AIM: Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), the antigenicity of the protein is better than spike (S) ...AIM: Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), the antigenicity of the protein is better than spike (S) protein.This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and to provide a basis for further researches on early diagnosis of SARS.METHODS: Full length cDNA of SCoV nucleocapsid (SCoVN) protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into Pichia pastoris (P.pastoris) GS115 (His^+ Mut^+) by electroporation. His^+Mut^+ recombinant strains were identified by PCR and cultivated on MM/MD plates. The influence of different factors on biomass and rSCoVN protein production during induction phase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, was subsequently studied. The expression level and activation were detected by SDS-PAGE and Western-blot respectively.RESULTS: All of the recombinants were His^+Mut^+ after transformation of P.pastoris with linearized plasmids. The BMMY medium was optimal for recombinant ScoVN (rSCoVN) protein expression and growth of the recombinant strains.The final optimal concentration of methanol was 20 ml_/L,the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains. The rSCoVN protein expressed in recombinant strains was about 8% of the total cell protein, 520 mg/L of rSCoVN protein was achieved,and a maximum cell A at 600 nm of 62 was achieved in shake flask culture. The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum.The biological activity of rSCoVN expressed in P.pastoris was about 4-fold higher than that expressed in Ecoliwhen the same rSCoVN protein quantity was used.CONCLUSION: Active recombinant severe acute respiratory syndrome (SARS) coronavirus nucleocapsid (rSCoVN) protein can be successfully expressed in recombinant methylotrophic yeast P.pastoris GSl15. The rSCoVN protein has a high specificity against SARS-CoVN-mAb and SARS positive sera, but has no cross-reaction with normal human serum.This provides a basis for further researches on the early diagnosis of SARS and the mechanism of SCoV.展开更多
[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucl...[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucleocapsid protein N gone sequence of PRRSV were designed or synthesized, and then inserted into CMV promoter downstream to clone into pSilencer 4,1 -CMV eukaryotic expression vector. The recombinant expression vector was identified by enzyme digestion and DNA sequencing. [ Result] The results showed that the siRNA interference recombinant plasmid vector pSilencer-N targeting nucleocapsid protein gone expression had been successfully constructed. [ Conclusion] This study lays a foundation for studies on the controlling PRRSV by RNA interference technique .展开更多
基金Supported by the Excellent School Incubation Plan of Ministry of Education,China
文摘AIM: Nucleocapsid (N) protein plays an important role in reproduction and pathological reaction of severe acute respiratory syndrome (SARS) coronavirus (SCoV), the antigenicity of the protein is better than spike (S) protein.This study was to find a highly specific and antigenic recombinant SCoV nucleocapsid (rSCoVN) protein, and to provide a basis for further researches on early diagnosis of SARS.METHODS: Full length cDNA of SCoV nucleocapsid (SCoVN) protein was amplified through polymerase chain reaction (PCR) and cloned into yeast expression vector pPIC3.5K to construct plasmid of pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into Pichia pastoris (P.pastoris) GS115 (His^+ Mut^+) by electroporation. His^+Mut^+ recombinant strains were identified by PCR and cultivated on MM/MD plates. The influence of different factors on biomass and rSCoVN protein production during induction phase, such as various induction media, dissolved oxygen (DO) and different final concentrations of methanol, was subsequently studied. The expression level and activation were detected by SDS-PAGE and Western-blot respectively.RESULTS: All of the recombinants were His^+Mut^+ after transformation of P.pastoris with linearized plasmids. The BMMY medium was optimal for recombinant ScoVN (rSCoVN) protein expression and growth of the recombinant strains.The final optimal concentration of methanol was 20 ml_/L,the DO had a significant effect on rSCoVN protein expression and growth of recombinant strains. The rSCoVN protein expressed in recombinant strains was about 8% of the total cell protein, 520 mg/L of rSCoVN protein was achieved,and a maximum cell A at 600 nm of 62 was achieved in shake flask culture. The rSCoVN protein had a high specificity against mouse-anti-SARS-CoVN-mAb and SARS positive sera, but had no cross-reaction with normal human serum.The biological activity of rSCoVN expressed in P.pastoris was about 4-fold higher than that expressed in Ecoliwhen the same rSCoVN protein quantity was used.CONCLUSION: Active recombinant severe acute respiratory syndrome (SARS) coronavirus nucleocapsid (rSCoVN) protein can be successfully expressed in recombinant methylotrophic yeast P.pastoris GSl15. The rSCoVN protein has a high specificity against SARS-CoVN-mAb and SARS positive sera, but has no cross-reaction with normal human serum.This provides a basis for further researches on the early diagnosis of SARS and the mechanism of SCoV.
基金Supported by Based on Cuttingedge technology and research Project of Henan Province(072300430060)The focus of Scientific andTechnological Project of Henan Province(072102130023)Colleges and Universities of Henan Province in Support of TechnologicalInnovation Plan~~
文摘[ Objective] The aim of this study was to investigate the construction and identification of siRNA expression vector targeting nucleocapsid protein N gone of PRRSV. [Method] Three siRNA oligonucleotides targeting nucleocapsid protein N gone sequence of PRRSV were designed or synthesized, and then inserted into CMV promoter downstream to clone into pSilencer 4,1 -CMV eukaryotic expression vector. The recombinant expression vector was identified by enzyme digestion and DNA sequencing. [ Result] The results showed that the siRNA interference recombinant plasmid vector pSilencer-N targeting nucleocapsid protein gone expression had been successfully constructed. [ Conclusion] This study lays a foundation for studies on the controlling PRRSV by RNA interference technique .
