共查询到18条相似文献，搜索用时 46 毫秒
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以RT PCR法从大鼠脑组织中克隆bcl XL 基因 ,将其定向插入带rep cap基因和neu基因的三功能腺相关病毒 (AAV)载体 ,转染HeLa细胞并以G4 18筛选 ,然后用腺病毒感染筛选后的细胞克隆 ,包装成重组腺相关病毒 .用带rep cap基因的C12细胞筛选和滴定产生重组腺相关病毒的细胞克隆 ,粗制细胞裂解物中病毒滴度最高只有 3× 10 5IU ml.从产病毒量较高的克隆大量制备重组病毒 ,经肝素柱高压液相亲和层析法纯化、浓缩病毒后获得了高达 4× 10 11IU ml的重组病毒 .为研究脑缺血动物模型中BCL XL 的抗脑细胞凋亡作用打下了基础 相似文献
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腺相关病毒(Adeno-associated virus,AAV)在基因治疗应用中具有很多优势,但是其生物学滴度的测定仍很繁琐,不同实验室使用各自的方法和参照,这些都影响了重组腺相关病毒(rAAV)载体在临床前和临床上的应用.反向末端重复序列(Inverted terminal repeats,ITR)是重组腺相关病毒载体中不可或缺的顺式作用元件,针对ITR2以及ITR2-CMV设计的qPCR检测方法可以快速、准确地得到rAAV2的基因组滴度,由于该方法可以广泛适用,因此对推动AAV滴度检测的标准化有重要意义. 相似文献
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一种高效快速浓缩腺相关病毒载体的方法 总被引：2，自引：0，他引：2
腺相关病毒载体(adeno associatedviralvector,AAVvector)是一种具有良好应用前景的基因治疗载体。研究中往往需要高滴度的重组AAV病毒(>105v g /细胞),为此利用完整的腺相关病毒颗粒具有耐受有机溶剂的特点,创造性地采用了一种用乙醇沉淀重组AAV病毒的方法,达到了高效、快速浓缩AAV载体的目的。结果表明,乙醇沉淀法可以高效浓缩AAV载体,并且对病毒的结构和活性没有明显影响。用这种方法可以方便地将低滴度的AAV病毒变成高滴度,解决动物体内实验中每点注射体积受到限制的问题。 相似文献
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目的:制备携带Bclxl基因的重组腺相关病毒,并鉴定其感染细胞有效性。方法:抽提BALB/c小鼠脾脏总RNA,通过RT-PCR获得cDNA,然后设计引物通过PCR获得Bclxl基因的编码序列。将得到的Bclxl编码序列插入腺相关病毒质粒pAAV-MCS中的多克隆位点,从而构建出重组腺相关病毒质粒pAAV-Bclxl。将质粒转染细胞后使用Western Blot和流式分析检测Bclxl蛋白的表达。进一步包装制备了携带Bclxl基因的重组腺相关病毒颗粒,用其感染HT1080细胞后用流式细胞仪分析了感染前后细胞中Bclxl基因的表达情况。结果:质粒转染后细胞中Bclxl蛋白的表达水平显著高于转染前,病毒感染后细胞中Bclxl蛋白的表达水平显著高于感染前。结论:携带Bclxl基因的重组腺相关病毒颗粒包装成功,能高效感染细胞,为后续的遗传改造树突细胞打下了基础。 相似文献
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在以病毒载体介导的基因治疗研究中,重组腺相关病毒(rAAV)因其疗效和安全性方面的优势,是最有临床应用前景的载体。但其转基因包装容量一般不能超过5.0kb,给需要转导大片段基因的应用带来了困难,限制了rAAV在基因治疗研究中的应用。随着对rAAV细胞转导生物学过程研究的不断深入,发现了一些可以突破rAAV包装容量限制的技术,如反式剪接和同源重组策略,为拓展该载体应用范围提供了可能性。另外,rAAV包装容量限制的特点还可以被用来减少生产过程中具有可复制能力的类病毒杂质的污染,为rAAV的临床安全性提供了保障。 相似文献
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目的:构建并制备携带靶向干扰Notch-1基因shRNA的重组腺相关病毒载体.方法:设计针对Notch-1的靶DNA序列,构建重组质粒pAAV-MCS2/siRNA-Notch-1,将该质粒和腺相关病毒包装质粒pAAV-RC,腺病毒辅助质粒pHelper共转染QBI-293A细胞,包装成带有Notch-1基因的重组腺相关病毒.结果:PCR鉴定分析结果表明成功构建针对Notch-1的siRNA重组腺相关病毒载体,滴定法测定重组病毒载体基因组得到滴度为1.2x 1012VP/L的高滴度腺相关病毒.结论:成功构建携带Notch-1基因短发夹状干扰RNA的腺相关病毒载体,为探索Notch-1基因在胶质母细胞瘤肿瘤干细胞中的作用提供实验基础. 相似文献
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可以稳定表达治疗基因而无明显不良反应的重组腺相关病毒 (rAAV) 载体被认为是最有发展前景的基因治疗载体。但如何建立可以有效去除rAAV载体内具有潜在致病危害的杂质、产品质量符合临床使用要求的纯化工艺是研究人员面临的巨大挑战。其中针对载体相关性杂质的纯化工艺尤为关键,因为该类杂质的性质与真正的rAAV载体极其相似,一旦存在便难以去除,且会引起严重不良反应。以下总结了该类杂质形成的过程及有别于rAAV载体的特点,并对可以防止其生成或将其与rAAV载体有效分离的技术手段进行了评价。 相似文献
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可以稳定表达治疗基因而无明显不良反应的重组腺相关病毒(rAAV)载体被认为是最有发展前景的基因治疗载体。但如何建立可以有效去除rAAV载体内具有潜在致病危害的杂质、产品质量符合临床使用要求的纯化工艺是研究人员面临的巨大挑战。其中针对载体相关性杂质的纯化工艺尤为关键,因为该类杂质的性质与真正的rAAV载体极其相似,一旦存在便难以去除,且会引起严重不良反应。以下总结了该类杂质形成的过程及有别于rAAV载体的特点,并对可以防止其生成或将其与rAAV载体有效分离的技术手段进行了评价。 相似文献
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ＬＤＬＲｐｌａｙｓａｖｉｔａｌｒｏｌｅｉｎｅｌｉｍｉｎａｔｉｎｇｐｌａｓｍａｃｈｏｌｅｓｔｅｒｏｌ.ＫｎｏｃｋｉｎｇｏｕｔＬＤＬＲｇｅｎｅｃａｎｃａｕｓｅｈｙｐｅｒｃｈｏｌｅｓｔｅｒｏｌｅｍｉａ.Ｐｅｒｓｏｎｓｗｉｔｈｆａｍｉｌｉａｌｈｙｐｅｒｃｈｏｌｅｓｔｅｒｏｌｅｍｉａ(ＦＨ)ｈａｖｅｂｅｅｎｆｏｕｎｄｔｏｈａｖｅｇｅｎｅｔｉｃｄｅｆｅｃｔｓｉｎＬＤＬＲｇｅｎｅ.ＩｎｔｒｏｄｕｃｉｎｇＬＤＬＲｇｅｎｅｔｏｅｘｐｅｒｉｍｅｎｔａｌａｎｉｍａｌｓｗｉｔｈｈｙｐｅｒｃｈｏｌｅｓｔｅｒｏｌｅｍｉａｃｏｕ… 相似文献
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Gene therapy holds promise for treating numerous heart diseases. A key premise for the success of cardiac gene therapy is the development of powerful gene transfer vehicles that can achieve highly efficient and persistent gene transfer specifically in the heart. Other features of an ideal vector include negligible toxicity, minimal immunogenicity and easy manufacturing. Rapid progress in the fields of molecular biology and virology has offered great opportunities to engineer various genetic materials for heart gene delivery. Several nonviral vectors (e.g. naked plasmids, plasmid lipid/polymer complexes and oligonucleotides) have been tested. Commonly used viral vectors include lentivirus, adenovirus and adeno-associated virus. Among these, adeno-associated virus has shown many attractive features for pre-clinical experimentation in animal models of heart diseases. We review the history and evolution of these vectors for heart gene transfer. 相似文献
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Qing Xie Thayumanasamy Somasundaram Smita Bhatia Weishu Bu Michael S. Chapman 《Acta Crystallographica. Section D, Structural Biology》2003,59(6):959-970
The atomic structure of adeno‐associated virus 2 (AAV‐2) has been determined to 3.0 Å resolution. AAV‐2 crystallized in space group P1, with unit‐cell parameters a = 249.7, b = 249.7, c = 644.8 Å, α = 90.0, β = 101.2, γ = 120.0°. The crystals contained three full virus particles in the asymmetric unit, allowing 180‐fold non‐crystallographic symmetry averaging. The particle orientations were determined using the self‐rotation function and found to have similar but resolvably different orientations. Approximate alignment of icosahedral and interparticle threefold screw symmetry led to a native Patterson that was interpretable in terms of approximate particle positions. Accurate positions required a Patterson correlation search that was constrained to be consistent with non‐crystallographic threefold projection symmetry evident in the diffraction intensities. Initial phases to 15.0 Å resolution were calculated by molecular replacement using the known structure of a distantly related homolog (23% sequence identity). Real‐space averaging was performed and phases were extended from 15.0 to 3.0 Å. An atomic model was fitted and refined using a simulated‐annealing real‐space procedure. 相似文献
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Stylianos Michalakis Christian Schön Elvir Becirovic Martin Biel 《The journal of gene medicine》2017,19(3)
The present review summarizes the current status of achromatopsia (ACHM) gene therapy‐related research activities and provides an outlook for their clinical application. ACHM is an inherited eye disease characterized by a congenital absence of cone photoreceptor function. As a consequence, ACHM is associated with strongly impaired daylight vision, photophobia, nystagmus and a lack of color discrimination. Currently, six genes have been linked to ACHM. Up to 80% of the patients carry mutations in the genes CNGA3 and CNGB3 encoding the two subunits of the cone cyclic nucleotide‐gated channel. Various animal models of the disease have been established and their characterization has helped to increase our understanding of the pathophysiology associated with ACHM. With the advent of adeno‐associated virus vectors as valuable gene delivery tools for retinal photoreceptors, a number of promising gene supplementation therapy programs have been initiated. In recent years, huge progress has been made towards bringing a curative treatment for ACHM into clinics. The first clinical trials are ongoing or will be launched soon and are expected to contribute important data on the safety and efficacy of ACHM gene supplementation therapy. 相似文献
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病毒载体与造血干细胞基因治疗 总被引：5，自引：0，他引：5
多种获得性和遗传性疾病累及造血细胞。造血干细胞是人类基因治疗的重要靶细胞。成功的造血干细胞基因治疗不仅需要高效基因转移,还需要治疗基因的长期、高水平表达。反转录病毒载体是造血干细胞基因治疗的常用载体,结合优化的造血干细胞转导条件,其介导的腺苷脱氨酶缺陷引起的严重联合免疫缺陷和X染色体连锁的严重联合免疫缺陷的基因治疗已经获得初步成功;其他整合型病毒载体如慢病毒和腺相关病毒载体,也在临床前造血干细胞基因治疗研究中得到广泛应用。从病毒载体、基因转移和基因表达等几个方面综述了造血干细胞基因治疗的临床前和临床研究的重要进展。 相似文献
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Edward B. Miller Brittney Gurda‐Whitaker Lakshmanan Govindasamy Robert McKenna Sergei Zolotukhin Nicholas Muzyczka Mavis Agbandje‐McKenna 《Acta Crystallographica. Section F, Structural Biology Communications》2006,62(12):1271-1274
Crystals of baculovirus‐expressed adeno‐associated virus serotype 1 (AAV1) capsids have been grown in the rhombohedral space group R32 (unit‐cell parameters a = 254.7 Å, α = 62.3°) and shown to diffract X‐rays to at least 2.5 Å resolution. The diffraction data were subsequently processed and reduced with an overall Rsym of 12.3% and a completeness of 89.0%. Based on the unit‐cell volume, rotation‐function and translation‐function results and packing considerations, there is one virus capsid (60 viral proteins) per unit cell and there are ten viral proteins per crystallographic asymmetric unit. The AAV1 capsid shares both the twofold and threefold crystallographic symmetry operators. The AAV1 data have been initially phased using a polyalanine model (based on the crystal structure of AAV4) to 4.0 Å resolution and the structure determination and refinement is in progress using tenfold noncrystallographic symmetry electron‐density averaging. 相似文献
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Odayme Quesada Brittney Gurda Lakshmanan Govindasamy Robert McKenna Erik Kohlbrenner George Aslanidi Sergei Zolotukhin Nicholas Muzyczka Mavis Agbandje‐McKenna 《Acta Crystallographica. Section F, Structural Biology Communications》2007,63(12):1073-1076
Crystals of baculovirus‐expressed adeno‐associated virus serotype 7 capsids diffract X‐rays to ∼3.0 Å resolution. The crystals belong to the rhombohedral space group R3, with unit‐cell parameters a = 252.4, c = 591.2 Å in the hexagonal setting. The diffraction data were processed and reduced to an overall completeness of 79.0% and an Rmerge of 12.0%. There are three viral capsids in the unit cell. The icosahedral threefold axis is coincident with the crystallographic threefold axis, resulting in one third of a capsid (20 monomers) per crystallographic asymmetric unit. The orientation of the viral capsid has been determined by rotation‐function searches and is positioned at (0, 0, 0) by packing considerations. 相似文献