Abstract: 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 1 359 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 ℃ 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.