High-yield expression of recombinant SARS coronavirus nucleocapsid protein in methylotrophic yeast Pichia pastoris
High-yield expression of recombinant SARS coronavirus nucleocapsid protein in methylotrophic yeast Pichia pastoris
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DOI：
10.3748/wjg.v10.i24.3602
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发表时间：
2004-12-15
影响因子：
4.3
通讯作者：
Xia, Ning-Shao
中科院分区：
文献类型：
--
作者：
Liu, Ru-Shi;Yang, Kun-Yu;Xia, Ning-Shao
目的：SARS冠状病毒核衣壳蛋白（Nucleocapsid，N）在SARS冠状病毒的复制和病理反应中起重要作用，其抗原性优于S蛋白。目的：寻找一种特异性强、抗原性高的重组SCoV核衣壳蛋白（rSCoVN），为SARS早期诊断的研究提供基础。方法：采用PCR方法扩增SCoV核衣壳蛋白（SCoVN）全长cDNA，克隆到酵母表达载体pPIC 3. 5 K中，构建pPIC 3. 5 K-SCoVN。将质粒线性化，然后通过电穿孔转化到巴斯德毕赤酵母（P. pastoris）GS115（His（-）Mut（+））中。His（+）Mut（+）重组菌株经PCR鉴定后，接种于MM/MD平板上培养。随后研究了不同诱导培养基、溶解氧（DO）和不同甲醇终浓度等因素对诱导期生物量和rSCoVN蛋白产量的影响。结果：线性化质粒转化毕赤酵母后，所有重组子均为His（+）Mut（+）。BMMY培养基是重组ScoVN（rSCoVN）蛋白表达和重组菌株生长的最佳培养基。甲醇的最终最适浓度为20 mL/L，DO对重组菌的生长和rSCoVN蛋白的表达有显著影响。重组菌表达的rSCoVN蛋白约占菌体总蛋白的8%，表达量为520 mg/L，摇瓶培养时600 nm处的最大细胞A值为62。rSCoVN蛋白与鼠抗SARS-CoVN-mAb和SARS阳性血清具有高度特异性，与正常人血清无交叉反应。在毕赤酵母中表达的rSCoVN的生物学活性比在E.结论：重组SARS冠状病毒核衣壳蛋白（rSCoVN）可在重组甲醇酵母GS115中成功表达。rSCoVN蛋白对SARS-CoVN-mAb和SARS阳性血清具有高度的特异性，但与正常人血清无交叉反应。这为进一步研究SARS的早期诊断和SCoV的致病机制奠定了基础。
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 E. coli when 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 GS115. 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.