摘要
作者最近证明intein的蛋白质剪接技术可用于双载体转B区缺失型FVIII(BDD-FVIII)基因,在此基础上,本研究将具有促FVIII分泌作用的L303E/F309S双突变和B结构域糖基化位点引入BDD-FVIII重链,观察重链变体对intein剪接的BDD-FVIII蛋白分泌和活性的影响。用分别融合Ssp DnaB intein的重链变体(DMN6HCIntN)和轻链(IntCLC)基因共转染培养的293细胞,用ELISA分析分泌至培养上清液中的剪接BDD-FVIII蛋白量,并用发色法检测培养上清液的凝血生物活性。结果显示,DMN6HCIntN和IntCLC共转基因细胞上清液中剪接BDD-FVIII的浓度为(149±23)ng.mL-1,活性为(1.12±0.14)u.mL-1,明显高于intein融合的野生型重链(HCIntN)与轻链(IntCLC)共转基因细胞[(99±14)ng.mL?1和(0.77±0.13)u.mL?1],提示重链变体能明显促进剪接BDD-FVIII蛋白的分泌和活性;另外,还检测到不依赖细胞机制的BDD-FVIII剪接。本研究为进一步动物体内双AAV载体转BDD-FVIII基因研究提供了依据。
We recently demonstrated that an intein-mediated protein splicing can be used to transfer B-domain-deleted FVIII（BDD-FVIII） gene by a dual-vector.In this study,we observed the effect of a variant heavy chain with six potential glycosylation sites of B domain and L303E/F309S mutations in its A1 domain,which were proven to be beneficial for FVIII secretion,on secretion of spliced BDD-FVIII.By transient co-transfection of cultured 293 cells with intein-fused variant heavy chain（DMN6HCIntN） and light chain（IntCLC） genes,the culture supernatant was analyzed quantitatively by ELISA for secreted spliced BDD-FVIII antigen and by a chromogenic assay for bioactivity.The data showed that the amount of spliced BDD-FVIII protein and coagulation activity in culture supernatant from DMN6HCIntN plus IntCLC co-transfected cells were up to（149 ± 23） ng.mL？1 and（1.12 ± 0.14） u.mL？1 respectively greater than that of intein-fused wild type heavy（HCIntN） and light chain（IntCLC） co-transfected cells [（99 ± 14） ng.mL？1 and（0.77 ± 0.13） u.mL？1] indicating that the variant heavy chain is able to improve the secretion of spliced BDD-FVIII and activity.A cellular mechanism-independent BDD-FVIII splicing was also observed.It provided evidence for ongoing animal experiment using intein-mediated dual-AAV vector technology for delivery of the BDD-FVIII genes.
出处
《药学学报》
CAS
CSCD
北大核心
2010年第11期1361-1366,共6页
Acta Pharmaceutica Sinica
基金
山东省自然科学基金(ZR2010CM061)
烟台市科技计划项目(2008152)
教育部留学回国人员科研启动基金项目(20071108)
鲁东大学科研基金项目(LZ20083305)
