摘要
目的 观察糖基化修饰对内含肽剪接的B区缺失型凝血因子Ⅷ（BDD-FⅧ）分泌的影响.方法 将含有6个潜在的天冬酰胺糖基化位点（N6）的FⅧ的B区226个氨基酸引入BDD-FⅧ重链,用双载体转融合内含肽的此重链和轻链基因（N6HCIntN和IntCLC）至培养的293细胞,用酶联免疫吸附试验（ELISA）和Coatest发色法分别检测基因共转染细胞分泌至培养上清的剪接BDD-FⅧ蛋白量和凝血生物活性.结果 共转染N6HCIntN和IntCLC基因细胞上清中BDD-FⅧ蛋白浓度为（123±18）ng/ml,凝血活性为（0.94±0.11）U/ml,明显高于共转染内含肽融合的无糖基化修饰重链（HCIntN）和IntCLC基因细胞上清的BDD-FⅧ蛋白浓度[（86±12）ng/ml]和凝血活性[（0.65±0.07）U/ml,均P＜0.05].分别转染N6HCIntN和IntCLC基因细胞混合培养后的上清中亦检测到剪接的BDD-FⅧ蛋白和凝血活性[（18±6）ng/ml,（0.15±0.05）U/ml].结论 糖基化修饰可促进内含肽剪接的BDD-FⅧ的分泌并表现出不依赖细胞机制的蛋白质剪接功能.
Objective To investigate the effect of glycosylation modification on secretion of intein spliced B-domain-deleted FⅧ （BDD-FⅧ）. Methods A total of 226 amino acid residues of FⅧ B domain with six potential asparagines-linked glycosylation sites （N6） were incorporated into heavy chain of BDD-FⅧ.By dual-vector co-transfer of heavy and light chain genes with fused intein （ N6HCIntN and IntCLC） into cultured 293 cells, the amounts of spliced BDD-FⅧ protein and coagulation activity in culture supernatant were observed by ELISA and Coatest method respectively. Results The amounts of spliced BDD-F Ⅷ protein and activity were up to （123 ± 18） ng/ml and （0.94 ±0. 11 ） U/ml in supernatant from cells cotransfected with N6HCIntN and IntCLC genes. And they were higher than those of cells co-transfected with intein-fused heavy （ HCIntN ） and light chain （IntCLC） genes [（ 86 ± 12） ng/ml and （0. 65 ± 0. 07 ）U/ml, both P 〈 0. 05]. Spliced BDD-FⅧ protein and activity could also be detected in the supernatant from mixed cells individually transfected with N6HCIntN and IntCLC genes [（ 18 ±6） ng/ml and （0. 15 ±0. 05） U/ml]. Conclusion It demonstrated that the glycosylation modified heavy chain can improve the secretion of intein spliced BDD-FⅧ and the protein splicing can occur independent of cellular mechanism.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2010年第36期2570-2574,共5页
National Medical Journal of China
基金
基金项目：教育部留学回国人员科研启动基金（20071108）
山东省自然科学基金（Y2005D14）
烟台市科技发展计划基金（2008152）
