Name: utilizing the antigen capsid-incorporation strategy for the development of adenovirus serotype 5-vectored vaccine approaches
Uploaded: 2015-05-06T14:00:00.000+00:00
Duration: 13 min 36 s
Description: Watch this Scientific Journal Video about Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches at JoVE.com

卫生部授予5T32AI7493-20和5R01AI089337-03部分这项工作是支持国家机构。该资助者在研究设计，数据收集和分析，发布决定，或准备的手稿没有作用。

阿拉巴马大学伯明翰分校机构动物使用及护理委员会批准使用小鼠作为下批准的协议号101109272描述。 1.遗传结构改良质粒PAD5 / H5-HVR1-KWAS-HVR5-他6与衣壳抗原掺入战略<ol><li>穿梭质粒的构建HVR1-KWAS-HVR5-他6 / pH5S <ol><li>交货含有合成的DNA序列HVR1-KWAS的限制酶位点AGEI之间hexon5基因的AccI质粒。 </li><li>消化6微克的合成片段（HVR1-KWAS）与酶AGEI（6单位）和的AccI（6单位）3小时，在37℃。解决在通过电泳在2％琼脂糖凝胶的消化片段，并根据制造商的方案用DNA凝胶提取试剂盒纯化该片段。 </li><li>根据插入的摩尔比为矢量在3:1，用T4 DNA连接酶和连接酶buffer结扎12纳克的片段（HVR1-KWAS）到100纳克先前构建的穿梭质粒的HVR5-他在一个10微升体积于室温2小时6 / pH5S 17，经由AGEI和AccI位的位点。 </li><li>变换1μl的总分10微升连接产物向50微升电感受态的DH5α细胞在电穿孔（在1800Ⅴ）。添加950微升SOC培养基，以转化DH5α细胞，并培育1小时，混合物在37℃下，以300转。传播100-200微升含有卡那霉素的卢里亚BERTANI（LB）琼脂1ml的混合物和孵化O / N在37℃。 <ol><li>筛选〜10个菌落通过PCR靶向片段HVR1-KWAS，混合的正向引物（TATGTGTGTCATGTATGCGT），反向引物（GGAGGCCCACTTGTCCAGCTC）和单个DH5α菌落成PCR主混合物溶液（根据制造商的协议）。通过参照设置有PCR主混合物溶液手动运行在热循环样本。 </li><li>使用正向引物（TATGTGTGTCATGTATGCGT）的测序片段HVR1-KWAS中筛选部分1.1.4.1克隆。 </li></ol></li></ol></li><li>质粒PAD5 /构建H5-HVR1-KWAS-HVR5-他6 <ol><li>消化6微克HVR1-KWAS-HVR5-他6 / pH5S用酶EcoRI位（6单位）和PmeI位（6单位）3小时，在37℃，并净化含有同源重组臂和双改性hexon5基因的片段，如在步骤1.1.2说明。线性化骨架质粒PAD5 /ΔH5（GL）16用SwaI。 </li><li>共转化100纳克从穿梭质粒的目的片段和100毫微克的线性PAD5 /ΔH5（GL）骨干到50微升电感受态细胞BJ5183为在电穿孔的同源重组（1800 V）。添加950微升SOC培养基，以转化细胞，并培育1小时，混合物在37℃，300转。传播100 - 200微升1ml的混合物在含卡那霉素（终50微克/毫升）对O / N培养在37℃的LB琼脂。 <ol><li>挑〜10微小菌落于3ml含有卡那霉素（最终50微克/毫升）对O / N培养在摇动器（250转，37℃）的液体LB。提取从培养质粒。使用PCR分析通过靶向片段HVR1-KWAS筛选的10个菌落，如图步骤1.1.4.1。 </li><li>筛选通过靶向骨干基因组的的pIX片段PCR分析相同的菌落，混合的正向引物（AGCTGTTGGATCTGCGCCAGCAGGTT），反向引物（CCAAACAGAGTCTGGTTTTTTATTTAT）和单个BJ5183菌落成PCR主混合物溶液（根据制造商的协议）。通过参照设置有PCR主混合物溶液手动运行样品上的热循环。 </li><li>指定PCR双阳性菌落作为PAD5 / H5-HVR1-KWAS-HVR5-他6。 </li></ol></li><li>改造100毫微克质粒PAD5 / H5-HVR1-KWAS-HVR5-他6中如图步骤1.1.4至DH5α细胞。 <ol><li>使用PCR分析来筛选〜10个菌落通过靶向片段HVR1-KWAS，如图步骤1.1.4.1。 </li><li>使用PCR分析通过靶向骨干基因组的pIX片段来筛选相同的菌落，如图步骤1.2.2.2。 </li><li>使用正向引物（TATGTGTGTCATGTATGCGT）的测序片段HVR1-KWAS在PAD5 / H5-HVR1-KWAS-HVR5-他6。 </li></ol></li></ol></li></ol> 2:改性Ad5的病毒载体的Ad5 / H5的-HVR1-KWAS-HVR5-他6 <ol><li>通过添加FBS（最后10％），100X非必需氨基酸（最终0.1mM的），200mM的L-谷氨酰胺（最终为2mM）和青霉素/链霉素溶液（最终1％）到DMEM高糖构成的完全培养基。保持人胚肾（HEK293）细胞在完全培养基中培养培养箱中（37℃和5％CO 2下85％湿度条件下）。 </li><li>病毒已经救援构造函数的Ad5 / H5-HVR1-KWAS-HVR5-他6 <ol><li>种子3.0×10 6的HEK293细胞在含有5ml完全培养基，并培养O / N中的一个的T-25烧瓶中，以达到与80％汇合的单层。 </li><li>线性15微克的PAD5 / H5-HVR1-KWAS-HVR5-他6在100微升体积用限制性酶PacI位（15个单位），在37℃3小时。 <ol><li>提取线性PAD5 / H5-HVR1-KWAS-HVR5-他6通过离心反应两次（10,000 xg离心1分钟）苯酚等体积的氯仿:异戊醇（比在25:24:1）在通风橱，并通过连续离心分离（10,000×g离心10分钟）的混合溶液（300微升的100％乙醇和10μl的乙酸钠）和700微升70％乙醇。弃上清在每个离心步骤。 </li></ol></li><li>重悬纯化的质粒在〜40微升蒸馏水或的Tris-EDTA（TE）缓冲液。通过测量光学德定量质粒DNAnsity（OD）在260nm处。 </li><li>染3微克线性化的质粒与在T-25烧瓶商用脂质体转染试剂，根据制造商的手册。在6小时后转染和随后的孵化，在孵化器的文化与变化完全培养基转染培养基。通过替换完全培养基每两至三天，直到单个病毒噬斑形式维持转染的细胞。 </li><li>当斑块发展到充分的细胞致病效应（CPE），刮去烧瓶中剩余的细胞在无菌罩，并收获细胞裂解物在4℃下离心分离介质，在300×g离心10分钟。暂停〜1ml含有2％FBS的培养基中的细胞沉淀，并通过冻融四次破细胞。收集含在4℃下离心裂解物以10,000rpm离心10分钟后获救病毒上清。 </li></ol></li><li>该病毒载体的Ad5 / H5-HVR1-KWAS-HVR5-他6大规模传播<ol><li>第传播病毒在一个T-75烧瓶中含有HEK293细胞在无菌罩通过用1/3感染到从T-25烧瓶中充分溶胞产物。允许完全CPE在培养培养箱内两到三天开发。收获裂解物上清液作为在步骤2.2.5说明。 </li><li>通过用1/2感染完整裂解物从T-75烧瓶中，根据不同的病毒传播条件传播在一到三颗T-175烧瓶中无菌罩病毒。允许完全CPE在培养培养箱内两到三天开发。收获裂解物上清液作为在步骤2.2.5说明。 </li><li>通过与感染的1/2，从以前的T-175瓶（S）全裂解液传播病毒在一打以上的T-175瓶的无菌罩。确定基于所述病毒传播条件和病毒的有需要的量烧瓶使用量。收获上清裂解当全CPE发生在两到三天的培养孵化器在步骤2.2.5规定。 </li></ol></li><li> PURIF中的Ad5 / H5-HVR1-KWAS-HVR5-他6氯化铯ication <ol><li>准备两个密度在5毫米的HEPES的CsCl溶液的:1.33克/毫升1.45克/毫升，而避免了与氯化铯接触。 </li><li>负载4毫升氯化铯（1.33微克/毫升）在无菌超速离心管，并轻轻装入4毫升氯化铯（1.45微克/毫升）对所述管的底部。负载4毫升裂解上清液慢慢在无菌罩梯度的顶部。 </li><li>离心管中，在110000×g离心3小时，在4℃至成熟/低病毒带从有缺陷的/更高的病毒带分开。使用3毫升注射器手持33克针收集低频段和5毫米的HEPES稀释乐队4ml的体积在无菌罩。 </li><li>载入另一个管的CsCl溶液的两种密度和病毒的稀释频带如步骤2.4.2中描述。离心管中，在11万XG O / N在4℃。如步骤2.4.3所述收集病毒带。 </li><li>注入的dialys收集病毒解决方案是由卡带3毫升注射器手持33克针在无菌罩。 </li><li>放置700毫升的1×透析缓冲液的盒（1L配方:100毫升10×PBS中，加入100毫升100％甘油和800毫升蒸馏水;过滤通过0.22微米的过滤器的缓冲器），并更换透析缓冲液，每3小时进行4次。使用针刺注射器吸出透析病毒解决方案。 </li></ol></li></ol> 3.验证获救病毒载体<ol><li>病毒载体滴定<ol><li>为病毒物理滴度，稀既病毒和透析缓冲液（背景对照）在1:10和1:20中病毒的裂解缓冲液（10％SDS在Tris-EDTA）中在小管，并孵育所有试管在56℃下为10分钟。 </li><li>打开一个BioPhotometer生物分光光度计和设置的吸在OD 260nm的模式。使用稀释透析缓冲液（1:10）通过点击"空白"底部，以平衡背景信号。在BioPhotometer生物分光光度计SCR型"10 + 90"EEN，并读取稀释病毒的（1:10）的信号。 </li><li>读的稀释的病毒（1:20）的信号通过以下步骤3.1.2所述的方法，而是在BioPhotometer生物分光光度计屏幕输入"5 + 95"。由1.1×10 12乘两个病毒读出的数字和计算平均滴度用单元为VP /毫升，基于从两个稀释两个单独滴度。 </li><li>对于病毒感染滴度（IP / ml）的，使用寇氏统计TCID 50方法14，以确定在后10天感染对HEK293细胞的感染深度（dpi）的</li></ol></li><li>抗原曝光显示评价的病毒载体<ol><li>大衣的病毒载体在ELISA板并继续在标准ELISA法14的其余步骤。使用人抗gp41的（2F5）单克隆抗体（mAb）和小鼠抗His标签单抗对应掺入的抗原14的检测。 </li><li>解决病毒载体在denatu红蛋白凝胶电泳（SDS-PAGE），并着手步骤一个标准的免疫印迹法14休息。使用人抗gp41的（2F5）单克隆抗体（mAb）和小鼠抗His标签单抗对应掺入的抗原14的检测。 </li></ol></li><li> 在载体上绕过的Ad5-positve血清的能力体外评价<ol><li>保持在培养培养箱中（37℃和5％CO 2条件下85％湿度条件下）的HeLa细胞在完全培养基中。构成通过添加FBS（最后10％），200 2mM L-谷氨酰胺（最终为2mM）和青霉素/链霉素溶液（终1％）到最低必需培养基Eagle（MEME）的完全培养基。 </li><li>种子HeLa细胞在6孔板以1×10 6个细胞/孔，并孵育在培养培养箱中（37℃和5％CO 2条件下85％湿度条件下）的板2小时。孵育的Ad5阳性血清（0.1微升或0微升）与病毒载体的5 IP /单元（的Ad5或的Ad5 / H5-HVR1-KWAS-HVR5-他6）在培养培养箱1小时之前加入的混合物在含有细胞的6孔平板上。 </li><li>在24小时后感染（HPI），在0.5ml裂解缓冲液一次用PBS裂解细胞冲洗细胞。离心以15,000×g离心5分钟，并收集上清。混合20微升用100μl的荧光素酶信号读取荧光素酶底物的上清液中，由于病毒含有荧光素酶基因。 </li></ol></li></ol>抢救性病毒载体免疫4.评价<ol><li>准备两个免疫组的Ad5和的Ad5 / H5-HVR1-KWAS-HVR5-他6。 <ol><li>注入小鼠（n = 8 /组）肌内病毒以1×10 10 VP /小鼠，在一个同源"初免-加强"免疫方案，以2周的间隔。 </li><li>在2周后每注射，小鼠流血无需麻醉由颊带刺血针的动物（5毫摩尖长）出血在1.5ml管中采集血液。 </li></ol></li><li>混合血液在管通过颠倒上下，孵育血液在RT下30分钟。旋管以10,000×g离心5分钟，在4℃和转让血清（顶​​层）至新的1.5 ml微量离心管。 <ol><li>旋新试管以10,000×g离心5分钟，在4℃和转让血清到新标记的1.5毫升管存储在-80℃。 </li></ol></li><li>由血清基于ELISA法评价的抗原特异性体液免疫<ol><li>他的稀释肽（现货1毫米）和HIV-1肽（股票在1毫米）分别在100。 </li><li> 0mM碳酸盐缓冲液（pH 9.5）中，以实现肽包被浓度为10μM。涂覆ELISA板与稀释的肽分别加入每孔100μl，并培养该板O / N在4℃。 </li><li>洗用PBST将板四次以200μl/孔和块1小时在PBST中的5％BSA封闭。适用的小鼠的血清在板1:在阻断的buf 100稀释FER，100μl/孔。孵育2小时在室温下孵育。 </li><li>洗用PBST将板四次。通过在RT中以100μl的封闭缓冲液温育30分钟/孔阻断板。 </li><li>以100μl/孔到两个涂覆有他的肽和HIV-1肽单独的板:（5000在封闭缓冲液1）应用的山羊抗小鼠IgG-HRP。孵育2小时，在室温。用PBST洗平板。 </li><li>读取平板在OD450 nm的HRP底物温育30分钟后。 </li></ol></li></ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunization+with+Hexon+Modified+Adenoviral+Vectors+Integrated+with+gp83+Epitope+Provides+Protection+against+Trypanosoma+cruzi+Infection.">Immunization with Hexon Modified Adenoviral Vectors Integrated with gp83 Epitope Provides Protection against Trypanosoma cruzi Infection.</a> PLoS neglected tropical diseases. 8, e3089 (2014).</li><li>Krause, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitopes+expressed+in+different+adenovirus+capsid+proteins+induce+different+levels+of+epitope-specific+immunity.">Epitopes expressed in different adenovirus capsid proteins induce different levels of epitope-specific immunity.</a> Journal of virology. 80, 5523-5530 (2006).</li><li>Omori, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+a+fiber+protein+in+an+adenovirus+vector+improves+in+vitro+gene+transfer+efficiency+to+the+mouse+microglial+cell+line.">Modification of a fiber protein in an adenovirus vector improves in vitro gene transfer efficiency to the mouse microglial cell line.</a> Neuroscience letters. 324, 145-148 (2002).</li><li>Dmitriev, I. P., Kashentseva, E. A., Curiel, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+adenovirus+vectors+containing+heterologous+peptide+sequences+in+the+C+terminus+of+capsid+protein+IX.">Engineering of adenovirus vectors containing heterologous peptide sequences in the C terminus of capsid protein IX.</a> Journal of virology. 76, 6893-6899 (2002).</li><li>Xue, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+a+recombinant+human+adenovirus+type+3+vector+containing+two+foreign+neutralizing+epitopes+in+hexon.">Construction and characterization of a recombinant human adenovirus type 3 vector containing two foreign neutralizing epitopes in hexon.</a> Virus research. 183, 67-74 (2014).</li><li>Tian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+human+adenovirus+serotype+3+packaged+by+serotype+7+hexon.">Construction and characterization of human adenovirus serotype 3 packaged by serotype 7 hexon.</a> Virus research. 160, 214-220 (2011).</li><li>Kim, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+adenovirus+vector+incorporating+carbohydrate+binding+domains+utilizes+glycans+for+gene+transfer.">An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.</a> PloS one. 8, e55533 (2013).</li><li>Vasconcelos, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen-induced+proapoptotic+phenotype+and+high+CD95+(Fas)+expression+accompany+a+suboptimal+CD8++T-cell+response:+reversal+by+adenoviral+vaccine.">Pathogen-induced proapoptotic phenotype and high CD95 (Fas) expression accompany a suboptimal CD8+ T-cell response: reversal by adenoviral vaccine.</a> PLoS pathogens. 8, e1002699 (2012).</li><li>Matthews, Q. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+incorporation+of+a+herpes+simplex+virus+type+1+thymidine+kinase+and+firefly+luciferase+fusion+into+the+adenovirus+protein+IX+for+functional+display+on+the+virion.">Genetic incorporation of a herpes simplex virus type 1 thymidine kinase and firefly luciferase fusion into the adenovirus protein IX for functional display on the virion.</a> Molecular imaging. 5, 510-519 (2006).</li></ol>

作者什么都没有透露。

上的Ad5修改为疫苗的发展传统的转基因策略的应用已经被降低主要是由于与Ad5的PEI 4,6相关联的瓶颈。这个瓶颈可以通过应用替代抗原衣壳-团策略（ 图1A）被部分地减小，因为这种策略可以通过的Ad5的NAb逃避中和通过与抗原的感兴趣取代的Ad5的中和表位，以及促进健壮免疫力的产生到结合抗原的感兴趣。在这项研究中，修改后的二价病毒载体的产生的Ad5 / H5-HVR1-KWAS-HVR5-他6<...

人腺病毒（AD）是一种中型，无包膜病毒含有双链DNA基因组的二十面体衣壳。广告属于腺病毒家族，与分类分为七组（A至G）。每个组都包含不同的血清型病毒。其中，腺病毒血清型5（Ad5的）从C组一直是最广泛研究和应用最广泛的用于矢量化方法，如基因治疗和疫苗接种。 传统的转基因策略已经被开发并应用于Ad5的修改，它的特点是由该病毒早期基因与基因的感兴趣的位移，并在宿主中的感兴趣基因的聚焦表达。例子是pENV9 /Ad5hrΔE3通过与猴免疫缺陷病毒1转基因替换早期基因3（E3）的建设，AdCMVGag通过与人类免疫缺陷的gag基因替换早期基因1（E1）的建设病毒（HIV）2，和广告LAC的Z用紫胶 ž基因3 E1更换施工。对Ad5的传统的转基因战略的广泛应用取决于以下优点:广泛用于Ad5的，对病毒和病毒传播的可行的基因工程，外源基因插入大量的住宿和4,5的Ad5的安全主机。然而，的Ad5向量临床治疗通过使用这种策略的功效降低一直是主要瓶颈，已经映射到主要与Ad5的PEI有关，因为Ad5的是广大儿童和成人4,6中如此普遍。 为了克服的Ad5的主要瓶颈，首要目标是开发一种替代策略规避的Ad5 PEI。先天免疫7，适应性免疫诸如中和抗体（NAb的）8-10和CD8 + T细胞应答10对Ad5的已示出的共同ntribute与Ad5 PEI，与Ad5的出现的NAb发挥贡献的主导作用与Ad5 PEI 10,11。此外，Ad5的NABS位于衣壳蛋白靶表位，包括主要的蛋白质六邻体，纤维和五邻体基底。其中，六邻体是的Ad5的NAbs 8,11-13的主要对象。基于这些发现，一个创新的衣壳抗原掺入战略已经出台。这种新颖的策略突出蛋白的感兴趣上的Ad5衣壳蛋白，其移位或屏蔽的Ad5中和表位的置换或掺入，导致降低识别由所述的NAb高效Ad5载体给药。值得注意的是，这一战略是有竞争力的，因为它也可以帮助主机引起强大的体液免疫和有效的细胞免疫功能通过直接抗原呈递的感兴趣的免疫系统4,14,15。基于这一战略，分子克隆和重组广告病毒载体抢救可分为结构分为四个主要步骤:（a）由任一聚合酶链式反应（PCR）或合成制备的基因的感兴趣的片段; （b）基因片段的连接到包含基因的感兴趣的片段和同源臂到腺病毒主链的穿梭质粒; （c）该同源重组通过共转化含基因的感兴趣的片段与线性化骨架质粒PAD5 /ΔH5（GL）16穿梭质粒; （四）线性化重组腺病毒质粒的转染营救与抗原的感兴趣并入重组Ad载体。 我们的团队和其他一些人已经扩展这种替代广告纳入战略针对不同传染病病原体广告载体疫苗的开发。我们报道了重组腺病毒载体的新一代广告-HVR1-LGS-他6 -V3通过将他标记的HIV-1抗原V3成的Ad5六邻体（hexon5）的HVR1区域。这个生成的载体引发STR具体到V3表位4翁体液免疫应答。我们还报道的Ad5 / HVR2-MPER-L15ΔE1的发展通过将HIV-1的近膜胞外区（MPER）到hexon5 2的HVR2区域。此外，周博士的研究小组已经使用这种广告策略结合的优势，开发广告3型（Ad3的）载体疫苗， 即 ，病毒载体R1SP70A3的通过将肠道病毒71型的中和表位SP70到Ad3的的HVR1的一代六邻体（hexon3）。 R1SP70A3产生了浓厚的NAbs和IFN-γ生产特定的抗原表位SP70，从而导致对肠病毒71型的挑战15率高保护。 对于技术参考的目的，我们的研究采取了定性衣壳抗原掺入战略的优势，专注于二价Ad5载体的Ad5 / H5-HVR1-KWAS-HVR5-他6的产生通过将一种HIV-1抗原进入HVR1一个DA他的标签为hexon5的HVR5。生成的病毒载体也被免疫评价。该衣壳抗原掺入策略可以被用来实现对不同传染病的Ad5疫苗向量方法的制定。

<table><tbody><tr><td>1x DPBS</td><td>Thermo Scientific</td><td>SH30256.01</td><td>for cell spliting&amp;nbsp;</td></tr><tr><td>10x DPBS</td><td>Thermo Scientific</td><td>SH30378.02</td><td>for dialysis buffer preparation</td></tr><tr><td>glycerol</td><td>SIGMA</td><td>G5516-1L</td><td>for dialysis buffer preparation</td></tr><tr><td>SDS</td><td>BIO-RAD</td><td>161-0301</td><td>for virus lysis buffer preparation</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Thermo Scientific</td><td>SH30910.03</td><td>component of culture medium</td></tr><tr><td>100x Non-Essential Amino Acids&amp;nbsp;</td><td>Thermo Scientific</td><td>SH30238.01</td><td>component of culture medium</td></tr><tr><td>200 mM L-glutamine</td><td>Cellgro</td><td>25-005-CI</td><td>component of culture medium</td></tr><tr><td>penicillin/streptomycin solution&amp;nbsp;</td><td>Cellgro</td><td>30-002-CI</td><td>component of culture medium</td></tr><tr><td>DMEM with high glucose</td><td>Thermo Scientific</td><td>SH30081.01</td><td>for HEK293 cell culture</td></tr><tr><td>Minimum Essential Medium Eagle</td><td>SIGMA</td><td>M5650</td><td>for HeLa cell culture</td></tr><tr><td>phenol:chloroform:isoamyl alcohol&amp;nbsp;</td><td>SIGMA</td><td>P3803-100ML</td><td>for large size of DNA purification</td></tr><tr><td>cesium chloride&amp;nbsp;</td><td>Research Products International Corp.</td><td>C68050</td><td>for virus purificiation</td></tr><tr><td>HEPES</td><td>Cellgro</td><td>25-060-CI</td><td>for CsCl solution preparation</td></tr><tr><td>HEK293</td><td>ATCC</td><td>51-0036</td><td>for virus rescue and upscale</td></tr><tr><td>HeLa</td><td>ATCC</td><td>CCL-2</td><td>for neutralization assay</td></tr><tr><td>T-25 flask</td><td>Thermo Scientific</td><td>156367</td><td>for cell culture&amp;nbsp;</td></tr><tr><td>T-75 flask</td><td>CORNING</td><td>430641</td><td>for cell culture&amp;nbsp;</td></tr><tr><td>T-175 flask</td><td>Thermo Scientific</td><td>159910</td><td>for cell culture&amp;nbsp;</td></tr><tr><td>Ultracentrifuge</td><td>BECKMAN</td><td>NA</td><td>for virus purification</td></tr><tr><td>Ultracentrifuge tube</td><td>BECKMAN</td><td>344059</td><td>for virus purification</td></tr><tr><td>dialysis cassette&amp;nbsp;</td><td>Thermo Scientific</td><td>66380</td><td>for virus dialysis</td></tr><tr><td>ELISA plate</td><td>Thermo Scientific</td><td>442404</td><td>for ELISA</td></tr><tr><td>human anti-gp41 (2F5) mAb</td><td>NIH AIDS Reagent Program</td><td>1475</td><td>for immunological assays</td></tr><tr><td>mouse anti-His tag mAb</td><td>GenScript</td><td>A00186</td><td>for immunological assays</td></tr><tr><td>SOC medium</td><td>CORNING</td><td>46-003-CR</td><td>for transformation</td></tr><tr><td>PCR master mix solution</td><td>QIAGEN</td><td>201445</td><td>for PCR</td></tr><tr><td>Animal lancet (point length at 5 mm)</td><td>MEDIpoint</td><td></td><td>for mice bleeding&amp;nbsp;</td></tr><tr><td>Biophotometer</td><td>Eppendorf</td><td></td><td>for virus physical titer titration</td></tr></tbody></table>

utilizing the antigen capsid-incorporation strategy for the development of adenovirus serotype 5-vectored vaccine approaches

腺病毒血清型5（Ad5的）已被广泛地修改传统的转基因方法，所述疫苗开发。在临床试验中，这些传统改性Ad5载体的疗效降低可能主要与相关Ad5的预存免疫性（PEI）的大多数人口中。要通过解决的Ad5 PEI的担忧推动的Ad5载体疫苗的开发，创新的衣壳抗原掺入战略已经就业。通过这一战略的优点，的Ad5向量，我们首先构建了六邻体穿梭质粒HVR1-KWAS-HVR5-他6 / pH5S通过亚克隆高变区（HVR）1六邻体成以前构建穿梭质粒HVR5-他6 / pH5S，其中有他的6标签纳入HVR5。合成这种含有HIV抗原决定簇ELDKWAS HVR1 DNA片段。 HVR1-KWAS-HVR5-他6 / pH5S然后线性化和共转化用线性骨架质粒PAD5 /ΔH5（GL），用于同源重组。此重组质粒PAD5 / H5-HVR1-KWAS-HVR5-他6转染到细胞中，以产生病毒载体的Ad5 / H5-HVR1-KWAS-HVR5-他6。这个载体被验证，以具有通过病毒物理滴度（VP / ml）的指示的定性健身，感染滴度（IP / ml）和相应的VP / IP的比率。两者的HIV抗原决定簇和His 6标签进行表面暴露在Ad5的衣壳，并保留其自己的表位特异性的抗原性。一中和试验表明该向量价由Ad5的阳性血清体外规避中和能力。免疫的小鼠表现出强劲的体液免疫特定的生成艾滋病毒抗原表位和他的6。证明型原理这项研究表明，与衣壳抗原掺入战略相关联的协议，可以切实通过改变不同的衣壳蛋白用于中的Ad5载体疫苗的产生。这个协议甚至可以为further修改为稀土血清型腺病毒载体疫苗的产生。

所述抗原衣壳-团策略（ 图1A）被用于生成二价的Ad5病毒载体的Ad5 / H5-HVR1-KWAS-HVR5-他6。首先，穿梭质粒HVR1-KWAS-HVR5-他6 / pH5S被亚克隆HVR1-KWAS片段插入先前构建穿梭质粒HVR5-他6/17 pH5S构建。其次，质粒PAD5 / H5-HVR1-KWAS-HVR5-他6月 14日通过"的EcoRI-HVR1-KWAS-HVR5-的His6-PmeI位"的片段与SwaI位线性化PAD5 /ΔH5之间的同源重组构建（GL ）16（ ?...

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Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

在这里，我们提出了一个协议，以产生证明性的原则，二价5型腺病毒（Ad5的）载体的Ad5 / H5-HVR1-KWAS-HVR5-他6通过利用衣壳抗原掺入策略。此载体被证明表现出定性的健身，能力逃脱的Ad5阳性血清在体外 ，和抗原性以及免疫原性的结合抗原。

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

Adenovirus serotype 5 (Ad5) has been extensively modified with traditional transgene methods for the vaccine development. The reduced efficacies of these ...

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10.3K 次观看.  University of Alabama at Birmingham. 在这里，我们提出了一个协议，以产生证明性的原则，二价5型腺病毒（Ad5的）载体的Ad5 / H5-HVR1-KWAS-HVR5-他6通过利用衣壳抗原掺入策略。此载体被证明表现出定性的健身，能力逃脱的Ad5阳性血清在体外 ，和抗原性以及免疫原性的结合抗原。 

视频: Utilizing the Antigen Capsid-Incorporation Strategy for the Development of Adenovirus Serotype 5-Vectored Vaccine Approaches

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties1. These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors2. A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol1,2. Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling3, i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically &gt;80% for most AAV serotypes), or peptide display4,5, i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

工程和演变的合成腺相关病毒（AAV），基因治疗载体，通过DNA家族改组

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

科研

免疫与感染

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

生产的非复制型犬2型腺病毒衍生载体与纯化

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

基于人类腺病毒5型高容量腺病毒载体的克隆及规模化生产

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

生物工程

An Efficient Method for Adenovirus Production

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types and organs. However, recombinant adenoviral technology is laborious, time-consuming, and expensive. Here, we present an improved protocol using the pAdEasy system to obtain purified adenoviral particles that can induce a strong green fluorescent protein (GFP) expression in transduced cells. The advantages of this improved method are faster preparation and decreased production cost compared to the original method developed by Bert Vogelstein. The main steps of the adenoviral technology are: (1) the recombination of pAdTrack-GFP with the pAdEasy-1 plasmid in BJ5183 bacteria; (2) the packaging of the adenoviral particles; (3) the amplification of the adenovirus in AD293 cells; (4) the purification of the adenoviral particles from cell lysate and culture medium; and (5) the viral titration and functional testing of the adenovirus. The improvements to the original method consist of (i) the recombination in BJ5183-containing pAdEasy-1 by chemical transformation of bacteria; (ii) the selection of recombinant clones by &#8220;negative&#8221; and &#8220;positive&#8221; PCR; (iii) the transfection of AD293 cells using the K2 transfection system for adenoviral packaging; (iv) the precipitation with ammonium sulfate of the viral particles released by AD293 cells in cell culture medium; and (v) the purification of the virus by one-step cesium chloride discontinuous gradient ultracentrifugation. A strong expression of the gene of interest (in this case, GFP) was obtained in different types of transduced cells (such as hepatocytes, endothelial cells) from various sources (human, bovine, murine). Adenoviral-mediated gene transfer represents one of the main tools for developing modern gene therapies.

Here, we present a protocol for adenovirus production using the pAdEasy system. The technology includes the recombination of the pAdTrack and pAdEasy-1 plasmids, the adenovirus packaging and amplification, the purification of the adenoviral particles from cell lysate and culture medium, the viral titration, and the functional testing of the adenovirus.

腺病毒生产的有效方法

Adenoviral transduction has the advantage of a strong and transient induction of the expression of the gene of interest into a broad variety of cell types ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

与腺相关病毒的向量的生产、纯化和质量控制

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

神经科学

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants

Gene delivery vectors derived from Adeno-associated virus (AAV) are one of the most promising tools for the treatment of genetic diseases, evidenced by encouraging clinical data and the approval of several AAV gene therapies. Two major reasons for the success of AAV vectors are (i) the prior isolation of various naturally occurring viral serotypes with distinct properties, and (ii) the subsequent establishment of powerful technologies for their molecular engineering and repurposing in high throughput. Further boosting the potential of these techniques are recently implemented strategies for barcoding selected AAV capsids on the DNA and RNA level, permitting their comprehensive and parallel in vivo stratification in all major organs and cell types in a single animal. Here, we present a basic pipeline encompassing this set of complementary avenues, using AAV peptide display to represent the diverse arsenal of available capsid engineering technologies. Accordingly, we first describe the pivotal steps for the generation of an AAV peptide display library for the in vivo selection of candidates with desired properties, followed by a demonstration of how to barcode the most interesting capsid variants for secondary in vivo screening. Next, we exemplify the methodology for the creation of libraries for next-generation sequencing (NGS), including barcode amplification and adaptor ligation, before concluding with an overview of the most critical steps during NGS data analysis. As the protocols reported here are versatile and adaptable, researchers can easily harness them to enrich the optimal AAV capsid variants in their favorite disease model and for gene therapy applications.

Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus AAV Capsid Variants

AAV peptide display library generation and subsequent validation through the barcoding of candidates with novel properties for the creation of next-generation AAVs.

通过工程、条形码和筛选腺相关病毒 （AAV） 衣壳变体分离下一代基因治疗载体

Gene delivery vectors derived from Adeno-associated virus (AAV) are one of the most promising tools for the treatment of genetic diseases, evidenced by ...

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

使用捕获测定法检测基于病毒样颗粒（VLP）的疫苗上显示的抗原中和敏感表位

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...

Construction of Adenoviral Vectors using DNA Assembly Technology

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5' end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3' end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

In this study, an infectious clone of human adenovirus type 7 (HAdV-7) was constructed, and an E3-deleted HAdV-7 vector system was established by modifying the infectious clone. This strategy used here can be generalized to make gene transfer vectors from other wild-type adenoviruses.

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an ...

通过单步肝素亲和层析纯化 AAV 载体

Isolation of Adeno-Associated Viral Vectors Through a Single-Step and Semi-Automated Heparin Affinity Chromatography Protocol

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. However, the commonly used methods to purify AAVs make use of cesium chloride or iodixanol density gradient ultracentrifugation. Despite their advantages, these methods are time-consuming, have limited scalability, and often result in vectors with low purity. To overcome these constraints, researchers are turning their attention to chromatography techniques. Here, we present an optimized heparin-based affinity chromatography protocol that serves as a universal capture step for the purification of AAVs.
This method relies on the intrinsic affinity of AAV serotype 2 (AAV2) for heparan sulfate proteoglycans. Specifically, the protocol entails the co-transfection of plasmids encoding the desired AAV capsid proteins with those of AAV2, yielding mosaic AAV vectors that combine the properties of both parental serotypes. Briefly, after the lysis of producer cells, a mixture containing AAV particles is directly purified following an optimized single-step heparin affinity chromatography protocol using a standard fast protein liquid chromatography (FPLC) system. Purified AAV particles are subsequently concentrated and subjected to comprehensive characterization in terms of purity and biological activity. This protocol offers a simplified and scalable approach that can be performed without the need for ultracentrifugation and gradients, yielding clean and high viral titers.

作者聚焦：腺相关病毒载体生产和纯化的改进方法

This manuscript describes a detailed protocol for the generation and purification of adeno-associated viral vectors using an optimized heparin-based affinity chromatography method. It presents a simple, scalable, and cost-effective approach, eliminating the need for ultracentrifugation. The resulting vectors exhibit high purity and biological activity, proving their value in preclinical studies.

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

Adeno-associated virus (AAV) has become an increasingly valuable vector for in vivo gene delivery and is currently undergoing human clinical trials. ...

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

摘要

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此视频中的章节

工程和演变的合成腺相关病毒（AAV），基因治疗载体，通过DNA家族改组

生产的非复制型犬2型腺病毒衍生载体与纯化

基于人类腺病毒5型高容量腺病毒载体的克隆及规模化生产

腺病毒生产的有效方法

与腺相关病毒的向量的生产、纯化和质量控制

通过工程、条形码和筛选腺相关病毒（AAV）衣壳变体分离下一代基因治疗载体

使用捕获测定法检测基于病毒样颗粒（VLP）的疫苗上显示的抗原中和敏感表位

Construction of Adenoviral Vectors using DNA Assembly Technology

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

利用衣壳抗原掺入战略的腺病毒血清型5载体疫苗方法的发展

摘要

探索更多视频

此视频中的章节

工程和演变的合成腺相关病毒（AAV），基因治疗载体，通过DNA家族改组

生产的非复制型犬2型腺病毒衍生载体与纯化

基于人类腺病毒5型高容量腺病毒载体的克隆及规模化生产

腺病毒生产的有效方法

与腺相关病毒的向量的生产、纯化和质量控制

通过工程、条形码和筛选腺相关病毒 （AAV） 衣壳变体分离下一代基因治疗载体

使用捕获测定法检测基于病毒样颗粒（VLP）的疫苗上显示的抗原中和敏感表位

Construction of Adenoviral Vectors using DNA Assembly Technology

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

通过单步法和半自动肝素亲和层析方案分离腺相关病毒载体

通过工程、条形码和筛选腺相关病毒（AAV）衣壳变体分离下一代基因治疗载体