摘要
目的:研究大鼠FVⅢ轻链(rL C)对不同长度人FVⅢ重链(hHC,包括hHC745和hHC1690)活性的影响。方法:将hHC745、hHC1690、人FVⅢ轻链(hLC)和rL C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。结果:在293T细胞中,hHC745和hHC1690与rL C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rL C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rL C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rL C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rL C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rL C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rL C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rL C共表达时的FVⅢ活性显著高于hHC745与rL C共表达时的活性。结论:rL C可以显著提高不同长度hF VⅢ重链(hHC745和hHC1690)的活性。
Objective:To explore the effects of the rat FVⅢlight chain(rLC)on the activity of human FVⅢheavy chain hHC745 and hHC1690.Methods:hHC745,hHC1690,human FVⅢlight chain(hLC)and rLC were cloned into adeno-associated virus serotype 8(AAV8)expression vectors with CB promoter(ubiquitous expression),respectively,and transfected into 293 T 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-CBhLC 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 FVⅢwas detected by activated partial thromboplastin time(aPTT)assay,and the antigen expression level of FVⅢwas determined by enzyme-linked immunosorbent assay(ELISA).The specific activity of FVⅢwas calculated based on the activity and the antigen expression level.Results:In 293 T cells,the coagulation activity of FVⅢwas significantly enhanced when hHC745 and hHC1690 combined with rLC compared with them combined with hLC.The FVⅢactivity 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 FVⅢantigen 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 FVⅢactivity of hHC745+rLC was significantly higher than that of hHC745+hLC,increasing to about 5.1 times,while,that of hHC1690+rLC in
作者
毛建华
沈艳
阮铮
奚晓东
MAO Jian-Hua;SHEN Yan;RUAN Zheng;XI Xiao-Dong(State Key Laboratory of Medical Genomics,Shanghai Institute of Hematology,Department of Hematology,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine;Research Center for Experimental Medicine,Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2022年第6期1817-1823,共7页
Journal of Experimental Hematology
基金
国家自然科学基金(81970112,81670127,81101721)
上海市卫生局项目(201940342)
中国诺和诺德血友病基金(2014.1-2015.12)。
