构建了一系列携带有人凝血因子Ⅸ突变衍生物基因（ｈⅨＲ３３８Ａ）及不同调控元件的腺相关病毒 表达载体，并经体外转导不同的细胞系，筛选、优化，挑取高表达质粒及其转导稳定细胞克隆，然后， 以一个携带有腺相关病毒包装蛋白的ｒＡＡＶ/ＨＳＶ杂合病毒包装系统介导进行重组腺相关病毒的大量制 备，建立了可进行产业化制备的技术路线及高滴度、大量制备重组腺相关病毒的技术方法，所获得的重 组病毒滴度达 １．２５ × １０１２病毒颗粒／ｍＬ，然后，利用这一重组病毒，对ｒｈＦⅨＲ３３８Ａ进行离体及在体表达， 获得了 ｒｈＦⅨＲ３３８Ａ在肌细胞系 Ｃ２Ｃ１２及血友病 Ｂ小鼠体内的高效表达，表达峰值分别达（２５５１．３２± ９２．１４）ｎｇ·（１０６细胞）－１·（２４ｈ）－１及４６３．２８ｎｇ·ｍＬ－１，与野生型Ⅸ因子的表达水平（２５６５．７６±６４．３６）ｎｇ· （１０６细胞）－１·（２４ ｈ）－１及４５３．９２ ｎｇ· ｍＬ－１无显著性差异．然而，其凝血活性却成倍增加，约为后者的 ２．４６倍.．将人凝血因子Ⅸ突变衍生物基因引入血友病 Ｂ基因治疗研究之中，同时，探讨了一个新的包装 系统──ｒＡＡＶ／ＨＳＶ杂合病毒包装系统──在重组ＡＡＶ载体大量制备中的应用 A series of adeno-associated virus expression vectors carrying human coagulation factor Ⅸ mutant derivative gene (hIXR338A) and different regulatory elements were constructed and transduced into different cell lines in vitro to screen, optimize and select high expression plasmids and their transduction Then, a series of rAAV / HSV hybrid virus packaging system carrying adeno-associated virus packaging protein was used to mediate the large-scale preparation of recombinant adeno-associated virus. The technical route of industrialized preparation and high titer, A large number of recombinant adeno-associated virus-producing technology, obtained recombinant virus titer of 1.25 × 1012 virus particles / mL, and then, using this recombinant virus, rhFIXR338A in vitro and in vivo expression obtained rhFIXR338A Mycobacterium cell C2C12 and hemophilia B mice showed high expression levels (2551.32 ± 92.14) ng · (106 cells) -1 · (24h) -1 and 463.28 ng · mL- 1, and The expression level of green type Ⅸ factor (2565.76 ± 64.36) ng · (106 cells) -1 · (24 h) -1 and 453.92 ng · mL-1 had no significant difference. However, its coagulation activity has doubled, about 2.46 times the latter. The gene mutation of human coagulation factor Ⅸ was introduced into the study of hemophilia B gene therapy. At the same time, a new packaging system ─rAAV / HSV hybrid virus packaging system ─ was explored in the mass preparation of recombinant AAV vector