Development and Evaluation of an Indirect Capripox Virus ELISA Based on Truncated P32 Protein expressed in E.coli

Document Type: Original Articles

Authors

1 Department of Pathobiology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Animal Viral Vaccine, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO),Karaj, Iran

3 Department of Microbiology Faculty of Veterinary medicine of Shahid Chamran, University of Ahvaz, Ahvaz, Iran

4 Department of Quality Control, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran.

Abstract

As notifiable diseases, lumpy skin disease (LSD), sheep pox (SPP) and goat pox (GTP) are associated with a profound effect on cattle, sheep and goat farming industries. Development of ELISA method could effectively help serodiagnosis of the infected animals. This study was aimed to develop an ELISA system based on recombinant full-length and truncated P32 protein (Tr.P32) of goat pox virus. The P32 protein was expressed in Rosetta strain of E. coli using pET24a+ vector and evaluated by SDS-PAGE and Western blotting. Then Tr.P32 was purified by Ni-NTA affinity chromatography under denaturing condition and used to develop a Capripoxvirus specific ELISA. The checker board titration and ROC analysis were used to optimize ELISA system and determination of diagnostic specificity and sensitivity, respectively. The diagnostic potential of developed ELISA was evaluated using positive and negative control sera, collected from goat, sheep and cattle. Results showed that expression level of full-length P32 recombinant protein was negligible; while, Tr.P32, a ~ 31 kDa recombinant protein, was expressed up to 0.270-0.300 mg/200 mL of culture media. The results of checkerboard titration revealed that 675 ng/well of Tr.P32 antigen and 1:10 dilution of control sera (anti GTPV HIS and healthy goat sera) caused maximum difference in absorbance between positive and negative goat sera. The recombinant Tr.P32 showed good reactions with antibodies against GTP virus (GTPV), SPP virus (SPPV) and LSD virus (LSDV), whereas no cross reactions with anti Orf virus antibody was detected. By comparison with neutralization index (NI), cut off, diagnostic sensitivity and specificity of the developed indirect-ELISA were estimated, 0.397, 94% and 96.6%, respectively. These finding indicated that ELISA system based on Tr.P32 protein potentially could be used in sero-surveillance of all Capripox viruses; however, further investigations are required.

Keywords