杭州浙大迪迅生物基因工程有限公司JACI：通过Bet v 1共享相同可变区域的管道克隆快速产生IgE、IgG1
发布日期：2019-06-26
原标题：通过与主要桦树花粉过敏原Bet v 1共享相同可变区域的管道克隆快速产生IgE、IgG1和IgG4
方法：文献中的可变区域序列（Levin等人，2014年）被用于通过管道克隆为IgE、IgG1和IgG4创建载体编码。这些自组装质粒随后在Expi293F系统中以30 ml的细胞培养体积表达。重组抗体通过亲和色谱纯化，并在SDS-PAGE中进行完整性测试。通过BCA蛋白质测定法测定浓度。通过斑点印迹杂交和ISAC112微阵列测定Bet v 1的特异性。
结果：Expi293F细胞中所有三种抗体类别的表达导致从0.9mg（IgE）至6和4.4mg（分别为IgG1和IgG4）的高产量。PIPE克隆正确地组装了IgE，IgG1和IgG4抗体，特异性识别Bet v 1。
结论：管道克隆是一种高效的生产Bet v 1重组抗体的方法。由于这些抗体属于不同的类别，但共享相同的可变区域，因此它们将是研究1型过敏和过敏原免疫治疗机制的有用工具。
延伸阅读
JACI
DOI: org/10.1016/j.jaci.2018.12.571
Abstract:
RATIONALE: Birch pollen allergy with the single major allergen Bet v 1 is a paradigm to study the molecular interplay of IgE and IgG1 or IgG4 in sensitization and allergen immunotherapy. However, there is a lack in the availability of such antibodies. We here aimed to fuel our antibody PIPEline by using Polymerase Incomplete Primer Extension (PIPE) cloning (Ilieva et al., 2017), the latest method for fast creation of different classes of antibodies sharing the same variable region.
METHODS: Variable region sequences from the literature (Levin et al.,2014) were used to create vectors coding for IgE, IgG1 and IgG4 via PIPE cloning. These self-assembled plasmids were then expressed in the Expi293F system at a cell culture volume of 30 ml. Recombinant antibodies were purified via affinity chromatography and integrity tested in SDS-PAGE. Concentration was determined via BCA protein assay.Specificity to Bet v 1 was determined by dot blot and ISAC112 microarray.
RESULTS: Expression of all three antibody classes in Expi293F cells resulted in high yields from 0.9 mg (IgE) to 6 and 4.4 mg (IgG1 and IgG4,respectively). PIPE cloning delivered correctly assembled IgE, IgG1 and IgG4 antibodies specifically recognizing Bet v 1.
CONCLUSIONS: PIPE cloning proves to be a highly efficient method for the production of recombinant antibodies against Bet v 1. As these antibodies belong to different classes but share the same variable region they will be useful tools for researching mechanisms underlying type 1 allergy as well as allergen immunotherapy.
First Author:
Jill A. Poole
Correspondence:
Nebraska Medical Center, Omaha, NE 68198.
All Authors:
Jill A. Poole, Charles S. Barnes, Jeffrey G. Demain, Jonathan A. Bernstein, Mahesh A. Padukudru, William J. Sheehan, Guillermo Guidos Fogelbach, James Wedner, Rosa Codina, Estelle Levetin, John R. Cohn, Steve Kagen, Jay M. Portnoy, Andre E.
——浙大迪迅 译
理由：用单一主要过敏原Bet v 1进行桦树花粉过敏是研究IgE和IgG1或IgG4在致敏和过敏原免疫治疗中的分子相互作用的范例。然而现在缺乏这种抗体。我们这里的目的是通过使用聚合酶不完全引物延伸（PIPE）克隆（Ilieva等，2017）来为我们的抗体管道提供燃料，这是快速创建共享相同可变区的不同类别抗体的最新方法。方法：文献中的可变区域序列（Levin等人，2014年）被用于通过管道克隆为IgE、IgG1和IgG4创建载体编码。这些自组装质粒随后在Expi293F系统中以30 ml的细胞培养体积表达。重组抗体通过亲和色谱纯化，并在SDS-PAGE中进行完整性测试。通过BCA蛋白质测定法测定浓度。通过斑点印迹杂交和ISAC112微阵列测定Bet v 1的特异性。
结果：Expi293F细胞中所有三种抗体类别的表达导致从0.9mg（IgE）至6和4.4mg（分别为IgG1和IgG4）的高产量。PIPE克隆正确地组装了IgE，IgG1和IgG4抗体，特异性识别Bet v 1。
结论：管道克隆是一种高效的生产Bet v 1重组抗体的方法。由于这些抗体属于不同的类别，但共享相同的可变区域，因此它们将是研究1型过敏和过敏原免疫治疗机制的有用工具。
延伸阅读
JACI
[IF:13.258]
Fast generation of IgE, IgG cloning sharing the same variable region to 1 and IgG4 by PIPE the major birch pollen allergen Bet v 1 DOI: org/10.1016/j.jaci.2018.12.571
Abstract:
RATIONALE: Birch pollen allergy with the single major allergen Bet v 1 is a paradigm to study the molecular interplay of IgE and IgG1 or IgG4 in sensitization and allergen immunotherapy. However, there is a lack in the availability of such antibodies. We here aimed to fuel our antibody PIPEline by using Polymerase Incomplete Primer Extension (PIPE) cloning (Ilieva et al., 2017), the latest method for fast creation of different classes of antibodies sharing the same variable region.
METHODS: Variable region sequences from the literature (Levin et al.,2014) were used to create vectors coding for IgE, IgG1 and IgG4 via PIPE cloning. These self-assembled plasmids were then expressed in the Expi293F system at a cell culture volume of 30 ml. Recombinant antibodies were purified via affinity chromatography and integrity tested in SDS-PAGE. Concentration was determined via BCA protein assay.Specificity to Bet v 1 was determined by dot blot and ISAC112 microarray.
RESULTS: Expression of all three antibody classes in Expi293F cells resulted in high yields from 0.9 mg (IgE) to 6 and 4.4 mg (IgG1 and IgG4,respectively). PIPE cloning delivered correctly assembled IgE, IgG1 and IgG4 antibodies specifically recognizing Bet v 1.
CONCLUSIONS: PIPE cloning proves to be a highly efficient method for the production of recombinant antibodies against Bet v 1. As these antibodies belong to different classes but share the same variable region they will be useful tools for researching mechanisms underlying type 1 allergy as well as allergen immunotherapy.
First Author:
Jill A. Poole
Correspondence:
Nebraska Medical Center, Omaha, NE 68198.
All Authors:
Jill A. Poole, Charles S. Barnes, Jeffrey G. Demain, Jonathan A. Bernstein, Mahesh A. Padukudru, William J. Sheehan, Guillermo Guidos Fogelbach, James Wedner, Rosa Codina, Estelle Levetin, John R. Cohn, Steve Kagen, Jay M. Portnoy, Andre E.
2019-05-08 Article
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