Vol. 12, Special Issue 11 (2023)

Author(s):
Aniket Thakare, Prabhakar A Tembhurne, Jivan Kesharakar, Deepali Gharat, Gayatri Chavan and Sudhakar Awandkar

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. Considering the recombinant VP7 ability for the detection of BTV antibodies, 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 clone was induced with 1mM IPTG at 30 °C at 220 rpm and rVP7 followed by the expression was confirmed by the 12.5% SDS-PAGE. The rVP7 protein was purified using denatured condition and affinity chromatography techniques and analyzed by SDS-PAGE analysis. The PCR revealed expected product of around 550bp. The expressed protein was extracted by sonication and freeze -thawed methods in denaturing lysis buffer, did not showed 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 in pre adsorbed serum with negative antigen against BTV using the Dot- ELISA. 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, the positive sample 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).