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 re展开更多
吡咯喹啉醌(PQQ)是具有重要生理功能的氧化还原酶辅酶,从化工污水中筛选到一株蛋白分泌量低的PQQ生产菌株FJNU-6。经形态学观察、生理生化测定和16S r DNA序列分析,甲基营养菌FJNU-6鉴定为脱氮生丝微菌(Hyphomicrobium denitrificans)...吡咯喹啉醌(PQQ)是具有重要生理功能的氧化还原酶辅酶,从化工污水中筛选到一株蛋白分泌量低的PQQ生产菌株FJNU-6。经形态学观察、生理生化测定和16S r DNA序列分析,甲基营养菌FJNU-6鉴定为脱氮生丝微菌(Hyphomicrobium denitrificans)。采用部分析因和中心组合实验方法对培养基进行优化,优化的培养基(g/L)为:甲醇12,(NH4)2SO41.68,KH2PO43.01,Na2HPO49.89,Mg SO4·7H2O 1.1,微量元素液和维生素液添加量分别为0.9 m L/L和1.1 m L/L,培养温度为30℃,最佳发酵培养时间为88 h。H.denitrificans FJNU-6在此优化条件下培养液中蛋白含量只有32.17 mg/L,而PQQ产量可达121.43 mg/L,比优化前提高了2.95倍。因此,H.denitrificans FJNU-6是一株具有工业化生产PQQ前景的新资源。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 re
文摘吡咯喹啉醌(PQQ)是具有重要生理功能的氧化还原酶辅酶,从化工污水中筛选到一株蛋白分泌量低的PQQ生产菌株FJNU-6。经形态学观察、生理生化测定和16S r DNA序列分析,甲基营养菌FJNU-6鉴定为脱氮生丝微菌(Hyphomicrobium denitrificans)。采用部分析因和中心组合实验方法对培养基进行优化,优化的培养基(g/L)为:甲醇12,(NH4)2SO41.68,KH2PO43.01,Na2HPO49.89,Mg SO4·7H2O 1.1,微量元素液和维生素液添加量分别为0.9 m L/L和1.1 m L/L,培养温度为30℃,最佳发酵培养时间为88 h。H.denitrificans FJNU-6在此优化条件下培养液中蛋白含量只有32.17 mg/L,而PQQ产量可达121.43 mg/L,比优化前提高了2.95倍。因此,H.denitrificans FJNU-6是一株具有工业化生产PQQ前景的新资源。
