Development of Antibtv Antibody Detection Platform Using Recombinant VP7 Protein
Loading...
Files
Date
2023-04-03
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
MAFSU, Nagpur
Abstract
Bluetongue (BT) is an arthropod-borne infectious, non-contagious viral disease of domestic and wild ruminants caused by the Bluetongue virus(BTV), which belongs to the genus Orbivirus and family Reoviridae. The diagnosis of Bluetongue, is based on serological testing as well as molecular techniques. The group specific VP7 protein is important for the detection of anti-BTV antibodies. The use of recombinant VP7 in different expression system had shown its ability for the detection of BTV antibodies. Hence, the study was planned to express the BTV partial VP7 gene in prokaryotic system and use it for detection of the anti-BTV antibodies. In the present study pET-32-a-BTV-trc.-VP7 recombinant clone in BL-21(DE3) was confirmed by the PCR. The recombinant colony was then induced with 1mM IPTG at 30 °C at 220 rpm and rVP7 expression was confirmed by the 12.5% SDS-PAGE indicating the expected band of around 36 kDa as compared with pre-stained protein marker. The induced protein was extracted using freeze -thawing and sonication. The rVP7 protein was then attempted to purify using denatured condition and affinity chromatography techniques using the batch purification method. The different fractions collected during the purification were used for the SDS-PAGE analysis. The sonication and freeze -thawed methods in denaturing lysis buffer did not show any rVP7 protein in supernatant, the rVP7 was found in the pellet might be due to the inclusion bodies. As the recombinant protein was expressed but remained in the pellet, therefore further attempts were made to use the crude lysate for the detection of antibodies against BTV using the Dot-ELISA. The known hyperimmune sera against BTV and field clinical serum samples, previously confirmed to be positive and known negative serum were used for recombinant protein analysis in order to detect the antibodies with an additional step of the pre-adsorption with the Bl-21 crude lysate. The Dot-ELISA revealed that all the positive serum samples were found to be positive, whereas the Bl-21 lysate negative antigen was very faintly visible, and the positive can be easily differentiated based on the colour intensity. The four negative serum samples tested did not showed any positivity. Hence, the present study concludes that the Dot ELISA platform based on the crude lysate of rVP7 expressing clone can be used for the detection of the anti-BTV antibodies with additional steps of pre-adsorption of testing sera with crude lysate of the negative antigen ( Bl-21 E. coli cells).