家蚕抗菌肽moricin在大肠杆菌中的融合表达及抗菌活性测定

Fusion Expression and Test of Antibacterial Activity of the Antibacterial Peptide Moricin of Bombyx mori in Escherichia coli

Moricin是家蚕中发现的一种抗菌肽,对革兰氏阴性和阳性菌都有较强的抗菌活性,具有良好的开发应用前景。为了建立一种大量表达和快速分离moricin的方法,采用PCR拼接法获得moricin基因后重组到表达载体pET-32M上,并在大肠杆菌(E.coli)中融合表达。表达产物存在于上清中为可溶状态,经Ni-NTA柱纯化获得融合蛋白,再经凝血酶(thrombin)酶切后过2次Ni-NTA柱获得纯度为90%以上的moricin,液相测定法表明纯化获得的moricin对大肠杆菌具有抗菌活性。

Moricin is a novel antibacterial peptide from the silkworm （ Bombyx mori） with activities against fungi end gram-positive and gram-negative bacteria, in order to establish an over-expressed and facilitated purification method, the moricin gene was obtained by a recursive PCR strategy using five overlap oligonucleotides and cloned into the expression vector pET-32M and expressed in Escherichia, coil BL21. The expressed product was soluble in supernatant and the fusion protein was purified by Ni-NTA, After digested with thrombin and purified with other 2 times of Ni-NTA, moricin with a purity of over 90 percent was obtained. Liquid turbidity analysis indicated that the purified moricin exhibited the antibacterial activity.