海马抗菌肽hkabf的重组表达纯化及初步活性研究-生物化学与分子生物学专业论文.docxVIP

海马抗菌肽HKABF的重组表达纯化及初步活性研究其活性不受NaCI和KCI高盐浓度的影响，但是其在仅15mM的CaCl2盐浓度下活 海马抗菌肽HKABF的重组表达纯化及初步活性研究 其活性不受NaCI和KCI高盐浓度的影响，但是其在仅15mM的CaCl2盐浓度下活 性就完全受到抑制。利用反向高效液相色谱鉴定纯化的重组HKABF，其色谱纯度 达85％以上。圆二色谱测定其二级结构可以将其归为CSaB型抗菌肽。 以上结果可以说明通过将酸性氨基酸融合到成熟抗菌肽上来进行抗菌肽的重组 表达是一种有效的表达方式，可以广泛应用于其他类似抗菌肽的表达。同样本研究 也可以说明本实验室发现的这种海马抗菌肽在海马孵化袋中可能发挥着重要的天然 免疫功能。本研究为此类抗菌肽在以后更深入的研究以及应用前景上奠定了很好的 基础。 关键词：抗菌肽，HKABF，重组表达，毕赤酵母，抗茵活性 Ⅱ 中山大学硕士学位论文Expression 中山大学硕士学位论文 Expression and purification of recombinant Hippocampus kuda Bleeker antimicrobial peptide HKABF for the analysis of its activities Abstract Antimicrobial peptides play all important role in eliminating pathogens infections as important component of the innate immune system．HKABF is an antimicrobial peptide discovered in the cDNA library construction of the bmoding pouch from Hippocampus kuda Bleeker．To validate this antimicrobial peptide for further research． we conducted this experiment to obtain this peptide by recombinant expression． However,in our previous lab work，the products expressed in E co／／BL21①E3)by fusing the peptide to pTRX had no antimicrobiai activity．Pichia p口s细r豇had also been used to express the mamre peptide directly but finally induced gene silence and could not stably inherit in previous work．In present work,we expressed the peptide in the Pichia pastoris expression system by fusing the mature peptide to the pro·region peptide which possess anionic charge and could neutralize the inherent toxicity．The fusion peptide could achieve high—level expression and was purified using Ni2+一chelating chromatographic．The resulting fusion peptide had no anfimicmbial activity but the mature HKABF peptide released by Enterokinase cleavage has obvious antimicmbial activity against Staphylococcus aureus． In our work,we finally obtained 15mg／1 purified HKABF peptide．The antimicrobial activit：and other characteristics were studied fur these purified HKABE Results exhibited that’r∞ombinant HKABF had antimicrobial activities against Ⅲ 海马抗苗肽HKABF的重组表达纯化及初步活性