![]() The full-length cDNA was then generated by long distance PCR using Platinum Taq DNA polymerase High Fidelity (Invitrogen, USA) with primers designed from the distal ends of both 5'- and 3'-RACE products : 5'-GTCGCCAGTAAATCCCAAGA-3' (sense, within the 5'-UTR) and 5'-GCACCTCAATCTCCACCACT-3' (antisense, 3'UTR). Total RNA from PMA-stimulated PBMCs was reverse transcribed using Improm II (Promega, USA). Miniprep were obtained from colonies grown in 5 ml LB-kanamycin broth and the clones were sequenced on the ABI-3730 Genetic Analyzer using the Big Dye terminator chemistry (Applied Biosystems, USA). The 5'- and 3'-RACE products were gel-purified using the S.N.A.P.™ Gel Purification Kit (Invitrogen, USA), TA-cloned into pCRII-TOPO (Invitrogen, USA) and seeded on kanamycin IPTG plates. ![]() ![]() Reverse transcription and polymerase chain reactions (PCR) were carried out according to the instruction manual of the SMART RACE cDNA Amplification Kit. For first-strand cDNA synthesis, and according to the sequence of bovine CD11a available, gene-specific primers were designed which were expected to give non overlapping ~1 kb RACE products: a sense primer for the 3'-RACE PCR : 5'-TGCAATGTRAGCTCTCCCATCTTC-3' (corresponding to nt 2572 to nt 2595) and an antisense primer for the 5'-RACE PCR : 5'-CCGGCCTCCTCTCTGCTCCCCATAG-3' (nt 1470 to nt 1446). We used SMART RACE technology (Clontech Laboratories Inc., USA) to obtain caprine CD11a (CaCD11a) 5'- and 3'-ends and RT-PCR to amplify full-length CaCD11a CDS. The PBMCs were obtained by density gradient centrifugation with Ficoll-Paque Plus (Amersham, USA) and maintained in RPMI 1640 supplemented with 10% foetal bovine serum (Gibco BRL, USA), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37☌ in a 5% CO 2 atmosphere. Total RNA from phorbol myristate acetate (PMA)-stimulated (25 ng/ml for 15 min) caprine ( Boer breed) peripheral blood mononuclear cells (PBMC) was extracted with TRIzol (Invitrogen, USA) as described by the manufacturer. Along with the caprine CD18-encoding cDNA, which is available for a few months, the sequence data provided here will allow the Capra hircus β 2-integrin CD11a/CD18 expression in vitro as a tool to examine the specificities of inflammation in the caprine species. As the relevance of the goat model for studying leukocyte traffic, diapedesis and pathologic tissue infiltration is well established in such important areas as mastitis or lentivirus-associated diseases, increasing our knowledge about caprine β 2 integrins is of great importance to offer new possibilities for research and to provide additional insights into those fields. ![]() The adhesion process mediated is a critical step of a wide range of immunological activities, including cytolysis of target cells, cross-interaction and cross-stimulation between lymphocytes, phagocytosis of complement-coated targets, neutrophils clearance from inflamed tissue, and the regulation of leukocyte traffic between the bloodstream and tissues. The heterodimer CD11a/CD18 is expressed on all leukocytes and mediates high affinity adhesion to a variety of cell types that express one or more of the β 2-integrins ligands, intercellular adhesion molecules (ICAM-1 to -5). The bulk of each integrin subunit is extracellular, where it typically functions as a receptor for extracellular matrix molecules or as a counterreceptor for surface proteins of apposed cells. Integrins consist of a 120 to 180 kDa α subunit (CD11a in this case) and a 90 to 110 kDa β subunit that are noncovalently associated single-pass transmembrane proteins. Lymphocyte function-associated antigen-1 (LFA-1, α Lβ 2, CD11a/CD18) is a member of the β 2-integrin subfamily of cell surface receptors.
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