mae和misgurin两种基因表达载体的构建及其在毕赤酵母中的表达
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摘要
MAE是本实验室设计的具有抗菌活性的杂合肽，它是以MagaininⅡ(残基4-14)和Melittin(残基2-13)为亲本，并对序列进行了优化改造后设计出的杂合肽。本文对杂合肽的高级结构进行了模拟，得到了该杂合肽高级结构的初步信息。并以已构建的载体pTYB2-mae为模板，通过PCR扩增出融合基因mae-imein-cbd，将其克隆于表达质粒pPIC9K中，通过鉴定并测序正确后，电转化真核表达宿主——毕赤酵母菌株GS115，通过营养缺陷型培养基筛选重组子，再利用G418抗性筛选出整合有多拷贝外源基因的重组子。按表达手册进行表达实验，挑选出具有高分泌表达水平的菌株，并采用不同的诱导时间、pH值、甲醇诱导浓度进行表达比较，优化出适宜的表达条件。采用优化的表达条件进行大量发酵，对上清液超滤浓缩脱盐后，采用几丁质基质进行亲和层析分离目的蛋白，溴化氰切割，琼脂孔穴法测抗菌活性。结果表明，纯化后的产物具有明显的抗菌活性。
Misgurin是一种从泥鳅中分离出来的，具有强抗菌活性而无溶血活性的抗菌肽，因而有望成为一种抗菌新药。本文根据GENEBANK登录的氨基酸序列，同时考虑构建和表达的需要，化学合成了misgurin基因和接头，采用一种新的策略，在体外将基因多拷贝同向串连，并将其克隆于表达质粒pPIC9K中，通过鉴定并测序正确后，电转化真核表达宿主——毕赤酵母菌株GS115，通过营养缺陷型培养基筛选重组子，再利用G418抗性筛选出整合有多拷贝外源基因的重组子。按表达手册进行初步表达实验，经SDS-PAGE检测表明，酵母重组子分泌出了目的蛋白。
MAE designed by our lab is a hybrid peptide with ideal antibacterial activity. It derives from MagaininII(amino acid residues 4-14) and Melittin (amino acid residues 2-13), and is optimized according to the relations between the structure and function. In this article, the advanced structure of hybrid peptide MAE is predicted with software. The fused gene mae-intein-cbd is amplified by PCR with the template of plasmid pTYB2, and then it is cloned into expression vector plasmid pPIC9K. After verified by restriction enzyme analyzing and sequencing, the vector is transferred into the eukaryotic host (yeast Pichia. Pastoris strains GS115) with electronic pulse. The recombinant is obtained through culturing on nutrition deficiency medium, and then the strains with multiple copy foreign gene is screened by G418 resistant ability. The strains with high expression level is selected according to the result of expression experiment with the methods introduced by expression guideline, and its expression condition is op
timized in three aspects, namely time of inducing, pH value and inducing concentration of methanol. The large-scale expression product is concentrated and desalted by ultra-filter, and the target protein is separated by affinity chromatography with chitin, and then cut by CNBr. The purified product appears evident antibacterial activity with method of agar diffusion.
Misgurin is a kind of pepetide separated from loach with high antibacterial activity as well as low hemolytic activity. As a result, it could be developed to be a new antibacterial agent. In this article, the misgurin gene and adaptor are synthesized according to the amino acid sequence reported in the GENEBANK and the need of construction and expression. Adopted a new strategy, multiple copy gene is ligated in the same direction. And then it is cloned into expression vector plasmid pPIC9K. After verified by restriction enzyme analyzing and sequencing, the vector is transfered into the eukaryotic host (yeast Pichia. Pastoris strains GS115) with electronic pulse. The recombinant is obtained through culturing on nutrition deficiency medium, and then the strains with multiple copy foreign gene is screened by G418 resistant ability.
The expression experiment is carried out with the methods introduced by expression guideline. According to the result of SDS-PAGE, the target protein is expressed successfully in the Pichia. Pastoris.
Misgurin是一种从泥鳅中分离出来的，具有强抗菌活性而无溶血活性的抗菌肽，因而有望成为一种抗菌新药。本文根据GENEBANK登录的氨基酸序列，同时考虑构建和表达的需要，化学合成了misgurin基因和接头，采用一种新的策略，在体外将基因多拷贝同向串连，并将其克隆于表达质粒pPIC9K中，通过鉴定并测序正确后，电转化真核表达宿主——毕赤酵母菌株GS115，通过营养缺陷型培养基筛选重组子，再利用G418抗性筛选出整合有多拷贝外源基因的重组子。按表达手册进行初步表达实验，经SDS-PAGE检测表明，酵母重组子分泌出了目的蛋白。
MAE designed by our lab is a hybrid peptide with ideal antibacterial activity. It derives from MagaininII(amino acid residues 4-14) and Melittin (amino acid residues 2-13), and is optimized according to the relations between the structure and function. In this article, the advanced structure of hybrid peptide MAE is predicted with software. The fused gene mae-intein-cbd is amplified by PCR with the template of plasmid pTYB2, and then it is cloned into expression vector plasmid pPIC9K. After verified by restriction enzyme analyzing and sequencing, the vector is transferred into the eukaryotic host (yeast Pichia. Pastoris strains GS115) with electronic pulse. The recombinant is obtained through culturing on nutrition deficiency medium, and then the strains with multiple copy foreign gene is screened by G418 resistant ability. The strains with high expression level is selected according to the result of expression experiment with the methods introduced by expression guideline, and its expression condition is op
timized in three aspects, namely time of inducing, pH value and inducing concentration of methanol. The large-scale expression product is concentrated and desalted by ultra-filter, and the target protein is separated by affinity chromatography with chitin, and then cut by CNBr. The purified product appears evident antibacterial activity with method of agar diffusion.
Misgurin is a kind of pepetide separated from loach with high antibacterial activity as well as low hemolytic activity. As a result, it could be developed to be a new antibacterial agent. In this article, the misgurin gene and adaptor are synthesized according to the amino acid sequence reported in the GENEBANK and the need of construction and expression. Adopted a new strategy, multiple copy gene is ligated in the same direction. And then it is cloned into expression vector plasmid pPIC9K. After verified by restriction enzyme analyzing and sequencing, the vector is transfered into the eukaryotic host (yeast Pichia. Pastoris strains GS115) with electronic pulse. The recombinant is obtained through culturing on nutrition deficiency medium, and then the strains with multiple copy foreign gene is screened by G418 resistant ability.
The expression experiment is carried out with the methods introduced by expression guideline. According to the result of SDS-PAGE, the target protein is expressed successfully in the Pichia. Pastoris.
引文
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