| 摘要: |
| 目的：探讨5′-非转录区(5′-UTR)序列改建对毕赤酵母表达外源蛋白LL-37的影响。方法：去除毕赤酵母分泌表达质粒pPIC9 5′-UTR内GGATCCAA序列，转化E.coli DH5α，构建改良的毕赤酵母分泌表达载体pPIC9-EDIT；PCR鉴定及测序确认后与酵母偏爱密码子编码的LL-37基因片段连接，构建改良的重组表达载体pPIC9-EDIT-LL-37；原生质球法转化毕赤酵母GS115，PCR扩增鉴定后诱导LL-37表达，筛选最佳表达条件，对表达产物进行凝胶电泳和Western印迹分析；比较转入pPIC9-EDIT-LL-37及pPIC9-LL-37后毕赤酵母表达产物的抑菌活性，间接测定改建前后LL-37蛋白表达量的变化。结果：PCR鉴定及测序证实pPIC9-EDIT改建成功，成功构建重组载体pPIC9-EDIT-LL-37；转化毕赤酵母后表达产物PCR鉴定出LL-37基因，最佳诱导甲醇浓度为0.5%，最佳诱导表达时间为72 h，电泳及Western印迹分析证实表达产物为LL-37；改建后LL-37蛋白表达量提高了约35倍。结论：pPIC9 5′-UTR序列的改建能明显提高毕赤酵母表达LL-37蛋白，值得进一步研究以应用于其他外源蛋白表达。 |
| 关键词: 抗菌肽LL-37 毕赤酵母 5′-非转录区 |
| DOI：10.3724/SP.J.1008.2007.01329 |
| 投稿时间：2007-09-10修订日期：2007-11-13 |
| 基金项目:江苏省南通市科学技术委员会资助项目（S40032）. |
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| Modification of 5′-UTR sequences of pPIC9 increases expression of antimicrobial peptide LL-37 |
| LU Jian-rong1*, WANG Hui-min2 ,WU Ping1 ,HUANG Song-ping1,CHANG Qiu-yue3, LING Yong-wu1, NI Xiao-rong1 |
| (1.Lab of Infectious Diseases, the Third People’s Hospital of Nantong, Nantong 226006, China; 2. Department of Clinical Laboratory, The Affiliated Hospital of Nantong University, Nantong 226001; 3. Nantong Blood Center, Nantong 226006) |
| Abstract: |
| Objective：To study the influence of 5′-untranslated region modification of pPIC9 on expression of LL-37 in Pichia pastoris. Methods: The sequence GGATCCAA was deleted from 5′-UTR of pPIC9 and the modified product was transformed into E.coli DH5α to construct a modified eukaryotic vector pPIC9-EDIT. After PCR and sequencing, pPIC9-EDIT was ligated with LL-37 sequence coded by the biased codon of yeast, the product was then transformed into E.coli DH5α to construct the recombinant expression vector pPIC9-EDIT-LL-37, the latter was transformed into P. pastoris GS115 by spheroplasting and the insert was confirmed by PCR. The bacteriolytic activity to E.coli.DH5α was analyzed to screen the highest expressing strain and to determine the best inducing time and concentration of methanol. The fermentation product was analyzed by Tricine-SDS-PAGE and Western blotting. The antibacterial activities of expression products of pPIC9-LL-37 and pPIC9-EDIT-LL-37 were compared, and the changes of LL-37 protein expression were determined before and after modification. Results: pPIC9-EDIT and pPIC9-EDIT-LL-37 were successfully constructed. Expression of LL-37 gene was confirmed by PCR in P. pastoris after pPIC9-EDIT-LL-37 transformation. The highest expressing strain was identified; the best inducing time was 72 h and the best concentration of methanol was 0.5%. Tricine-SDS-PAGE and Western blotting analysis showed that the expression product was LL-37. The expression level of LL-37 protein increased by 35 times after modification.Conclusion: Modification of pPIC9 5′-UTR can obviously improve expression of LL-37 protein in P. pastoris; it is worth to be used in the research of other heterogenous protein. |
| Key words: antimicrobial peptide LL-37 Pichia pastoris 5′-untranslated regions |
