Prokaryotic expression and identification of rabies virus nucleocapsid protein.

In order to realize efficient expression of nucleoprotein (N) from rabies virus (RV) in the prokaryotic system, this paper refers to the RV N gene sequence published by GenBank (accession number 1489853) . Without changing the amino acid sequence of the RV N protein, the codon of the gene is optimized, and the N gene is chemically synthesized and cloned into the prokaryotic expression vector pET28a (+). The recombinant plasmid pET28a (+)/RV N is constructed and transformed into E. coli BL21 (DE3) competent cells for expression. The recombinant RV N protein is purified by His-tag nickel column. The purified RV N protein is then identified by SDS-PAGE and analyzed by Western blot. The results show that the recombinant plasmid is identified by double enzyme digestion, and a band appears at 1359 bp, which is completely consistent with the corresponding known gene sequence after sequence tests. When the recombinant bacteria are induced in an IPTG concentration of 0.1 mmol/L at 30 °C for 6 hours, the RV N protein expression is the highest. The RV N recombinant protein is obtained through the purification of the nickel column. The concentration of the recombinant RV N protein measured by BCA method is 1.783 mg/mL. An obvious western blot appears at 51 kD, which is consistent with the expected target band. It shows that RV N recombinant protein is successfully expressed in E. coli BL21 (DE3) competent cells, which lays a foundation for the subsequent rabies detection and the development of a new vaccine. [ABSTRACT FROM AUTHOR]