Jm. Bumstead et al., NITROCELLULOSE IMMUNOBLOTTING FOR IDENTIFICATION AND MOLECULAR GENE CLONING OF EIMERIA-MAXIMA ANTIGENS THAT STIMULATE LYMPHOCYTE-PROLIFERATION, Clinical and diagnostic laboratory immunology, 2(5), 1995, pp. 524-530
An immunoblotting technique was used to identify lymphostimulatory ant
igens within sized polypeptide fractions Of Eimeria maxima sporozoites
, Six fractions contained polypeptides that specifically stimulated th
e proliferation of immune lymphocytes in an in vitro assay, and polycl
onal antisera were made in rabbits against these fractions, cDNA clone
s, isolated with antisera against a lymphostimulatory fraction of arou
nd 70 kDa, mere found to encode four different antigens including a cl
assical hsp70, a molecule homologous to an endoplasmic reticulum chape
ronin (BIP/GRP), and a calcium-dependent serine/threonine protein kina
se that appears homologous to a recently described molecule from Plasm
odium falciparum, The protein kinase cDNA clone was overexpressed in E
scherichia coli, and the recombinant antigen was found to induce both
antibody and lymphoproliferative responses in chickens when administer
ed subcutaneously, Thus, immunoblotting, in combination with in vitro
lymphoproliferation assays, can be used as an initial screen for the i
dentification of lymphostimulatory antigens from a complex pool of pol
ypeptides, and a combination of cDNA cloning, expression, and immuniza
tion allows assessment of the lymphostimulatory activity of individual
polypeptides. These studies should facilitate further evaluation of a
ntigens that are potential candidates for inclusion in a recombinant v
accine against poultry coccidiosis.