Expression of the ORF2 of Norovirus in Escherichia coli and the Product Analysis

Abstract:

Norovirus (NV) was one of new borne viruses, which was found firstly in the USA in 1972 and not reported in China until 1995. The main food-borne viral pathogens affect people badly and cause the epidemic acute gastroenteritis in all age groups worldwide. In this study, the genomic RNA was extracted from the non-bacterial gastroenteritis samples. A 1623 bp fragment, containing the complete coding sequence of the ORF2 gene, was amplified from the NV samples by RT-PCR. Sequencing analysis showed that it belongs to GII and shared more than 99% homology with the corresponding sequences published in the GenBank(DQ419908 and DQ369797). The ORF2 gene was then in-frame fused to the prokaryotic expression vector pET-28a, and the resultant recombinant plasmid was transformed into Escherichia coli BL21 (DE3) competent cells. A 62 kD fusion protein, named rVP1, was expressed after IPTG induction. Rabbits were immunized by the purified rVP1. Western Blot results showed that the high titer antibody can specifically recognize the VP1 in the clinical samples from hospital. The data suggested that the rVP1 has good immunogenicity and reactiongenicity. The minor protein in the expression product was analyzed by Western Blot and double?immunodiffusion?test. The data showed that the minor proteins were the fragments of rVP1.

Key words:Norovirus, VP1, Prokaryotic expression, Expression product analysis