重组狗凝血因子Ⅷ的慢病毒介导体外表达的研究  

Expression of Recombinated Canine Factor Ⅷ In Vitro Mediated by Lentiviral Vector

作　　者：孙海英[1] 程海[1] 李振宇[1] 杜冰[1] 曾令宇[1] 鹿群先[1] 何徐彭[1] 潘秀英[1] 徐开林[1] 

机构地区：[1]徐州医学院附属医院血液科,徐州221002

出　　处：《中国实验血液学杂志》2007年第4期845-848,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目;编号30370606;江苏省高校自然科学研究项目;编号04KJB320145

摘　　要：本研究的目的是制备携带狗凝血因子Ⅷ(cFⅧ)基因的慢病毒载体,探讨慢病毒载体能否介导cFⅧ在体外的有效表达。用构建携带cFⅧ基因的慢病毒载体pTK161(含PUB启动子)和pTK162(含2OH1启动子),同时构建含绿色荧光蛋白(GFP)的慢病毒载体pTK161′(含PUB启动子)和pTK162′(含2OH1启动子)的方法,分别与包装质粒ΔNRF、包膜蛋白质粒VSV-G共转染293T包装细胞,将包装好的病毒颗粒再感染293T细胞,检测病毒滴度和培养细胞上清中cFⅧ活性。结果显示:经限制性酶切鉴定,成功构建了pTK161、pTK162、pTK161′和pTK162′正向连接载体;pTK161′和pTK162′的病毒滴度分别为1.54×106U/ml和2.83×106U/ml;pTK161、pTK162感染靶细胞后24小时细胞上清中即可检测到cFⅧ的表达,72小时表达量达高峰。pTK162载体cFⅧ表达活性接近正常狗血浆FⅧ活性,而且明显高于pTK161(p<0.05),6周后cFⅧ表达活性仍达最高值的1/4。结论:构建的自身失活慢病毒载体可携带较大片段的cFⅧ基因,并在体外可获得有效表达;本研究为慢病毒载体介导的血友病A的基因治疗研究提供实验依据。The study was purposed to prepare the recombinant lenfiviral vector pTK161 and pTK162 carrying B- domain-deleted canine factor （BDDcFⅧ） gene, and to investigate whether the canine FⅧ （cⅧ） can be expressed in vitro. The BDDcFⅧ gene was ligated behind PUB and 2OH1 promotors to create lentiviral vectors pTK161 and pTK162. Meantime lentiviral vectors pTK161＇ and pTK162＇ were produced by cloning a green fluorescent protein （GFP） into pTK151 and pTK152, which was driven by PUB and 2OH1 promotors respectively. Vector supernatant were prepared by using transfer calcium phosphate mediated-cotransfection of 293T cells. The virus vector, ANRF packaging- plasmid, and VSV-G envelope-plasmid was assayed by titers and cFⅧ activity in cell culture supernatant after infection into 293T cells, pTK161, pTK162, pTK161＇ and pTK162＇were identified by restriction enzyme analyzing. The results showed that the lentiviral vectors pTK161 ,pTK162, pTK161＇ and pTK162＇ were successfully constructed, and the titers of pTK161 ＇and pTK162＇ reached to 1.54 × 10^6 U/ml and 2.83 × 10^6 U/ml; the activity of cFⅧ could be detected at 24 hours after infection of 293T cells by pTK161 and pTK162, and achieved the highest level at 72 hours later. The higher level of cFⅧ activity was achieved by transfected with pTK162 than that of pTK161 （p 〈 0.05 ）, which closed to the cFⅧ activity in normal dog plasma. 1/4 of the highest level could be detected 6 weeks later. It is concluded that the prepared HIVl-based lentiviral vectors can infect 293T cells to express cFⅧ effectively. The results provide the basis for further studying HIV-1-based lentiviral vector gene therapy for hemophilia A.

关 键 词：慢病毒 狗凝血因子Ⅷ 血友病A 基因治疗 

分 类 号：Q782[生物学—分子生物学] R554.1[医药卫生—血液循环系统疾病]