关键词: 新城疫病毒; NP蛋白; 原核表达; 纯化

Abstract: The nucleocapsid protein (NP) gene of Newcastle disease virus (NDV) was cloned into a prokaryotic expression vector, and then using E.coli BL21(DE3) to express this protein. NP gene was amplified from cRNA which was reverse transcription of RNA of NDV La Sota strain, then the gene was cloned into pET-30a. PCR,digestion and sequencing were used to identify positive plasmid, which named pET-NDV NP; the identified recombined plasmid was expressed by E.coli BL21(DE3) and induced by IPTG, the expressed products were purified by Ni-NTA Ni⁺ affinity column; SDS-PAGE was used to analyze the expressed protein; Western blotting was used to identify the reaction activity. The results showed that the sequence of NP gene was consistent with predicted size, 1470 bp. PCR, digestion and sequencing results showed that the recombinant plasmid pET-NDV NP was correct. NP protein was successfully expressed by E.coli BL21(DE3), then the recombinant protein was purified by Ni-NTA Ni⁺ affinity column and the concentration of the purified protein was 3 mg/mL. The recombinant protein could react with the antibody of NDV NP protein using the method of Western blotting assay. In summary, the recombinant plasmid pET-NDV NP containing NP gene was constructed and recombinant protein was successfully expressed in E.coli BL21(DE3) and purified.

Key words: Newcastle disease virus (NDV); NP protein; prokaryotic expression; purification