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载体构建及病毒包装服务

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  • 载体构建及病毒包装服务
     
          和元上海的技术团队拥有十年以上的病毒载体开发和包装服务经验。我们依靠坚持不懈的努力和精益求精的态度,推动了病毒载体在国内的技术转化和应用推广。随着生物医学的飞速发展和基因ZL的日益进步,病毒载体越来越多应用于机制研究和临床前实验。本着服务于客户、服务于科研的宗旨,和元生物将病毒包装服务的稳定和质量作为公司首要目标。在材料质量、操作水平、质量控制等方面都建立了严格的内部标准,力争为客户提供高质量病毒和最准确的结果!
          目前,公司以“打造一个平台,推动一个行业,让基因ZL造福人类”为发展战略,坚持以基因ZL重组病毒载体研发及生产为核心,专注于为生物医药领域提供优质的产品和技术服务,致力于发展成为辐射的重组病物生产CRO服务ZX和基因药物生产CDMO/CMO基地,同时孵化基因ZL药物,成为国内JZYL细分行业的标杆企业!
         和元上海慢病毒(LV)、腺病毒(ADV)、腺相关病毒(AAV)、逆转录病毒(RV)的生产和指控采取了国际学术界公认的标准流程,所获得的病毒完全可以满足各类实验的使用要求:
          慢病毒(和元)的滴度在108~109TU/ml
          腺病毒(和元)的滴度在1010~1011pfu/ml
          腺相关病毒AAV(和元)的滴度在1012~1013v.g./ml
          逆转录病毒(和元)滴度在107~108TU/ml

    一、病毒载体及病毒包装介绍
       
          病毒载体是当前生物医学研究中重要的基因转导工具。一方面病毒载体经过改造具有更强的使用安全性和更快的分子克隆速度;另一方面,病毒的感染能力得到了定向的人工进化:ZL靶点筛选者希望得到更快捷,更广谱的转导工具,而基因ZL却需要更安全更特异的感染特性。在不断涌现的新工具新方法面前,研究者常常会无所适从,既难舍对已有系统的信赖与熟悉,又倾心于新工具的蓬勃前景。
         目前生物医学研究中常用的病毒载体包括慢病毒,逆转录病毒,腺相关病毒,腺病毒。表1简单的总结了四种病毒载体的基本生物学特性。和元慢病毒感染范围广,表达稳定长效,已经成为分子生物操作中的常规载体,越来越多的分子生物学技术与方法借助于慢病毒载体得以应用。腺病毒载体作为研究和使用时间最长的载体,虽然存在一些不足,但其特点依然鲜明,即感染速度快,基因装载量大。逆转录载体因其特殊的感染特性,仍在干细胞和血液细胞等分裂细胞研究中占有一席之地。而势头强劲的腺相关病毒因其极高的滴度和低转基因安全性,成为基因ZL研究的新宠儿。
     
    表1 不同病毒的生物学特性比较
     病毒特性 病毒类型
    慢病毒(LV) 逆转录病毒(RV) 腺相关病毒(AAV) 腺病毒(ADV)
    包膜
    颗粒直径 90-100 nm 90-100 nm 20-30 nm 60-90nm
    基因组 dsRNA dsRNA ssDNA dsDNA
    表达起始时间 48-72h 48-72h 72-96h 24-48h
    表达持续时间 > 2 months  > 2 months  > 6 months  < 1 month
    整合方式 随机高频整合 随机高频整合 定向低频整合 非整合

    1.腺相关病毒
         和元腺相关病毒(adeno-associated virus,AAV)是一类细小病毒,基因组为单链DNA,对分裂细胞和非分裂细胞均具有感染能力。 AAV的血清型是指目前已发现的不同衣壳蛋白的数十种AAV在体内产生不同的抗血清类型,它们对相同的组织和细胞具有不同的感染效率。
         重组腺相关病毒rAAV是利用AAV2型基因组与不同的衣壳蛋白结合产生的混合体病毒载体,一般标记为rAAV2/N (N为不同的衣壳血清型)。纽恩生物通过长期的研发,构建了一系列用于基因表达和基因干扰操作的载体。
     
    表2  不同血清型AAV的组织嗜亲性
    血清型 适用组织 胞外吸附分子
    AAV1 肌肉,心脏,神经 未知
    AAV2 体外,神经 硫酸乙酰肝素蛋白多糖
    AAV5 肺,眼睛,神经 N-唾液酸
    AAV6 肺,心脏 未知
    AAV7 肌肉,肝脏 未知
    AAV8 肝脏,肌肉,眼睛 未知
    AAV9 肺,肝脏,肌肉,心脏 未知
    rh10 肋膜,神经 未知

    病毒感染实例如下:
     
          
    2.慢病毒
         慢病毒(Lentivirus): 是一类改造自人免疫缺陷病毒(HIV)的病毒载体。它是一种RNA病毒,可利用逆转录酶将外源基因整合到基因组中实现稳定长时表达。目前广泛用于哺乳动物细胞和整体的基因转化操作,具有能同时感染分裂期与非分裂期的细胞等特性。

    病毒感染实例如下:
     
    3. 逆转录病毒
         逆转录病毒(Retrovirus) :改自鼠白血病病毒(MMLV),是一种RNA病毒,可以利用逆转录酶将外源基因整合到基因组中实现稳定长时表达。逆转录病毒特异性地感染分裂细胞,对造血干细胞和神经干细胞在体外和体内感染具有良好的效果。
     
    病毒感染实例如下:
     
    4. 腺病毒
         腺病毒 (Adenovirus) :是一种DNA病毒,具有广泛的细胞和组织感染能力。感染过程剧烈,适于在短时间内进行基因高表达的实验。与其它病毒载体相比,腺病毒ZD优势在于插入片段较长,表达活性强;可进行扩增操作。
     
     病毒感染实例如下:

    二、客户代表性文章列表
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    [2] Science. (IF=34.661).Ding L,et.al.(2014).Medial prefrontal activity during delay period contributes to learning of a working memory task.[腺相关病毒,光敏感,神经系统,前额叶]
    [3] Nature Medicine. (IF=30.357).Zhu LJ,et.al.(2014).CAPON-nNOS coupling can serve as a target for developing new anxiolytics.[腺相关病毒,体内注射,过表达]
    [4] Nature Medicine. (IF=30.357).Cao X,et.al.(2013).Astrocyte-derived ATP modulates depressive-like behaviors.[腺相关病毒,神经系统,皮层,干扰]
    [5] Cell. (IF=28.71).Bian WJ,et.al.(2015).Coordinated Spine Pruning and Maturation Mediated by Inter-Spine Competition for Cadherin/Catenin Complexes.[腺相关病毒,神经]
    [6] Cancer Discovery. (IF=19.783).Sun TT,et.al.(2016).A novel IncRNA GClnc1 promotes gastric carcinogenesis and may act as a modular scaffold of WDR5 and KAT2A complexes to specify the histone modification pattern.[腺病毒,lncRNA,胃癌]
    [7] Nature Neuroscience. (IF=16.724).Yang HB,et.al.(2015).Laterodorsal tegmentum interneuron subtypes oppositely regulate olfactory cue-induced innate fear.[腺相关病毒,光遗传]
    [8] Nature Neuroscience. (IF=16.724).Zheng JJ,et.al.(2013).Oxytocin mediates early experience-dep Nature medicine endent cross-modal plasticity in the sensory cortices.[腺相关病毒,神经系统,感觉皮层]
    [9] Cell Research. (IF=14.812).Xie C,et.al.(2016).Genome editing with CRISPR/Cas9 in postnatal mice corrects PRKAG2 cardiac syndrome.[腺相关病毒,心脏应用]
    [10] Cell Research. (IF=14.812).Gao M,et.al.(2014).Generation of transgenic golden Syrian hamsters.[慢病毒,转基因仓鼠]
    [11] Nature Communications. (IF=11.329).Wei PF,et.al.(2015).Processing of visually evoked innate fear by a non-canonical thalamic pathway.[腺相关病毒,神经,丘脑]
    [12] Nature Communications. (IF=11.329).Zhu YJ,et.al.(2015).Control of response reliability by parvalbumin-expressing interneurons in visual cortex.[腺相关病毒,视觉皮质]
    [13] Nature Communications. (IF=11.329).Han G,et.al.(2016).Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice.[腺相关病毒,肌肉注射,基因ZL]
    [14] Nature Communications. (IF=11.329).Yao XH,et.al.(2016).Electrical coupling regulates layer 1 interneuron microcircuit formation in the neocortex.[慢病毒,神经系统,干扰]
    [15] Embo Journal. (IF=9.643).Ren SQ,et.al.(2013).PKCλ is critical in AMPA receptor phosphorylation and synaptic incorporation during LTP.[腺相关病毒,神经系统,大脑皮层]
    [16] Embo Molecular Medicine. (IF=9.547).Guan Y,et.al.(2016).CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse.[腺病毒,CRISPR/Cas9,血友病]
    [17] PNAS. (IF=9.423).Li J,et.al.(2015).Synaptic P-Rex1 signaling regulates hippocampal longterm depression and autism-like social behavior.[腺病毒,体内感染]
    [18] PNAS. (IF=9.423).Du H,et.al.(2016).Dopaminergic inputs in the dentate gyrus direct the choice of memory encoding.[腺相关病毒,光遗传,大脑海马区域记忆]
    [19] Developmental Cell. (IF=9.338).Cheng TL,et.al.(2014).MeCP2 Suppresses Nuclear MicroRNA Processing and Dendritic Growth by Regulating the DGCR8/Drosha Complex.[慢病毒,腺相关病毒,体内体外]
    [20] Autophagy. (IF=9.108).Guo WJ,et.al.(2015).Small molecule-driven mitophagy-mediatedNLRP3 inflammasome inhibition is responsible for the prevention of colitis-associated cancer.[慢病毒,肿瘤细胞,干扰]
    [21] Autophagy. (IF=9.108).Zhao XJ,et.al.(2015).Elaiophylin, a novel autophagy inhibitor, exerts antitumor activity as a single agent in ovarian cancercells.[自噬]
    [22] Diabetes. (IF=8.784).Xiao YZ,et.al.(2015).Activation of ERK1/2 ameliorates liver steatosis in leptin receptor deficient (db/db)mice via stimulating ATG7-dependent autophagy.[腺病毒,整体注射,肝脏]
    [23] Clinical Cancer Research. (IF=8.738).Shi Y,et.al.(2014).Primate-specific miR-663 functions as a tumor suppressor by targeting PIK3CD and predicts the prognosis of human glioblastoma.[慢病毒,肿瘤细胞感染]
    [24] Biomaterials. (IF=8.387).Luo X,et.al.(2015).CAD based design sensitivity analysis and shape optimization of scaffolds for bio-root regeneration in swine.[慢病毒,生物牙根研究]
    [25] Biomaterials. (IF=8.387).Wang X,et.al.(2013).Promotion of dentin regeneration via CCN3 modulation on Notch and BMP signaling pathways.[慢病毒,牙髓干细胞,体外感染,基因表达与干扰]
    [26] ELIFE. (IF=8.303).Chen M,et.al.(2015).Morphine disinhibits glutamatergic input to VTA dopamine neurons and promotes dopamine neuron excitation.[腺相关病毒,神经系统,腹侧被盖区]
    [27] ELIFE. (IF=8.303).Ni KM,et.al.(2016).Selectively driving cholinergic fibers optically in the thalamic reticular nucleus promotes sleep.[腺相关病毒,丘脑]
    [28] Oncogene. (IF=7.932).Wen SY,et.al.(2013).miR-506 acts as a tumor suppressor by directly targeting the hedgehog pathway transcription factor Gli3 in human cervical cancer.[慢病毒,肿瘤细胞,miRNA过表达]
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    [30] Redox Biology. (IF=6.235).Huang GY,et.al.(2016).Signaling pathways involved in HSP32 induction by hyperbaric oxygen in rat spinal neurons.[慢病毒,大鼠脊髓神经元,干扰]
    [31] Diabetologia. (IF=6.206).Ma X,et.al.(2015).RBP4 functions as a hepatokine in the regulation of glucose metabolism by the circadian clock in mice.[腺相关病毒,肝脏,干扰]
    [32] Cancer Letters. (IF=5.992).Fang ZY,et.al.(2016).A feed-forward regulatory loop between androgen receptor and PlncRNA-1 promotes prostate cancer progression.[CCK-8,Luciferase assay,前列腺癌细胞]
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    [34] Journal of Neuroscience. (IF=5.924).Liu YG,et.al.(2015).Ascl1 Converts Dorsal Midbrain Astrocytes into Functional Neurons In Vivo.[腺相关病毒,神经系统]
    [35] Free Radical Biology and Medicine. (IF=5.784).Cheng X,et.al.(2016).Lipoxin A4 attenuates LPS-induced mouse acute lung injury via Nrf2-mediated E-cadherin expression in airway epithelial cells.[质粒过表达,肺部]
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    [37] Mol Neurobiol. (IF=5.397).Hu G,et.al.(2016).Triptolide Promotes the Clearance of α-Synuclein by Enhancing Autophagy in Neuronal Cells.[慢病毒,神经元感染]
    [38] Molecular Neurobiology. (IF=5.397).Guo J,et.al.(2015).Overexpression of Fibulin-5 Attenuates Ischemia/Reperfusion Injury After Middle Cerebral Artery Occlusion in Rats.[腺病毒,大鼠大脑中动脉]
    [39] FASEB Journal. (IF=5.299).Wang Y,et.al.(2016).Down-regulation of Risa improves insulin sensitivity by enhancing autophagy.[腺病毒,小鼠原代肝细胞,过表达,自噬]
    [40] Brain Pathology. (IF=5.256).Xu QF,et.al.(2014).MiR-22 is Frequently Down‐regulated in Medulloblastomas, and Inhibits Cell Proliferation via the Novel Target PAPST1.[慢病毒,肿瘤细胞感染]
    [41] Scientific Reports. (IF=5.228).Xiong YS,et.al.(2015).Early treatment of minocycline alleviates white matter and cognitive impairments after chronic cerebral hypoperfusion.[逆转录病毒,脑缺血,白质,认知障碍]
    [42] Scientific Reports. (IF=5.228).Shan LL,et.al.(2015).Light exposure before learning improves memory consolidation at night.[腺相关病毒,海马,过表达]
    [43] Scientific Reports. (IF=5.228).Tao Y,et.al.(2015).Astroglial β-Arrestin1-mediated Nuclear Signaling Regulates the Expansion of Neural Precursor Cells in Adult Hippocampus.[腺相关病毒,胶质细胞]
    [44] Scientific Reports. (IF=5.228).Gu Y,et.al.(2015).Endogenous IL-6 of mesenchymal stem cell improves behavioral outcome of hypoxic-ischemic brain damage neonatal rats by supressing apoptosis in astrocyte.[慢病毒,MSC感染]
    [45] Scientific Reports. (IF=5.228).Chen X,et.al.(2016).GSK-3β downregulates Nrf2 in cultured cortical neurons and in a rat model of cerebral ischemia-reperfusion.[慢病毒,神经系统,过表达,干扰]
    [46] Scientific Reports. (IF=5.228).Lyu JW,et.al.(2016).Reciprocal regulation of autism-related genes MeCP2 and PTEN via microRNAs.[慢病毒,神经系统,干扰]
    [47] Scientific Reports. (IF=5.228).Zhu YB,et.al.(2016).Astrocyte-derived phosphatidic acid promotes dendritic branching.[慢病毒,星形胶质细胞,干扰]
    [48] Scientific Reports. (IF=5.228).Yu X,et.al.(2016).Differences in vulnerability of neurons and astrocytes to heme oxygenase-1 modulation: Implications for mitochondrial ferritin.[慢病毒,神经元感染]
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    [53] Frontiers in Molecular Neuroscience. (IF=5.154).Gao QQ,et.al.(2015).Post-training activation of Rac1 in the basolateral amygdala is required for the formation of both short-term and long-term auditory fear memory.[腺相关病毒,杏仁核]
    [54] Biochimica et Biophysica Acta-Molecular Cell Research. (IF=5.128).Xiang L,et.al.(2014).Knock-down of glutaminase 2 expression decreases glutathione, NADH, and sensitizes cervical cancer to ionizing radiation.[慢病毒,宫颈癌细胞,RNA干扰]
    [55] Clinical Science. (IF=5.016).He T,et.al.(2015).Pigment epithelium-derived factor regulates microvascular permeability through adipose triglyceride lipase in sepsis.[慢病毒,色素上皮衍生因子,干扰]
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    [57] Journal of Neuroinflammation. (IF=4.667).Lin WP,et.al.(2016).Heme oxygenase-1 promotes neuron survival through down-regulation of neuronal NLRP1 expression after spinal cord injury.[腺相关病毒,原代神经元]
    [58] Cellular Physiology and Biochemistry. (IF=4.652).Zhou X,et.al.(2015).Human Umbilical Cord-Derived Mesenchymal Stem Cells Improve Learning and Memory Function in Hypoxic-Ischemic Brain-Damaged Rats via an IL-8-Mediated Secretion Mechanism Rather than Differentiation Pattern Induction.[脐带来源间充质干细胞]
    [59] Cellular Physiology and Biochemistry. (IF=4.652).Huo Y,et.al.(2015).Amino-Nogo Inhibits Optic Nerve Regeneration and Functional Recovery via the Integrin αv Signaling Pathway in Rats.[腺相关病毒,眼科应用]
    [60] Frontiers in Cellular Neuroscience. (IF=4.609).Huang GH,et.al.(2016).Porf-2 inhibits neural stem cell proliferation through Wnt/β-catenin pathway by its GAP domain.[腺相关病毒,shRNA,mcherry,海马区域神经干细胞]
    [61] Fertility and Sterility. (IF=4.426).Wang Y,et.al.(2015).MicroRNAome in decidua: a new approach to assess the maintenance of pregnancy.[慢病毒,人类胚胎干细胞,microRNA干扰]
    [62] International Journal of Neuropsychopharmacology. (IF=4.333).Wang YX,et.al.(2014).Protein kinase Mζ is involved in the modulatory effect of fluoxetine on hippocampal neurogenesis in vitro.[腺相关病毒,海马,定位注射]
    [63] Journal of Biological Chemistry. (IF=4.258).Tian T,et.al.(2013).Exosome Uptake through Clathrin-mediated Endocytosis and Macropinocytosis and Mediating miR-21 Delivery.[慢病毒,间充质干细胞]
    [64] Journal of Biological Chemistry. (IF=4.258).Cai R,et.al.(2012).SUMO-specific Protease 1 Regulates Mitochondrial Biogenesis through PGC-1α.[腺相关病毒,心肌细胞感染]
    [65] Journal of Biological Chemistry. (IF=4.258).Zhang B,et.al.(2016).Increased Activity of Src Homology 2 Domain Containing Phosphotyrosine Phosphatase 2 (Shp2) Regulates Activity Dependent AMPA Receptor Trafficking.[腺相关病毒,小鼠海马神经元]
    [66] International Journal of Biological Sciences. (IF=3.982).Li Y,et.al.(2016).Glycogen Synthase Kinase 3β Influences Injury Following Cerebral Ischemia/Reperfusion in Rats.[慢病毒,脑缺血]
    [67] International Journal of Biological Sciences. (IF=3.982).Liu B,et.al.(2015).Gadd45b is a Novel Mediator of Neuronal Apoptosis inIschemic Stroke.[慢病毒,体内注射]
    [68] International Journal of Biological Sciences. (IF=3.982).Kuang L,et.al.(2013).Knockdown of Nrf2 Inhibits the Angiogenesis of Rat Cardiac Micro-vascular Endothelial Cells under Hypoxic Conditions.[慢病毒,心肌血管内皮细胞,过表达]
    [69] Cell Cycle. (IF=3.952).Meng ZS,et.al.(2015).Tumorigenicity Analysis of Heterogeneous Dental Stem Cells and Its Self-Modification for Chromosome Instability.[慢病毒,牙髓干细胞感染]
    [70] Journal of Neurochemistry. (IF=3.842).Luo DJ,et.al.(2014).Knockdown of Phosphotyrosyl phosphatase activator (PTPA) induces apoptosis via mitochondrial pathway and the attenuation by simultaneous tau hyperphosphorylation.[慢病毒,体外神经元细胞,过表达]
    [71] Journal of Neurochemistry. (IF=3.842).Wang B,et.al.(2014).Destabilization of Survival Factor MEF2D mRNA by Neurotoxin in Models of Parkinson’s Disease.[腺相关病毒,脑黑质]
    [72] Stem Cells International. (IF=3.687).Jia Q,et.al.(2016).The Regulatory Effects of Long Noncoding RNA-ANCR on Dental Tissue-Derived Stem Cells.[慢病毒,lncRNA干扰,牙齿组织来源的干细胞]
    [73] Febs Letters. (IF=3.519).Li Q,et.al.(2013).MicroRNA-222 promotes tumorgenesis via targeting DKK2 and activating the Wnt/β-catenin signaling pathway.[慢病毒,胶质瘤,microRNA表达]
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