Cloning, structural analysis, and expression of the pig IgE ε chain

Abstract

As a step in the evolutionary studies of immunoglobulin E (IgE) and for the purpose of developing new reagents that will facilitate a more detailed analysis of IgE-mediated inflammatory reactions in a large animal model, we here present the cloning of the epsilon chain of IgE in the domestic pig (Sus scrufa). A partial cDNA clone for the epsilon chain of pig IgE was isolated by polymerase chain reaction (PCR) amplification using degenerate primers directed against conserved regions in the second (CH2) and the fourth (CH4) constant domains of IgE. cDNA derived from mRNA isolated from the spleen and lymph nodes of a pig actively sensitized with a protein extract from the nematode Ascaris suum was used as template. Screening of a spleen cDNA library with the partial cDNA clone as probe resulted in isolation of a clone that contained the entire coding region. The nucleotide sequence was determined and was found to conform with the previously identified mammalian epsilon-chain sequences. The highest degree of similarity was found to sheep IgE. A DNA construct encoding a baculovirus signal sequence, a histidine hexapeptide, and the CH2-CH3-CH4 domains of the pig IgE epsilon chain was obtained by PCR amplification. The construct was ligated into the baculovirus expression vector pVL1392. Infection of High Five insect cells with recombinant baculovirus resulted in expression and secretion of a soluble 6 x His-CH2-CH3-CH4 protein product.

Heavy Chain V Region Diversity in the Duck-Billed Platypus (Ornithorhynchus anatinus): Long and Highly Variable Complementarity-Determining Region 3 Compensates for Limited Germline Diversity

“…The remaining three clones have one cysteine in the CDR2 and one in the CDR3, suggesting a cysteine bridge formation between the CDR2 and CDR3. The role of a single additional cysteine residue is unknown; however, we favor the explanation that these clones are nonfunctional byproducts of the variability-generating mechanism, as free unpaired cysteine residues often lead to uncontrolled complex formations by binding to free cysteines in other molecules (41).…”

The evolution of the Th2 immune responses and its relationships with parasitic diseases and allergy

“…En la inmunidad Th2, la IgE es tal vez la de aparición más reciente en la escala evolutiva (figura 1); como se ha mencionado, se ha detectado sólo en los mamíferos (8)(9)(10). En los monotremas se confirmó la presencia del locus c épsilon, lo cual sugiere que la divergencia de la IgE de otro gen más primitivo sucedió antes de la división de los mamíferos en sus tres ramas evolutivas (27).…”

“…Según la información disponible, la inflamación alérgica mediada por IgE es exclusiva de los mamíferos (8)(9)(10); este anticuerpo es filogenética-mente cercano a la IgY, una inmunoglobulina detectada en reptiles y aves, que interviene en reacciones de tipo anafiláctico en respuesta a antígenos de origen protozoario (11).…”

Lepidopteran cells, an alternative for the production of recombinant antibodies?

“…18,40,[43][44][45][46] It is very easy and fast to generate recombinant viruses expressing H or L bearing specific mutations and to determine the impact of these mutations on the specificity or activity of a given antibody. 21,22,41 This strategy has been successfully used to analyze the catalytic activity of the 6D9 monoclonal antibody and the specificity of the PAC1 antibody that binds to the integrin αII b β3 on activated platelets.…”

“…Several immunoglobulin isotypes from many different species, including murine IgG, 2,3,[21][22][23][24][25] chimeric IgG, [26][27][28][29][30] human IgG, [31][32][33][34][35][36][37][38][39] IgA, [40][41][42] IgM 41 and IgE, [43][44][45] as well as pig Igs 30,45 have been successfully expressed in insect cells. These molecules are all correctly processed (signal peptide cleaved), glycosylated and assembled (H2L2) with a production rate varying from 0.75 to 100 mg/l, depending on the antibody ( …”