L303E/F309S突变促进细胞分泌内含肽连接的B区缺失型凝血VIII因子

L303E/F309S Mutations Enhance Secretion of Intein-Ligated B-Domain-Deleted Coagulation Factor VIII

凝血VⅢ因子(FVⅢ)尽管与凝血V因子具有相似的结构,但其分泌的低效性不仅限制了重组产品在甲型血友病患者中的广泛应用,也困扰基于转FVⅢ基因的基因治疗。为了提高FVⅢ的分泌效率,在我们以前用内含肽(intein)的蛋白质剪接功能介导的双载体转B区缺失型FVⅢ(BDD-FVⅢ)基因研究的基础上,探讨了重链L303E/F309S双突变对剪接BDD-FVⅢ蛋白分泌的影响。PCR突变法将L303E/F309S双突变引入我们以前构建的融合Ssp DnaB内含肽的野生型重链(HCIntN),得到融合内含肽的重链突变基因(DMHCIntN),与融合内含肽的轻链(IntCLC)基因共转染培养的293细胞,用ELISA检测了培养上清中的重链多肽和剪接的BDD-FVⅢ蛋白浓度,用Coatest法分析了培养上清的凝血生物活性。结果显示,单独转DMHCIntN和DMHCIntN与IntCLC共转染细胞上清中的分泌的重链多肽浓度分别为(35±12)ng/ml和(178±19)ng/ml,高于单独转HCIntN细胞和HCIntN与IntCLC共转细胞[(14±6)ng/ml和(127±23)ng/ml];共转DMHCIntN和IntCLC细胞上清中剪接的BDD-FVⅢ蛋白浓度和活性分别为(128±24)ng/ml和(1.01±0.15)U/ml,明显高于共转HCIntN和IntCLC细胞[(90±12)ng/ml和(0.71±0.14)U/ml];另外,单独转DMHCIntN和IntCLC的细胞合并培养后的上清也检测到剪接的BDD-FVⅢ蛋白[(20±3)ng/ml]和活性[(0.17±0.07)U/ml]。结果表明,双突变可提高重链的分泌效率并明显被轻链顺式提高,伴之以剪接BDD-FVⅢ的分泌增加,表明内含肽对双载体转BDD-FVⅢ基因功效的改善并表现出不依赖细胞机制的BDD-FVⅢ剪接作用。为进一步动物体内实验运用内含肽的双AAV转重链双突变BDD-FVⅢ基因研究奠定了基础。

The coagulation factor Ⅷ （FⅧ） has a similar structure with factor V but its inefficient secretion not only hampered the wide use of recombinant FⅧ product for treatment of hemophilia A but adversely affected the FⅧ transgene-based gene therapy for its low levels of expression. Our previous work demonstrated that an intein＇s protein splicing can be used in delivery of the B-domain-deleted FⅧ （BDD-FⅧ） gene by a dual vector system. In this study, the effect of L303E/F309S mutations within FⅧ heavy chain on secretion of an intein-spliced BDD-FⅧ protein was investigated. A PCR directed mutagenesis was performed to produce the intein-fused heavy chain containing L303E/F309S mutations （DMHCIntN） from the Ssp DnaB intein-fused wildtype heavy chain （HCIntN） constructed previously. By co-transfection of the cultured 293 cells with DMHCIntN and intein-fused light chain （IntCLC） genes, the amount of secreted heavy chain polypeptide and spliced intact BDD- FⅧ protein and coagulation activity in the culture supernatant were respectively determined by ELISA and Coatest assay. The data showed that the amount of heavy chain in supernatant from cells transfected with DMHCIntN alone and both DMHCIntN plus IntCLC were （35±12） ng/ml and （178±19） ng/ml greater than that of HCIntN alone and HCIntN plus IntCLC cotransfecteion [（14±6） ng/ml and （127±23） ng/ml]. The amount of spliced BDD-FⅧ and coagulation activity in supernatant from DMHCIntN and IntCLC cotransfected cells were （128±24） ng/ml and （1.01 ±0.15） U/ml respectively also higher than HCIntN and IntCLC cotransfection [（90± 12） ng/ml and （0.71±0.14） U/ml]. The spliced BDD-FⅧ and its activity were also detected in supernatant of mixed cells transfected with DMHCIntN and IntCLC individually [（20±3） ng/ml and （0.17±0.07） U/ml]. The results demonstrated that secretion of the mutated heavy chain can be markedly improved by the light chain in cis with an increased secretion of spliced FⅧ, and intein can increase efficacy of dual-vector delivery of the doubly mutated BDD-FⅧ gene with splicing independently of any cellular mechanisms. It encourages our ongoing study in animal model in vivo by using inteinbased dual-AAV vector to transfer doubly mutated BDD-FⅧ gene.