Cloning and characterization of an Eimeria necatrix gene encoding a gametocyte protein and associated with oocyst wall formation

Particle and Fibre Toxicology

Jianping Tao¹  Lili Wu¹  Jingui Li¹  Wenjie Jin¹  Jinjun Xu¹  Shijie Su¹  Changjing Deng¹  Yulan Zhu¹  Liqin Cao¹  Dandan Liu¹ 

[1] 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

关键词: Immunolocalization;    Cloning and expression;    Gene;    Gametocyte protein;    Eimeria;   

Others  :  823003
DOI  :  10.1186/1756-3305-7-27

 received in 2013-08-25, accepted in 2014-01-02,  发布年份 2014

【 摘 要 】

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.

【 授权许可】

   
2014 Liu et al.; licensee BioMed Central Ltd.

【 图 表 】

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