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• W4311577025 abstract "To explore the effects of the rat FVIII light chain (rLC) on the activity of human FVIII heavy chain hHC745 and hHC1690.hHC745, hHC1690, human FVIII light chain (hLC) and rLC were cloned into adeno-associated virus serotype 8 (AAV8) expression vectors with CB promoter (ubiquitous expression), respectively, and transfected into 293T cells using a dual-chain strategy of co-expression of HC and LC. The cultured cell supernatant was collected at 48 hours after transfection. The plasmids (pAAV8-CB-hHC745, pAAV8-CB-hHC1690, pAAV8-CB-hLC and pAAV8-CB-rLC) were hydrodynamically injected into hemophilia A (HA) mice via lateral tail vein. Forty-eight hours after injection, the peripheral blood of the mice was collected through retroorbital venous plexus and the plasma was obtained by centrifugation. The activity of FVIII was detected by activated partial thromboplastin time (aPTT) assay, and the antigen expression level of FVIII was determined by enzyme-linked immunosorbent assay (ELISA). The specific activity of FVIII was calculated based on the activity and the antigen expression level.In 293T cells, the coagulation activity of FVIII was significantly enhanced when hHC745 and hHC1690 combined with rLC compared with them combined with hLC. The FVIII activity of hHC745+rLC was about 4.6 times higher than that of hHC745+hLC, while hHC1690+rLC was about 2.9 times higher than that of hHC1690+hLC. The data from ELISA showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.1 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 2.8 times compared with hHC1690+hLC. In HA mice administrated with hydrodynamic injection, the FVIII activity of hHC745+rLC and hHC1960+rLC was significantly higher than that of hHC745+hLC and hHC1690+hLC at comparable expression level. The FVIII activity of hHC745+rLC was significantly higher than that of hHC745+hLC, increasing to about 5.1 times, while, that of hHC1690+rLC increasing to about 2.1 times than hHC1690+hLC. ELISA results also showed that there was no significant difference in FVIII antigen expression when hHC745 and hHC1690 combined with rLC and hLC. The specific activity of hHC745+rLC increased to about 4.2 times compared with hHC745+hLC, while that of hHC1690+rLC increased to about 1.8 times compared with hHC1690+hLC. In addition, the activity of hHC1690 combined with rLC was significantly higher than that of hHC745 combined with rLC.rLC can significantly enhance the coagulation activity of FVIII when co-expressed with hHC of different length including hHC745 and hHC1690.腺相关病毒载体血清型8递送的大鼠FVIII 轻链对不同长度人FVIII 重链活性的促进作用.研究大鼠FVIII 轻链（rLC）对不同长度人FVIII 重链（hHC, 包括hHC745和hHC1690）活性的影响。.将hHC745、hHC1690、人FVIII 轻链（hLC）和rLC分别克隆至含CB启动子的腺相关病毒血清型8（AAV8）表达载体中，采用HC和LC共表达的双链策略，转染293T细胞，收取转染后48 h的细胞培养上清；质粒经尾静脉高压注入血友病A（HA）小鼠，注射后48 h通过眼眶采血收集小鼠的外周血血浆。应用活化部分凝血活酶时间（aPTT）法检测FVIII的活性，酶联免疫吸附实验（ELISA）检测FVIII的抗原表达量，计算比活性。.在293T细胞中，hHC745和hHC1690与rLC共表达时的FVIII活性显著高于与hLC共表达时的活性，分别提高到约4.6倍和2.9倍；ELISA结果表明hHC745和hHC1690与hLC和rLC共表达时的FVIII抗原表达无统计学差异；根据aPTT测得的FVIII活性与ELISA测得的FVIII抗原表达量计算的比活性结果显示，hHC745与rLC共表达时的比活性提高到与hLC共表达时的比活性的约4.1倍，而hHC1690与rLC共表达时的比活性提高到与hLC共表达时的约2.8倍。在高压注射的HA小鼠中，hHC745和hHC1690与rLC共表达时的FVIII活性显著高于与hLC共表达时的活性，活性分别提高到约5.1倍和2.1倍；ELISA结果同样显示，hHC745和hHC1690与hLC和rLC共表达时FVIII抗原表达无统计学差异；比活性结果表明，hHC745和hHC1690与rLC共表达时的比活性较与hLC共表达时的比活性显著提高，分别提高到约4.2倍和1.8倍；hHC1690与rLC共表达时的FVIII活性显著高于hHC745与rLC共表达时的活性。.rLC可以显著提高不同长度hFVIII 重链（hHC745和hHC1690）的活性。." @default.
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• W4311577025 date "2022-12-01" @default.
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• W4311577025 title "[Promoting Effect of Rat FVIII Light Chain Delivered by Adeno-Associated Virus Vector Serotype 8 on the Activity of Human FVIII Heavy Chain with Different Length]." @default.
• W4311577025 doi "https://doi.org/10.19746/j.cnki.issn.1009-2137.2022.06.029" @default.
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