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| 抗菌肽模式肽PGYa的分子设计、克隆及在毕赤酵母中的分泌表达 | |
| 引用本文： | 张建峰,蒋宗勇,唐兆新,王贵平,余永鹏,魏文康,李春玲.抗菌肽模式肽PGYa的分子设计、克隆及在毕赤酵母中的分泌表达[J].农业生物技术学报,2008,16(1):154-157. |
| 作者姓名： | 张建峰 蒋宗勇 唐兆新 王贵平 余永鹏 魏文康 李春玲 |
| 作者单位： | 1. 广东省农业科学院兽医研究所,广州,510640;华南农业大学兽医学院,广州,510642 2. 广东省农业科学院,广州,510640 3. 华南农业大学兽医学院,广州,510642 4. 广东省农业科学院兽医研究所,广州,510640 |
| 基金项目： | 广东省农业科学院博士启动基金 |
| 摘 要： | 在α-螺旋抗菌肽序列比较和两亲性分析的基础上，提取序列模板，计算机辅助（螺旋轮法）设计出新型抗菌肽模式肽PGYa(Peptide以Gly开头，以Tyr-NH2结尾)，然后选用毕赤酵母偏爱密码子，设计合成了PGYa基因（rPCR法）。所合成的基因全长为94bp，并在其N端引入kex2裂解位点，以保证表达抗菌肽具有天然N端。基因克隆入pPICZα-A质粒，构建分泌型酵母表达载体pPICZα-A-PGYa。在AOX1 (醇氧化酶)启动子调控下，PGYa蛋白获得分泌表达，其表达量达到132 mg/L。初步抑菌（E.coli DH5α）活性显示：PGYa有较好的抗菌活性。 |
| 关 键 词： | α螺旋抗菌肽模式肽 两亲性 毕赤酵母 分泌表达 |
| 文章编号： | 1006-1304（2008）01-0154-04 |
| 收稿时间： | 2007-05-22 |
| 修稿时间： | 2007-06-27 |
Molecular Design and Cloning of Model Antimicrobial Peptide PGYa and Its Secreted Expression in Pichia pastoris |
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| ZHANG Jian-feng,JIANG Zhong-yong,TANG Zao-xin,WANG Gui-ping,YU Yong-peng,WEI wen-kang,LI Chun-lin.Molecular Design and Cloning of Model Antimicrobial Peptide PGYa and Its Secreted Expression in Pichia pastoris[J].Journal of Agricultural Biotechnology,2008,16(1):154-157. | |
| Authors: | ZHANG Jian-feng JIANG Zhong-yong TANG Zao-xin WANG Gui-ping YU Yong-peng WEI wen-kang LI Chun-lin |
| Abstract: | Based on sequence analogy and amphipathicity analyse of α-helical antimicrobial peptides, a sequence template was extracted. The model Antimicrobial Peptide PGYa(peptide beginning with Gly and ending with Tyr-NH2) was designed by virtue of the template and computer-aided design subsequently. Using the optimal condon of Pichia pastoris, PGYa gene with 94bp length were achieved through a recursive PCR strategy. Especially a Kex2 signal cleavage site was fused in 5’end of the antibacterial peptide gene to make sure the expression protein with nature N-terminal. The modified antibacterial peptide gene was then cloned into the pPICZα-A vector to construct the recombinant expression vector pPICZα-A- PGYa, and transformed into yeast host strain X-33. Under the control of the promoter AOX1(alcohol oxidase 1), the PGYa protein with a molecular weight of 2.8 KD was expressed in supernatant and the amount of secreted pertein can reach 132 μg/mL. Preliminary antibacterial assays showed that PGYa has a potent antibacterial activity against E.coli DH5α. |
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