Bumstead J M, Dunn P P, Tomley F M
Institute for Animal Health, Compton, Newbury, Berkshire, United Kingdom.
Clin Diagn Lab Immunol. 1995 Sep;2(5):524-30. doi: 10.1128/cdli.2.5.524-530.1995.
An immunoblotting technique was used to identify lymphostimulatory antigens within sized polypeptide fractions of Eimeria maxima sporozoites. Six fractions contained polypeptides that specifically stimulated the proliferation of immune lymphocytes in an in vitro assay, and polyclonal antisera were made in rabbits against these fractions. cDNA clones, isolated with antisera against a lymphostimulatory fraction of around 70 kDa, were found to encode four different antigens including a classical hsp70, a molecule homologous to an endoplasmic reticulum chaperonin (BiP/GRP), and a calcium-dependent serine/threonine protein kinase that appears homologous to a recently described molecule from Plasmodium falciparum. The protein kinase cDNA clone was overexpressed in Escherichia coli, and the recombinant antigen was found to induce both antibody and lymphoproliferative responses in chickens when administered subcutaneously. Thus, immunoblotting, in combination with in vitro lymphoproliferation assays, can be used as an initial screen for the identification of lymphostimulatory antigens from a complex pool of polypeptides, and a combination of cDNA cloning, expression, and immunization allows assessment of the lymphostimulatory activity of individual polypeptides. These studies should facilitate further evaluation of antigens that are potential candidates for inclusion in a recombinant vaccine against poultry coccidiosis.
采用免疫印迹技术在巨型艾美耳球虫子孢子的分级多肽组分中鉴定淋巴刺激抗原。六个组分含有在体外试验中能特异性刺激免疫淋巴细胞增殖的多肽，并以此制备了兔多克隆抗血清。用针对约70 kDa淋巴刺激组分的抗血清分离出的cDNA克隆，被发现编码四种不同抗原，包括一种典型的热休克蛋白70、一种与内质网伴侣蛋白（BiP/GRP）同源的分子，以及一种钙依赖性丝氨酸/苏氨酸蛋白激酶，该激酶似乎与最近描述的恶性疟原虫分子同源。蛋白激酶cDNA克隆在大肠杆菌中过量表达，重组抗原皮下注射给鸡时，被发现能诱导抗体和淋巴细胞增殖反应。因此，免疫印迹与体外淋巴细胞增殖试验相结合，可作为从复杂多肽库中鉴定淋巴刺激抗原的初步筛选方法，而cDNA克隆、表达和免疫相结合则可评估单个多肽的淋巴刺激活性。这些研究应有助于进一步评估作为抗家禽球虫病重组疫苗潜在候选抗原。
