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首页> 外文期刊>Parasites Vectors >Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation
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Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation

机译：编码配子体蛋白并与卵囊壁形成有关的Eimeria necatrix基因的克隆和鉴定

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Background Gametocyte proteins of Eimeria (E.) spp. are important components of the oocyst wall and some have been used to develop transmission-blocking vaccines against avian coccidiosis. Methods Total RNA isolated from E. necatrix gametocytes was utilized as templates for RT-PCR amplification and sequencing of cDNA encoding a gametocyte protein using gene-specific primers. The cDNA was cloned into the bacterial expression vector pET28a(+) and expressed in E. coli BL21 cells. The antigenicity of the recombinant gametocyte protein and its localization in different E. necatrix life-cycle stages were determined by western blot and indirect immunofluorescence analyses, respectively. Results A 731-nucleotide sequence of cDNA [GenBank: KF649255] of E. necatrix had 97.7% identity to that of Etgam22 of E. tenella. The cDNA ORF encoded a 186-amino acid protein containing a histidine-proline-rich region. The recombinant gametocyte protein (rEnGAM22) was predominately expressed in the insoluble inclusion body and recognized by antiserum from chickens immunized with oocysts of E. necatrix, E. maxima and E. tenella. A specific antibody to the rEnGAM22 protein recognized the wall-forming bodies in macrogametocytes and the walls of oocysts and sporocysts. Conclusions The gene cloned from E. necatrix gametocytes is an ortholog to Etgam22 of E. tenella and presents a potential target for future recombinant subunit vaccines against coccidiosis.
机译：背景Eimeria（E.）spp。的配子体蛋白质。是卵囊壁的重要组成部分，有些已被用于开发抗禽球虫病的传递阻断疫苗。方法以分离自大肠杆菌的配子细胞中的总RNA为模板，使用基因特异性引物进行RT-PCR扩增和编码配子细胞蛋白的cDNA测序。将该cDNA克隆到细菌表达载体pET28a（+）中，并在大肠杆菌BL21细胞中表达。分别通过蛋白质印迹和间接免疫荧光分析来确定重组配子细胞蛋白的抗原性和其在不同E. necatrix生命周期阶段的定位。结果necatrix的cDNA [GenBank：KF649255]的731个核苷酸序列与tenella的Etgam22的cDNA有97.7％的同一性。 cDNA ORF编码包含富含组氨酸-脯氨酸的区域的186个氨基酸的蛋白质。重组配子细胞蛋白（rEnGAM22）主要在不溶性包涵体中表达，并被抗中性肠球菌，最大肠球菌和大肠球菌卵囊免疫的鸡的抗血清识别。 rEnGAM22蛋白的特异性抗体识别巨配子细胞的壁形成体以及卵囊和孢子囊的壁。结论从猪中肠球菌配体细胞中克隆的基因是与大肠杆菌中Etgam22的直系同源基因，为将来的抗球虫病重组亚单位疫苗提供了潜在的靶标。

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