原標題：通過與主要樺樹花粉過敏原Bet v 1共享相同可變區(qū)域的管道克隆快速產(chǎn)生IgE、IgG1和IgG4
　　理由：用單一主要過敏原Bet v 1進行樺樹花粉過敏是研究IgE和IgG1或IgG4在致敏和過敏原免疫治療中的分子相互作用的范例。然而現(xiàn)在缺乏這種抗體。我們這里的目的是通過使用聚合酶不完全引物延伸（PIPE）克隆（Ilieva等，2017）來為我們的抗體管道提供燃料，這是快速創(chuàng)建共享相同可變區(qū)的不同類別抗體的最新方法。
　　方法：文獻中的可變區(qū)域序列（Levin等人，2014年）被用于通過管道克隆為IgE、IgG1和IgG4創(chuàng)建載體編碼。這些自組裝質(zhì)粒隨后在Expi293F系統(tǒng)中以30 ml的細胞培養(yǎng)體積表達。重組抗體通過親和色譜純化，并在SDS-PAGE中進行完整性測試。通過BCA蛋白質(zhì)測定法測定濃度。通過斑點印跡雜交和ISAC112微陣列測定Bet v 1的特異性。
　　結(jié)果：Expi293F細胞中所有三種抗體類別的表達導致從0.9mg（IgE）至6和4.4mg（分別為IgG1和IgG4）的高產(chǎn)量。PIPE克隆正確地組裝了IgE，IgG1和IgG4抗體，特異性識別Bet v 1。
　　結(jié)論：管道克隆是一種高效的生產(chǎn)Bet v 1重組抗體的方法。由于這些抗體屬于不同的類別，但共享相同的可變區(qū)域，因此它們將是研究1型過敏和過敏原免疫治療機制的有用工具。
延伸閱讀
JACI
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.
　　創(chuàng)建過敏性疾病的科研、科普知識交流平臺，為過敏患者提供專業(yè)診斷、治療、預防的共享平臺。