The display of full-length antibody on the cell surface was achieved by fusing a transmembrane domain of the platelet-derived growth factor receptor (PDGFR) to the C-terminus of the heavy chain constant region. We also incorporated a furin cleavage site between the constant region and PDGFR transmembrane domain to obtain secreted antibodies. As a result, antibodies can be expressed simultaneously on the cell surface in a membrane-anchored version for screening and selecting through fluorescence-activated cell sorting (FACS) analysis, as well as in conditioned medium in a secreted version for function analysis.
View Article and Find Full Text PDFInflammatory response plays an important role in the pathogenesis of secondary damage after traumatic brain injury (TBI). The inflammasome is a multiprotein complex involved in innate immunity and a number of studies have suggested that the inflammasome plays a critical role in a host inflammatory signaling. Nucleotide-binding domain, leucine-rich repeat, pyrin domain containing 3 (NLRP3) is a key component of the NLRP3-inflammasome, which also includes apoptotic speck-containing protein (ASC) with a cysteine protease (caspase)-activating recruitment domain and pro-caspase1.
View Article and Find Full Text PDFDespite numerous researches and improvements in the past few years, the precise mechanisms underlying secondary brain injury after trauma remain obscure. Iron is essential for almost all types of cells, including nerve cells. However, excess of iron has been proved to contribute to the brain injury following trauma in animal models.
View Article and Find Full Text PDFBackground: Traumatic brain injury (TBI) can cause gastrointestinal dysfunction and increase intestinal permeability. Nuclear factor kappa B (NF-κB) has been shown to be associated with these intestinal events, but it is not well known how NF-κB is activated in the intestine after TBI. Based on previous studies, we hypothesize that myeloid differentiation primary response protein 88 (Myd88) may have an important role in NF-κB activation in the intestine, which mediates the inflammation and ultimately results in acute intestinal mucosal injury.
View Article and Find Full Text PDFHepatitis B immunoglobulin (HBIG) is important in the management of hepatitis B virus (HBV) infection. Aiming to develop recombinant monoclonal antibodies as an alternative to HBIG, we report the successful identification of HBV surface antigen (HBsAg)-specific antibodies from a full-length human antibody library displayed on mammalian cell surface. Using total RNA of peripheral blood mononuclear cells of a natively immunized donor as template, the antibody repertoire was amplified.
View Article and Find Full Text PDFNan Fang Yi Ke Da Xue Xue Bao
August 2011
Objective: To construct a rheumatoid arthritis-specific full-length fully human mammalian display antibody libraries.
Methods: Peripheral blood lymphocytes were isolated from patients with rheumatoid arthritis. The repertoires of kappa light chain (LCκ) and heavy chain variable region (VH) of the antibodies were amplified by RT-PCR.
Nan Fang Yi Ke Da Xue Xue Bao
February 2011
Objective: To construct a mammalian cell surface display library of full-length human antibodies.
Methods: The total RNA was isolated from human peripheral blood mononuclear cells (PBMCs), and the genes encoding the heavy chain variable regions and kappa light chains (VH and Cκ) of the antibodies were amplified by RT-PCR. The amplified VH and Cκ gene sequences were separately inserted into the vector pDGB-HC-TM.
Acta Biochim Biophys Sin (Shanghai)
March 2011
A unique four-way ligation strategy was developed for rapid construction of a full-length antibody library. A mammalian expression vector was constructed that contained dual mammalian expression cassettes and sequences recognized by the unique restriction enzymes BsmBI, BstXI, and SfiI. Both full-length light-chain and variable domain of heavy-chain genes were inserted into the vector in one step by four-way ligation, and full-length bivalent antibodies were displayed on mammalian cell surfaces.
View Article and Find Full Text PDFActa Biochim Biophys Sin (Shanghai)
August 2010
Development of a versatile mammalian display system is essential for the selection of functional human antibodies with high affinities. Here we described a novel strategy for rapid construction of full-length antibody libraries that could be efficiently expressed on mammalian cell surfaces. The universal vector pDGB-HC-TM was constructed by inserting multiple cloning site unique sequences recognized by restriction endonucleases BsmBI, SfiI, and BstXI for the pop-in and pop-out of genes of interest.
View Article and Find Full Text PDFObjective: To construct human renal cell carcinoma patient-specific full-length antibody library using mammalian cell surface display technique.
Methods: Peripheral blood mononuclear cells (PBMC) were isolated from patients with renal cell carcinoma. The repertoires of kappa light chain (LCkappa) and heavy chain variable region (VH) of antibody were amplified by RT-PCR.
Nan Fang Yi Ke Da Xue Xue Bao
January 2010
Objective: To study the effects of UV irradiation on DNA ligation and transformation efficiency of the expression vector into competent bacterial cells.
Methods: The expression vector was digested with the restriction enzyme SfiI, and the purified target DNA fragments were exposed to UV light at different wavelengths. Ligation and transformation experiments with the exposed fragments were carried out and the colony number and transformation efficiency were assessed.