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Recombinant adeno-associated virus(rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1(rHSV-1) designated HSV1-rc/△UL2, which expressed adeno-associated virus type2(AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein(GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/△UL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit(TU) or 4.28×104 particles per cell. Therefore, compared with the conventional transfection method, the yield of rAAV using this "one proviral cell line, one helper virus" strategy was increased by two orders of magnitude. Large-scale production of rAAV can be easily achieved using this strategy and might meet the demands for clinical trials of rAAV-mediated gene therapy. 相似文献
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采用定点诱变技术,将R338A点突变引入人凝血因子Ⅸ基因,并构建于AAV载体上,以rHSV/AAV杂合辅助病毒系统介导制备rAAV-hFIX重组病毒,然后,经肌肉注射对血友病B小鼠进行治疗实验,观察该突变基因在小鼠体内的表达、活性以及机体对该突变衍生物的免疫反应与治疗效果.结果显示:(i)治疗小鼠体内可检测到hFIX-R338A突变衍生物的存在,并持续15周以上;(ii)突变衍生物hFIX-R338A在小鼠血浆中的凝血活性达(34.2±5.23)%,显著高于野生型Ⅸ因子凝血活性((14.27±3.4)%);(iii)治疗小鼠体内未检测到抗Ⅸ因子突变衍生物抗体的存在;(iv)未发现与治疗相关的局部及全身性毒副作用.提示:以AAV介导人凝血因子Ⅸ高活性突变衍生物hFLX-R338A基因治疗可能成为替代野生型Ⅸ因子进行血友病B基因治疗的一个更为有效的途径. 相似文献
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用SARS冠状病毒全基因组芯片杂交方法分析SARS-CoV 总被引:2,自引:1,他引:2
为从临床样品中检测和分析SARSCoV病毒打基础,并为分析SARSCoV病毒的复制和转录等机理提供一种有效方法。以SARS冠状病毒TOR2株序列作为标准设计和制备一种覆盖SARS冠状病毒全基因组的寡聚核苷酸芯片,探针长度为70nt,每相邻的探针序列重复25nt,共660条。用该芯片分析了细胞培养的SARSCoV病毒总RNA、7个SARSCoV病毒的基因克隆片段。对RNA样品用随机引物进行反转录PCR获得cDNA。对DNA用随机引物扩增和dUTPcy3标记。结果用这种芯片杂交检测SARSCoV病毒RNA可见阳性信号呈全基因组分布,并且有多处连续的阳性信号点;用正常人的白细胞RNA为对照,杂交未出现明显阳性信号。检测7个SARSCoV病毒基因克隆片段,在该片段相应的探针区段出现连续阳性信号点。这种方法可有效地检测和分析样品中SARS冠状病毒全基因组的信息。 相似文献
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肌注腺伴随病毒基因治疗血友病B的安全性研究 总被引:1,自引:0,他引:1
研究了肌注腺伴随病毒(adeno-associated virus,AAV)介导凝血Ⅸ因子基因治疗血友病B的安全性,道德采用PCR、反转录PCR(RT-PCR)分别检测rHSV/AAV包装系统产生的重组腺伴随病毒AAV-mFⅨ,和病毒感染细胞后所得上清再次感染的BHK细胞中的HSV(herpes simplex virus,HSV)和野生型AAV。同时观察HSV引起的细胞毒作用。结果表明:纯化后的AA-mFⅨ中HSV≤1个病毒基因组(viral genome,v.g.)/10^8个病毒基因组AAV-mFⅨ,且不具备感染活性;没有野生型AAV。其次,采用PCR、RT-PCR1免疫组化等方法检测了AAV-mFⅨ在体内的分布和表达时间;通过抗AAV的抗体检测和病理切片等方法观察了AAV-mFⅨ在体内引起的免疫反应和病理变化。结果表明:AAV-mFⅨ仅分布在注射点肌肉组织内,表达可持续200天以上;抗体水平低,各主要脏器均未发生明显的病理变化。AAV介导的凝血因子Ⅸ系统是安全的。 相似文献
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为了得到一种可以包装AAV2/5和表达绿色荧光蛋白的重组单纯疱疹病毒,设计并构建了一个由AAV2rep基因和AAV5cap基因嵌合而成的rep2cap5基因,然后,利用一套携带HSV1基因组的粘粒系统(cos6、cos28、cos14、cos56、cos48),将rep2cap5基因插入cos6粘粒上HSV1基因组片段的UL2基因中,而将EGFP的表达单位插入cos56粘粒上HSV1基因组片段的UL44基因中,用这2个重组粘粒与其它3个粘粒(cos14、cos28、cos48)共转染BHK-21细胞获得了重组病毒HSV1-r2c5-EGFP并进行了空斑纯化。HSV1-r2c5-EGFP病毒能够在BHK-21细胞连续传代,并且可以观察到几乎所有的感染细胞都能产生绿色荧光。用PCR方法以及Southern杂交方法表明所获得的HSV1-r2c5-EGFP中携带有rep2cap5基因,用HSV1-r2c5-EGFP感染携带报告基因LacZ的AAV载体细胞株,获得了具有感染性的重组AAV2/5-LacZ。结果表明,所获得的重组单纯疱疹病毒HSV1-r2c5-EGFP可提供AAV2/5载体包装所需的全部辅助功能,是一种能简便、高效制备重组AAV2/5病毒的通用性辅助病毒。 相似文献
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A novel and highly efficient production system for recombinant adeno-associated virus vector 总被引:9,自引:0,他引:9
Recombinant adeno-associated virus (rAAV) has proven to be a promising gene delivery vector for human gene therapy. However, its application has been limited by difficulty in obtaining enough quantities of high-titer vector stocks. In this paper, a novel and highly efficient production system for rAAV is described. A recombinant herpes simplex virus type 1 (rHSV-1) designated HSV1-rc/AUL2, which expressed adeno-associated virus type2 (AAV-2) Rep and Cap proteins, was constructed previously. The data confirmed that its functions were to support rAAV replication and packaging, and the generated rAAV was infectious. Meanwhile, an rAAV proviral cell line designated BHK/SG2, which carried the green fluorescent protein (GFP) gene expression cassette, was established by transfecting BHK-21 cells with rAAV vector plasmid pSNAV-2-GFP. Infecting BHK/SG2 with HSV1-rc/AUL2 at an MOI of 0.1 resulted in the optimal yields of rAAV, reaching 250 transducing unit (TU) or 4.28×104 particles per cell. Therefore, compared 相似文献
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可溶性肿瘤坏死因子受体II-脂联素球部融合蛋白的真核表达及生物学活性检测 总被引:1,自引:1,他引:1
本研究设计和构建了一种人肿瘤坏死因子受体II胞外区与人脂联素球部的融合基因sTNFRII-gAD,且相应的融合蛋白在哺乳动物细胞BHK-21S的无血清培养体系中实现了表达,并对该融合蛋白进行了初步鉴定。首先,用RT-PCR方法从人的外周血淋巴细胞总RNA中扩增人肿瘤坏死因子II型受体胞外区基因片段,与脂联素球部基因片段融合,克隆至pAAV2neo表达载体中,构建成pAAV2neo-sTNFRII-gAD。随后,用pAAV2neo-sTNFRII-gAD转染BHK-21S细胞获得G418抗性细胞BHK-21S/pAAV2neo-sTNFRII-gAD;然后,将原来含有血清的培养液换成无血清的化学成分限定的培养液,细胞从贴壁培养方式转换成悬浮培养方式;最后,收集BHK-21S/pAAV2neo-sTNFRII-gAD无血清悬浮培养24h后的培养上清,进行sTNFRII-gAD融合蛋白的鉴定分析。酶切鉴定和测序结果显示,所构建的pAAV2neo-sTNFRII-gAD质粒结构正确,sTNFRII-gAD序列与预期一致;分别用抗人肿瘤坏死因子受体II和抗人脂联素球部的单克隆抗体检测pAAV2neo-sTNFRII-gAD瞬时转染的BHK-21S细胞,免疫荧光呈现阳性;免疫印迹分析在pAAV2neo-sTNFRII-gAD稳定转染的BHK-21S细胞上清中检测到sTNFRII-gAD融合蛋白的表达,并以单体、三聚体和三聚体以上的多聚体形式存在。活性测定结果表明,sTNFRII-gAD融合蛋白具有显著抑制TNFα杀伤L929细胞的活性。因此,本研究为下一步大量制备sTNFRII-gAD融合蛋白用于体内外功能研究提供了良好基础。 相似文献