کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
8418055 | 1545712 | 2013 | 9 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
A mammalian expression system for high throughput antibody screening
ترجمه فارسی عنوان
یک سیستم بیان کننده پستانداران برای غربالگری آنتی بادی بالا
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کلمات کلیدی
HTPscFvEBNA-1IRESSingle chain Fv - Fv تنها زنجیره ایMammalian expression - بیان پستاندارانHigh throughput - توان بالاinternal ribosome entry site - سایت ورودی ریبوزوم داخلیCMV - سیتومگالوویروسhigh throughput screening - غربالگری بالاFab - فابfragment antigen-binding - قطعه آنتیژن اتصالPhage display - نمایش فاژی
موضوعات مرتبط
علوم زیستی و بیوفناوری
بیوشیمی، ژنتیک و زیست شناسی مولکولی
بیوتکنولوژی یا زیستفناوری
چکیده انگلیسی
We describe herein a method to enable high throughput (HTP) screening of libraries of soluble proteins such as phage-derived clones of IgG, scFv-Fc, or other Fc-fusion proteins expressed in mammalian cells via adenovirus transduction. DNA fragments of antibody single chains (scFvs) and fragment antigen-binding (Fabs) from the positive clones of the third round of bacteriophage panning against a target antigen were batch reformatted into scFv-Fc or IgG in an oriP bearing entry vector and then recombined to an adenovirus vector through Gateway technology. The resulting antibody gene-containing adenovirus libraries were added to 96-well plates seeded with mammalian cells at a ratio of 0.7 infectious viral particles per well to establish clonality. Protocol optimization improved the expression of scFv-Fc and IgGs up to 100 μg/mL in 96-well plates, which is sufficient for most antibody characterizations. In addition, 78% of the wells that were positive for protein expression contain only one sequence, indicating successful establishment of clonality in a majority of wells. We have established and optimized a mammalian expression system that produces soluble protein variants in a HTP manner. The system will facilitate developing multiple downstream screening methodologies.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Immunological Methods - Volume 395, Issues 1â2, 30 September 2013, Pages 45-53
Journal: Journal of Immunological Methods - Volume 395, Issues 1â2, 30 September 2013, Pages 45-53
نویسندگان
Linda Xu, Xiaofang Jin, G. Jonah Rainey, Herren Wu, Changshou Gao,