Generation of two polyclonal antibodies for western blot detection of hepatitis A virus in plants
Abstract
In recombinant systems, the availability of good quality antibodies is required to detect Hepatitis A virus (HAV) proteins. Following this line, the cDNA sequences coding for two hepatitis A virus structural proteins, VP2 and VP3, were cloned into the bacterial expression vector pTrcHis yielding plasmids pTrcVP2 and pTrcVP3. Recombinant Escherichia coli XL1-blue strains harboring these two constructs were grown in liquid media and after IPTG gene induction, both fused recombinants molecules were detected by SDS-PAGE as 34 kDa and 24 kDa bands respectively. Recombinant VP2 and VP3 proteins containing N-terminal hexa-histidine tags were purified under denaturing conditions by immobilized metal affinity chromatography yielding 8 mg and 10 mg of refolded protein per 300 mL of bacterial culture respectively. Immunization of rabbits against purified recombinant proteins allowed the production of high titer polyclonal sera with immunological reactivity detecting hepatitis A virus proteins using western-blot analysis. According to our results, these two polyclonal sera constitute a valuable tool to follow the production of HAV proteins in transgenic plants, where a putative expression of HAV proteins higher than 0.25% of the total soluble protein could be detected with the use of these sera by western blot assay.
