Lillehoj H S, Choi K D, Jenkins M C, Vakharia V N, Song K D, Han J Y, Lillehoj E P
Immunology and Disease Resistance Laboratory, Livestock and Poultry Sciences Institute, BARC-East, U.S. Department of Agriculture, Beltsville, MD 20705, USA.
Avian Dis. 2000 Apr-Jun;44(2):379-89.
A rabbit antiserum against an 18- to 27-kD native protein fraction (F3) from Eimeria acervulina merozoites identified a cDNA (3-1E) containing a 1086-base pair insertion with an open reading frame of 170 amino acids (predicted molecular weight, 18,523). The recombinant 3-1E cDNA expressed in Escherichia coli produced a 60-kD fusion protein and a 23-kD protein after factor Xa treatment of the fusion protein. Both proteins were reactive with the F3 antiserum by western blot analysis. A rabbit antiserum against a synthetic peptide deduced from the amino acid sequence of the 3-1E cDNA reacted with a 27-kD recombinant 3-1E protein expressed in Sf9 insect cells and a 20-kD native protein expressed by E. acervulina sporozoites and Eimeria tenella sporozoites and merozoites. By immunofluorescence staining, a monoclonal antibody produced against the recombinant 3-1E protein reacted with sporozoites and merozoites of E. acervulina, E. tenella, and Eimeria maxima. Spleen lymphocytes from E. acervulina-immune chickens showed antigen-specific proliferation and interferon (IFN)-gamma production upon stimulation with the recombinant 3-1E protein, indicating that the protein activates cell-mediated immunity during coccidiosis. Immunization of chickens with either the E. coli- or Sf9-expressed recombinant 3-1E protein with adjuvant, or direct injection of the 3-1E cDNA, induced protective immunity against live E. acervulina. Simultaneous injection of the recombinant 3-1E protein, or the 3-1E cDNA, with cDNAs encoding chicken IFN-gamma or interleukin (IL)-2/15 further enhanced protective immunity. These results indicate that the recombinant E. acervulina 3-1E cDNA or its polypeptide product may prove useful as vaccines against avian coccidiosis.
针对来自堆型艾美耳球虫子孢子18至27千道尔顿天然蛋白组分（F3）的兔抗血清鉴定出一个cDNA（3 - 1E），其含有一个1086碱基对的插入片段，开放阅读框为170个氨基酸（预测分子量为18,523）。在大肠杆菌中表达的重组3 - 1E cDNA产生了一个60千道尔顿的融合蛋白，该融合蛋白经因子Xa处理后产生一个23千道尔顿的蛋白。通过蛋白质免疫印迹分析，这两种蛋白均与F3抗血清发生反应。针对从3 - 1E cDNA氨基酸序列推导的合成肽的兔抗血清，与在Sf9昆虫细胞中表达的27千道尔顿重组3 - 1E蛋白以及堆型艾美耳球虫子孢子、柔嫩艾美耳球虫子孢子和裂殖子表达的20千道尔顿天然蛋白发生反应。通过免疫荧光染色，针对重组3 - 1E蛋白产生的单克隆抗体与堆型艾美耳球虫、柔嫩艾美耳球虫和巨型艾美耳球虫的子孢子和裂殖子发生反应。来自堆型艾美耳球虫免疫鸡的脾淋巴细胞在用重组3 - 1E蛋白刺激后显示出抗原特异性增殖和γ干扰素（IFN - γ）产生，表明该蛋白在球虫病期间激活细胞介导的免疫。用佐剂处理的大肠杆菌或Sf9表达的重组3 - 1E蛋白免疫鸡，或直接注射3 - 1E cDNA，均可诱导针对活的堆型艾美耳球虫的保护性免疫。将重组3 - 1E蛋白或3 - 1E cDNA与编码鸡IFN - γ或白细胞介素（IL）-2/15的cDNA同时注射可进一步增强保护性免疫。这些结果表明，重组堆型艾美耳球虫3 - 1E cDNA或其多肽产物可能作为抗禽球虫病疫苗具有应用价值。
