title: [Expression of the recombinant SARS coronavirus nucleocapsid protein in Pichia pastoris and identification of its bioactivity].
abstract: The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.
authors.affiliation: Department of Biochemistry and Molecular Biology Life Science College, Hunan Normal University, Changsha 410081, China.
authors.forename: Ru-Shi
authors.initials: RS
authors.lastname: Liu
authors.forename: Yi-Lan
authors.initials: YL
authors.lastname: Qiu
authors.forename: Kun-Yu
authors.initials: KY
authors.lastname: Yang
authors.forename: Zhi-Hong
authors.initials: ZH
authors.lastname: Zhang
authors.forename: Liang
authors.initials: L
authors.lastname: Liang
authors.forename: Jun
authors.initials: J
authors.lastname: Zhang
authors.forename: Ning-Shao
authors.initials: NS
authors.lastname: Xia
mesh_terms: D003001:Cloning, Molecular; D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics*
chemical_list: D000086462:Coronavirus Nucleocapsid Proteins; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins