Production of Recombinant Simian Type-D Retrovirus p27 Gene and Evaluation of its Diagnostic Potential

doi:10.3969/j.issn.1674-5817.2016.04.005

Abstract

Abstract: Objective To Clone and express p27 gene of simian type-D retrovirus and evaluate its diagnostic potential. Method The p27 gene was amplified by RT-PCR and cloned into the prokaryotic expressive vector pET-28b(+) after sequencing. Recombinant plasmid was transformed into E.coli Rosetta DE3) and recombinant protein was analysed by SDS-PAGE and Western blot. The protein was purified affinity chromatography with Ni-NTA, and its diagnostic value was evaluated by ELISA. Results The highest soluble expression level of recombinant protein was obtained after being induced at 37℃ for 4 h, with the concentration of Isopropyl-β-D-thiogalactoside (IPTG) was 0.5 mmol/L, and the recombinant protein had immunological activity. After purified, the concentration of protein was 2.043 g/L, and the purity was 93.3%. The indirect ELISA was developed using the purified recombinant protein, detection of 3 standard positive sera and 22 negative sera showed the detection rate were 100%. Conclusion Recombinant p27 protein from prokaryotic expression system had perfect antigenicity, which would can be used as the candidate antigen of simian type-D retrovirus.

Key words: Simian type-D retrovirus, p27 gene, Prokaryotic expression, Serological detection

CLC Number:

Q95-33

ZHOU Jie, YANG Yan-fei, HU Jian-hua, TAO Ling-yun, GAO Cheng. Production of Recombinant Simian Type-D Retrovirus p27 Gene and Evaluation of its Diagnostic Potential[J]. Laboratory Animal and Comparative Medicine, 2016, 36(4): 270-275.

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