%전기인%산성구3
Factor Ⅷ%Protein splicing%Transgenes%Acidic region-3
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.
目的 觀察具有促進凝血因子Ⅷ(fⅧ)重鏈分泌的痠性區3(AR-3)對蛋白質剪接作用連接的全長fⅧ分泌的影響.方法 以雙載體轉融閤內含肽的全長fⅧ重鏈和輕鏈基因,併將AR-3融閤于重鏈基因,瞬時共轉培養的293細胞,用Western印跡觀察轉基因細胞內的蛋白質剪接;用酶聯免疫吸附試驗(ELISA)和Coatest法定量分析分泌至培養上清中的剪接的全長fⅧ蛋白和由其產生的生物活性.結果 共轉基因細胞內可見明顯的剪接fⅧ蛋白形成,培養上清中剪接的fⅧ蛋白量和活性分彆為(1 12±18)ng/ml和(0.76±0.13)U/ml,明顯高于共轉未融閤AR-3的重鏈與輕鏈基因細胞[(64±11)ng/ml和(0.37±0.05)U/ml],而且,混閤培養的分彆單獨轉AR-3融閤重鏈和輕鏈基因細胞的上清中亦檢測到剪接的fⅧ蛋白及活性[(27±7)ng/ml和(0.16±0.05)U/ml].結論 AR-3可通過改善內含肽剪接的全長fⅧ的分泌增彊轉fⅧ基因的效果.
목적 관찰구유촉진응혈인자Ⅷ(fⅧ)중련분비적산성구3(AR-3)대단백질전접작용련접적전장fⅧ분비적영향.방법 이쌍재체전융합내함태적전장fⅧ중련화경련기인,병장AR-3융합우중련기인,순시공전배양적293세포,용Western인적관찰전기인세포내적단백질전접;용매련면역흡부시험(ELISA)화Coatest법정량분석분비지배양상청중적전접적전장fⅧ단백화유기산생적생물활성.결과 공전기인세포내가견명현적전접fⅧ단백형성,배양상청중전접적fⅧ단백량화활성분별위(1 12±18)ng/ml화(0.76±0.13)U/ml,명현고우공전미융합AR-3적중련여경련기인세포[(64±11)ng/ml화(0.37±0.05)U/ml],이차,혼합배양적분별단독전AR-3융합중련화경련기인세포적상청중역검측도전접적fⅧ단백급활성[(27±7)ng/ml화(0.16±0.05)U/ml].결론 AR-3가통과개선내함태전접적전장fⅧ적분비증강전fⅧ기인적효과.
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.
