家蚕抗菌肽CMIV基因结构改造及表达产物的研究

参照天然抗菌肽ＣＭＩＶ组分的氨基酸序列，作了近５０％的改动，根据大肠杆菌偏爱的密码子，设计并人工合成了抗菌肽基因片段．将人工合成的抗菌肽类ＣＭＩＶ基因先重组到测序载体ｐＵＣ１１８上，经过序列分析，发现克隆于载体ｐＵＣ１１８上的基因片段与设计的序列完全一致．再将该基因片段重组到表达载体ｐＥＴ２８（ａ）上，抗菌肽以融合蛋白的形式表达．融合蛋白经镍－金属离子胶亲和层析纯化后，再用ＣＮＢｒ裂解，最终产物具有与天然抗菌肽相同的生物学活性

Studies on the Changes of CMIV Gene Structure and the Expression Products in E.coli

According to the amino acid sequence of CMIV from Bombyx mori ,with slightly modification,the CMIV like gene was designed favorable for the E.coli codons.The sequence of amino acid was changed about 50% and the synthesized gene was first subcoloned into the vector pUC118.After the sequence was analysed,it was then subcoloned into the fusion expression vector pET28(a) and transformed into E.coli BL21(DE3).Recombinant CMIV like gene expression was driven by the T7 promoter on the vector upon addition of IPTG and high level of expression was achieved.The fusion protein ws purified by Ni chelating agarose and treated with CNBr.The recombinant protein has nearly the same specific activity as the native antibacterial peptide from hemolymph of the Bombyx mori pupae induced by E.coli infection.