NITROCELLULOSE IMMUNOBLOTTING FOR IDENTIFICATION AND MOLECULAR GENE CLONING OF EIMERIA-MAXIMA ANTIGENS THAT STIMULATE LYMPHOCYTE-PROLIFERATION

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.