克隆和鉴定柔嫩艾美耳球虫编码配子体蛋白的基因及其与卵囊壁形成的关系。
Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation.
机构信息
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Ministry of Education Key Lab for Avian Preventive Medicine, Key Lab of Jiangsu Preventive Veterinary Medicine, College of Veterinary Medicine, Yangzhou University, 12 East Wenhui Road, Yangzhou, Jiangsu 225009, People's Republic of China.
出版信息
Parasit Vectors. 2014 Jan 15;7:27. doi: 10.1186/1756-3305-7-27.
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.
背景
艾美耳球虫（Eimeria）属的配子体蛋白是卵囊壁的重要组成部分，其中一些已被用于开发抗禽球虫病的传播阻断疫苗。
方法
用 RT-PCR 扩增和测序 cDNA 编码配子体蛋白，所用模板为从柔嫩艾美耳球虫配子体中分离的总 RNA，所用基因特异性引物。将 cDNA 克隆到细菌表达载体 pET28a(+)中，并在大肠杆菌 BL21 细胞中表达。通过 Western blot 和间接免疫荧光分析分别确定重组配子体蛋白的抗原性及其在不同柔嫩艾美耳球虫生活史阶段的定位。
结果
柔嫩艾美耳球虫 cDNA [GenBank：KF649255] 的 731 个核苷酸序列与柔嫩艾美耳球虫 Etgam22 的同源性为 97.7%。cDNA ORF 编码一个 186 个氨基酸的蛋白，含有组氨酸-脯氨酸丰富区。重组配子体蛋白（rEnGAM22）主要表达在不溶性包涵体中，并被来自用柔嫩艾美耳球虫、巨型艾美耳球虫和毒害艾美耳球虫卵囊免疫的鸡的抗血清识别。针对 rEnGAM22 蛋白的特异性抗体识别大配子体中的壁形成体和卵囊及孢子囊的壁。
结论
从柔嫩艾美耳球虫配子体中克隆的基因是柔嫩艾美耳球虫 Etgam22 的同源物，为未来抗球虫病重组亚单位疫苗提供了潜在的靶标。
Zotero 插件