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Abstract To obtain a recombinant Glycoprotein G1 of Akabane virus for diagnostic purpose, the gene fragment coding the high antigenic domain of AKAV G1 protein, was amplified from the AKAV genome by RT-PCR after analyzing glycoprotein G1 with bio-software. The amplified product was cloned into pMD18-T vector and sequenced. Then the gene fragment was subcloned into pET-28a (+) vector directionally. The recombinant plasmid was transformed into E. coli BL21 (DE3). SDS-PAGE and Western-blot analysis indicated that the fusion protein was insoluble and approximately 44 ku in molecular weight and had immunological activity. After purified by affinity chromatography, the concentration of purified protein was 2mg/ml and the purity was 92.6%. An indirect enzyme linked immunosorbent assay (ELISA) was developed using the purified recombinant protein. The optimal reaction conditions of ELISA were determined: the coating concentration of the purified recombinant protein was 20g/ ml; the dilution fold of the serum samples was 1:200. There was no cross reaction with positive sera of other six infectious diseases. Bovine serum samples from Yun Nan (77) and Inner Mongolia (70) had been detected by serum neutralizing (SN) test before were detected by this ELISA. In accordance with SN test, the positive threshold of the ELISA was determined as 0.493 and 0.488 in two districts respectively. Comparing to SN test, the speciality of the ELISA was 73% and 86.9% respectively. The agreement ratio between the two methods was 80%(52/65) and 85.9%(55/64) respectively.
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Received: 15 November 2007
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