重组鸡Cathelicidins类抗菌肽的融合表达及其对金黄色葡萄球菌抗菌活性的研究

›› 2013, Vol. 40 ›› Issue (6): 19-26.

重组鸡Cathelicidins类抗菌肽的融合表达及其对金黄色葡萄球菌抗菌活性的研究

吴静^1,2, 梁永利³, 李玉峰¹, 马秀丽¹, 姜亦飞¹

1. 山东省农业科学院家禽研究所, 山东济南 250023;
2. 山东大学生命科学学院, 山东济南 250100;
3. 山东省实验动物中心, 山东济南 250002

收稿日期:2012-11-30 出版日期:2013-06-20 发布日期:2013-06-20
作者简介:吴静(1978-),女,山东人,助理研究员,博士,主要从事家禽天然免疫相关分子方面的研究。
基金资助:
山东省自然科学基金(ZR2009DQ018);山东省优秀中青年科学家科研奖励基金(2007BSC03011);山东省农科院高技术自主创新基金(2006YCX026);山东省优秀中青年科学家科研奖励基金(2007BS06012)。

Fusion Expression of Recombinant Cathelicidins Genes in Escherichia coli and Study on its Antibacterial Activity against Staphylococcus aureus in vivo

WU Jing^1,2, LIANG Yong-li³, LI Yu-feng¹, MA Xiu-li¹, JIANG Yi-fei¹

1. Poultry Institute of Shandong Academy of Agricultural Sciences, Jinan 250023, China;
2. School of Life Sciences, Shandong University, Jinan 250100, China;
3. Shandong Experimental Animal Center, Jinan 250002, China

Received:2012-11-30 Online:2013-06-20 Published:2013-06-20

摘要/Abstract

摘要： Cathelicidins抗菌肽在家禽天然免疫系统中发挥重要作用,本研究将已构建的含Cathelicidins抗菌肽成熟肽的阳性克隆菌株Rosetta(DE3)/pET30-CathL1S、pET30-CathL2S、pET30-CathL3S分别在37℃、1.0 mmol/L IPTG的条件下进行诱导表达。SDS-PAGE和Western blotting分析结果表明,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质量分别为7、8、7.5 ku。琼脂糖孔穴扩散法检测结果显示,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、鸭疫里默氏杆菌及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。以雏鸡为实验模型研究重组抗菌肽对金黄色葡萄球菌的体内抗菌活性,结果表明,注射中、高剂量CathL-1抗菌肽试验组(5、10 mg/kg),在试验结束后取肝脏组织匀浆液涂布于TMP平板,未分离到葡萄球菌。各抗菌肽试验组平均增重均高于感染对照组,其中低剂量抗菌肽试验组(2.5 mg/kg)体内抑菌效果虽与普通抗生素相当,但其增重效果尤为明显。

关键词: 抗菌肽; Cathelicidins; 融合表达; 纯化; 抑菌活性; 金黄色葡萄球菌

Abstract: Cathelicidins comprised a family of antimicrobial peptides sharing a highly conserved cathelin domain, they played a critical role in the innate immune system of chicken. In this study, the constructed positive clones of Rosetta(DE3)/pET30-CathL1S, pET30-CathL2S and pET30-CathL3S were cultured and induced to express target protein by addition of 1.0 mmol/L IPTG in LB. SDS-PAGE and Western blotting analysis demonstrated that Cathelicidins genes were expressed in the form of fusion protein, except for CathL-1 mature peptide, and the apparent molecular weight of expression products were 7, 8, 7.5 ku, 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. Young chickens as animal model were used to test antibacterial activity and the result indicated that 5 mg/kg recombinant CathL-1 could completely protect the chicken from infection of Staphylococcus aureus(ATCC 6538). Low dose of CathL-1 could apparently increase weight of chickens.

Key words: antimicrobial peptide; Cathelicidins; fusion expression; purification; antibacterial activity; Staphylococcus aureus

中图分类号:

Q785

吴静, 梁永利, 李玉峰, 马秀丽, 姜亦飞. 重组鸡Cathelicidins类抗菌肽的融合表达及其对金黄色葡萄球菌抗菌活性的研究[J]. , 2013, 40(6): 19-26.

WU Jing, LIANG Yong-li, LI Yu-feng, MA Xiu-li, JIANG Yi-fei. Fusion Expression of Recombinant Cathelicidins Genes in Escherichia coli and Study on its Antibacterial Activity against Staphylococcus aureus in vivo[J]. , 2013, 40(6): 19-26.

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