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本研究采用具有促分泌作用的猪FVIII分子的A1和A3区,替换人FVIII分子的相应结构,构建人/猪嵌合体BDD-FVIII(BDD-hpFVIII),利用Ssp DnaB intein的蛋白质反式剪接功能,双载体培养的COS-7细胞瞬时共转嵌合体BDD-hpFVIII基因。免疫印迹法观察了转基因细胞内的蛋白表达和BDD-hpFVIII的剪接,采用ELISA和Coatest法分别观察了分泌至培养上清液中剪接的嵌合体BDD-hpFVIII蛋白和生物活性。结果显示,基因共转染细胞内可见明显的剪接BDD-hpFVIII蛋白,分泌至上清液的剪接BDD-hpFVIII蛋白量为(340±64)ng·mL-1,活性为(2.52±0.32)u·mL-1,明显高于双载体共转人BDD-FVIII基因细胞[质量浓度为(93±22)ng·mL-1,活性为(0.72±0.13)u·mL-1],提示intein介导的双载体共转BDD-hpFVIII基因可明显促进剪接蛋白的分泌。另外,分别转intein融合BDD-hpFVIII重链和轻链基因的细胞混合培养后上清液中仍可检测到剪接的BDD-hpFVIII蛋白及其活性,分别为(44±9)ng·mL-1和(0.32±0.07)u·mL-1,提示intein可进行不依赖细胞机制的BDD-hpFVIII剪接。本研究为旨在提高分泌的动物体内应用intein的双载体转BDD-hpFVIII基因奠定了基础。
In this study, the human and porcine chimeric BDD-FVIII (BDD-hpFVIII) was constructed by replacing A1 and A3 regions of porcine FVIII molecule with secreted human FVIII molecules and using the protein splicing function of Ssp DnaB intein , Double-vector cultured COS-7 cells co-transiently co-chimeric BDD-hpFVIII gene. The expression of BDD-hpFVIII protein and the splicing of BDD-hpFVIII were observed by immunoblotting. The spliced chimeric BDD-hpFVIII protein secreted into the culture supernatant was assayed by ELISA and Coatest method. The results showed that BDD-hpFVIII protein was spliced in transfected cells. The amount of BDD-hpFVIII spliced in supernatant was (340 ± 64) ng · mL-1 and the activity was (2.52 ± 0.32) u · mL-1, which was significantly higher than that of BD-FVIII co-transfected cells [mass concentration (93 ± 22) ng · mL-1 and activity of (0.72 ± 0.13) μ · mL-1] The double vector co-transfected BDD-hpFVIII gene can significantly promote the splicing protein secretion. In addition, the spliced BDD-hpFVIII protein and its activity were still detected in the supernatants of cells mixed with intein-fused BDD-hpFVIII heavy and light chain genes, respectively (44 ± 9) ng · mL -1 And (0.32 ± 0.07) u · mL-1, respectively, suggesting that intein may be involved in cell-free BDD-hpFVIII splicing. This study lays the foundation for the application of intein-mediated BDD-hpFVIII gene in ex vivo animal models.