Cloning and expression of Neospora caninum dense-granule 7 in E. coli

Neospora caninum is an obligate intracellular protozoan which induces abortion, still birth and neuromuscular disorders in cattle and is an important problem in dairy and beef industry worldwide. The dense granule protein 7 (GRA7) of N. caninum is an immune-dominant protein shared by both tachyzoite and bradyzoites. This study was conducted to produce recombinant GRA7 of N. caninum using a plasmid with high level of expression of this protein in E. coli. For this purpose, a segment of N. caninum DNA corresponding to GRA7 was amplified using PCR. After sequencing, this fragment was cloned into expression vector pMAL-c2X under the control of the lac promoter. Expression of this plasmid in E. coli strain TG1 was identified by western blotting. In this study, pMAL-c2X had a strong promoter to produce high level expression of NcGRA7. This result revealed that this recombinant protein with pMAL-c2X vector may be suitable for developing of diagnostic procedures.

[1]  A. Hemphill,et al.  Differential expression of cell surface- and dense granule-associated Neospora caninum proteins in tachyzoites and bradyzoites. , 1998, The Journal of parasitology.

[2]  J. Dubey,et al.  Review of Neospora caninum and neosporosis in animals. , 2003, The Korean journal of parasitology.

[3]  M. H. Hajikolaei,et al.  Serological study of Neospora caninum infection in cattle from Ahvaz area, Iran , 2008 .

[4]  L. Sibley,et al.  Comparison of the major antigens of Neospora caninum and Toxoplasma gondii. , 1999, International journal for parasitology.

[5]  J. Fernández,et al.  Serological diagnosis of Bovine Neosporosis in farms from Tucacas, Falcon state, Venezuela. , 2013 .

[6]  P. Conrad,et al.  Neosporosis in dairy cattle. , 2012, The Japanese journal of veterinary research.

[7]  L. F. Gondim,et al.  IMPROVED PRODUCTION OF NEOSPORA CANINUM OOCYSTS, CYCLICAL ORAL TRANSMISSION BETWEEN DOGS AND CATTLE, AND IN VITRO ISOLATION FROM OOCYSTS , 2002, The Journal of parasitology.

[8]  N. Müller,et al.  In Vitro Induction of Neospora caninum Bradyzoites in Vero Cells Reveals Differential Antigen Expression, Localization, and Host-Cell Recognition of Tachyzoites and Bradyzoites , 2004, Infection and Immunity.

[9]  M. Khalili,et al.  Prevalence of antibodies to Neospora caninum in cattle in Kerman province, South East Iran , 2008 .

[10]  D. Buxton,et al.  An IgG Avidity ELISA to Discriminate between Recent and Chronic Neospora Caninum Infection , 1999, Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc.

[11]  S. Liddell,et al.  IMMUNIZATION OF MICE WITH PLASMID DNA CODING FOR NcGRA7 OR NcsHSP33 CONFERS PARTIAL PROTECTION AGAINST VERTICAL TRANSMISSION OF NEOSPORA CANINUM , 2003, The Journal of parasitology.

[12]  J. Dubey,et al.  Serological diagnosis of bovine neosporosis by Neospora caninum monoclonal antibody-based competitive inhibition enzyme-linked immunosorbent assay , 1996, Journal of clinical microbiology.

[13]  X. Xuan,et al.  Dense-Granule Protein NcGRA7, a New Marker for the Serodiagnosis of Neospora caninum Infection in Aborting Cows , 2007, Clinical and Vaccine Immunology.

[14]  J. Dubey,et al.  Effect of polyclonal antisera developed against dense granule-associated Neospora caninum proteins on cell invasion and development in vitro by N. caninum tachyzoites , 1999, Parasitology.

[15]  A. Tabar,et al.  Seroprevalence of Neospora caninum Infection in Rural and Industrial Cattle in Northern Iran , 2009 .

[16]  M. Reichel,et al.  Progress in the serodiagnosis of Neospora caninum infections of cattle. , 2000, Parasitology today.

[17]  R. Miller,et al.  One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[18]  P. Conrad,et al.  A Modified Agglutination Test for Neospora caninum: Development, Optimization, and Comparison to the Indirect Fluorescent-Antibody Test and Enzyme-Linked Immunosorbent Assay , 1998, Clinical Diagnostic Laboratory Immunology.

[19]  M. Maleki,et al.  Seroepidemiology of Neospora caninum infection in dairy cattle herds in Mashhad area, Iran. , 2006, Veterinary parasitology.

[20]  Hiroshi Suzuki,et al.  Serodiagnosis of Neospora caninum infection in cattle by enzyme-linked immunosorbent assay with recombinant truncated NcSAG1. , 2003, Veterinary parasitology.

[21]  J. Mineo,et al.  Evaluation of serological tests for the diagnosis of Neospora caninum infection in dogs: optimization of cut off titers and inhibition studies of cross-reactivity with Toxoplasma gondii. , 2007, Veterinary parasitology.

[22]  J. Dubey,et al.  In vitro isolation and characterization of a Neospora sp. from aborted bovine foetuses , 1993, Parasitology.

[23]  B. Na,et al.  Protective efficacy of vaccination with Neospora caninum multiple recombinant antigens against experimental Neospora caninum infection. , 2005, The Korean journal of parasitology.

[24]  J. Dubey,et al.  Diagnosis of bovine neosporosis. , 2006, Veterinary parasitology.

[25]  M. Igarashi,et al.  Expression of recombinant dense granule protein 7 of Neospora caninum and evaluation of its diagnostic potential for canine neosporosis , 2006 .

[26]  A. Fernández-García,et al.  Characterisation of NcGRA7 and NcSAG4 proteins: Immunolocalisation and their role in the host cell invasion by Neospora caninum tachyzoites , 2010, Acta Parasitologica.

[27]  N. Salehi,et al.  Molecular and Pathological Study of Bovine Aborted Fetuses and Placenta from Neospora caninum Infected Dairy Cattle , 2009 .

[28]  Diana J L Williams,et al.  Diagnosis and seroepidemiology of Neospora caninum-associated bovine abortion. , 2002, International journal for parasitology.

[29]  D. Lindsay,et al.  Neonatal Neospora caninum infection in dogs: isolation of the causative agent and experimental transmission. , 1988, Journal of the American Veterinary Medical Association.

[30]  R. Z. Machado,et al.  Neospora caninum infection in birds: experimental infections in chicken and embryonated eggs , 2007, Parasitology.

[31]  D. Lindsay,et al.  A review of Neospora caninum and neosporosis. , 1996, Veterinary parasitology.

[32]  I. Bjerkås,et al.  Unidentified cyst-forming Sporozoon causing encephalomyelitis and myositis in dogs , 2004, Zeitschrift für Parasitenkunde.

[33]  Á. Zaballos,et al.  The NcGRA7 gene encodes the immunodominant 17 kDa antigen of Neospora caninum , 2006, Parasitology.

[34]  M. Takiguchi,et al.  Analysis of energy generation and glycolysis pathway in diminazene aceturate-resistant Babesia gibsoni isolate in vitro. , 2012, The Japanese journal of veterinary research.

[35]  D. Williams,et al.  Experimental studies on the transmission of Neospora caninum between cattle. , 2001, Research in veterinary science.

[36]  G. Habibi,et al.  Serological prevalence of Neospora caninum in healthy and aborted dairy cattle in Mashhad, Iran. , 2004, Veterinary parasitology.

[37]  J. Wastling,et al.  A multiple antigen ELISA to detect Neospora-specific antibodies in bovine sera, bovine foetal fluids, ovine and caprine sera. , 1998, Veterinary parasitology.

[38]  J P Dubey,et al.  Neosporosis in animals--the last five years. , 2011, Veterinary parasitology.

[39]  L. Lecordier,et al.  Role of secretory dense granule organelles in the pathogenesis of toxoplasmosis. , 1996, Current topics in microbiology and immunology.