乌骨鸡Cathelicidins 类抗菌肽基因在大肠杆菌中的融合表达与抑菌活性

Cathelicidins抗菌肽是含高度保守cathelin结构域的一大类抗菌肽，在家禽天然免疫系统中发挥重要作用。通过PCR方法，从含乌骨鸡Cathelicidins抗菌肽基因（CathL\|1，\|2，\|3）cDNA完整序列的质粒pMD\|CathL中分别扩增Cathelicidins\|1，\|2，\|3成熟肽的编码序列，将其分别克隆至原核表达载体pET\|30a（+）中，构建其重组表达质粒pET30\|CathL1S，pET30\|CathL2S，pET30\|CathL3S，经测序验证的重组质粒转化大肠杆菌Rosetta（DE3）菌株。阳性克隆菌株在37℃，10 mmol·L-1 IPTG的条件下进行诱导表达。SDS\|PAGE和Western\|blot分析表明，Cathelicidins成熟肽片段均在大肠杆菌中以融合形式成功表达，表达产物的表观分子量分别为70，80，75 kD。琼脂糖孔穴扩散法检测显示表达产物对多种革兰氏阴性菌和阳性菌均具有很好的抑制活性，其对金黄色葡萄球菌（Staphylococcus aureus）、枯草芽孢杆菌（Bacillus subtilis）、藤黄微球菌（Micrococcus luteus）、大肠杆菌（Escherichia coli）、鸡白痢沙门氏菌C79\|13 （Salmonella pullorum）、巴氏杆菌、鸭疫里默氏杆菌、绿脓杆菌等供试菌株均有较强的抑制作用，尤其对抗生素耐药菌株有明显的抑制作用。

Fusion expression of Silkie Cathelicidins genes in Escherichia coli and detection of their antibacterial activity

Cathelicidins comprise a family of antimicrobial peptides sharing a highly conserved cathelin domain， they play a critical role in the innate immune system of chicken. In order to investigate the antibacterial activity of recombinant Cathelicidins， the putative mature peptide coding fragments of CathL\|1， CathL\|2， CathL\|3 genes were amplified from the plasmid pMD\|CathL containing Cathelicidins cDNA sequences， and then subcloned into pET\|30a（+） plasmid respectively. The recombinant vectors named pET30\|CathL1S， pET30\|CathL2S and pET30\|CathL3S were transformed into the competent cell of E.coli Rosetta（DE3）. The positive clones were cultured and induced to express target protein by addition of 10 mmol·L-1 IPTG in LB. SDS\|PAGE and Western blot analysis demonstrated that cathelicidins genes were expressed in the form of fusion protein， and the apparent molecular weight of expression products were 70 kD， 80 kD， 75 kD， respectively. The recombinant Cathelicidins were able to kill both Gram negative bacteria and Gram positive bacteria by the means of agar well diffusion assay （AWDA）， especially for the antibiotic\|resistant strains.