CAJ | 학술논문

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West-ern-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（Micrococ-cus 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。
Cathelicidins항균태시함고도보수cathelin결구역적일대류항균태，재가금천연면역계통중발휘중요작용。통과PCR방법，종함오골계Cathelicidins 항균태기인（ CathL-1，-2，-3） cDNA 완정서렬적질립pMD-CathL중분별확증Cathelicidins-1，-2，-3성숙태적편마서렬，장기분별극륭지원핵표체재체pET-30a（＋）중，구건기중조표체질립pET30-CathL1S，pET30-CathL2S，pET30-CathL3S，경측서험증적중조질립전화대장간균Rosetta（DE3）균주。양성극륭균주재37℃，1.0 mmol· L-1 IPTG적조건하진행유도표체。 SDS-PAGE화West-ern-blot분석표명，Cathelicidins성숙태편단균재대장간균중이융합형식성공표체，표체산물적표관분자량분별위7.0，8.0，7.5 kD。경지당공혈확산법검측현시표체산물대다충혁란씨음성균화양성균균구유흔호적억제활성，기대금황색포도구균（Staphylococcus aureus）、고초아포간균（Bacillus subtilis）、등황미구균（Micrococ-cus luteus）、대장간균（ Escherichia coli）、계백리사문씨균C79-13（ Salmonella pullorum）、파씨간균、압역리묵씨간균、록농간균등공시균주균유교강적억제작용，우기대항생소내약균주유명현적억제작용。
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 recom-binant Cathelicidins , the putative mature peptide coding fragments of CathL-1, CathL-2, CathL-3 genes were ampli-fied from the plasmid pMD-CathL containing Cathelicidins cDNA sequences , and then subcloned into pET-30a(+) plasmid respectively .The recombinant vectors named pET 30-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 ex-press 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 .