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银杏抗菌肽蛋白基因原核表达及其重组蛋白的体内外抑菌作用
研究 Δ
*
#
刘 蕾 ,何光志 ,王文佳(贵州中医药大学基础医学院,贵阳 550025)
中图分类号 R915;Q81 文献标志码 A 文章编号 1001-0408(2019)18-2513-06
DOI 10.6039/j.issn.1001-0408.2019.18.12
摘 要 目的:获取银杏抗菌肽(GBA)重组蛋白,并考察其体内外抗菌活性,为解决病原菌耐药问题、规模化生产植物源性新型抗
菌剂提供实验基础。方法:根据Genebank中公布的GBA基因序列(FJ865399),利用基因技术构建重组质粒pET32a(+)-GBA,再
以大肠杆菌原核表达获取重组蛋白,并采用凝胶电泳和Western blotting法对所得蛋白进行纯化及鉴定。采用纸片扩散法考察所
获重组蛋白对大肠杆菌、金黄色葡萄球菌、绿脓杆菌、鼠伤寒沙门氏菌的药物敏感度;采用肉汤稀释法测定重组蛋白最小抑菌浓度
(MIC)和最低杀菌浓度(MBC);考察重组蛋白对金黄色葡萄球菌感染模型小鼠的保护作用。结果:成功表达并纯化获得目标重组
蛋白(大小约32 kDa)。该重组蛋白对金黄色葡萄球菌中度敏感,对其他3种细菌低度敏感;对金黄色葡萄球菌的MIC为(50.00±
5.00)mg/mL、MBC为(138.33±12.58)mg/mL,MIC显著高于其他3种细菌(P<0.05)。高剂量重组蛋白(8.0 g/kg)能显著降低金黄
色葡萄球菌导致的小鼠死亡率(P<0.05),对小鼠的保护效果与阳性药物青霉素接近。结论:所获重组蛋白对金黄色葡萄球菌的
抑制效果明显,对大肠杆菌和绿脓杆菌有一定的抑制作用,对鼠伤寒沙门氏菌抑制作用较弱;该蛋白高剂量给药对金黄色葡萄球
菌感染小鼠具有明显的保护作用。
关键词 银杏;抗菌肽;载体质粒;重组蛋白;原核表达;抑菌作用;体内;体外;小鼠
Study on Gene Prokaryotic Expression of Ginkgo biloba Antimicrobial Peptide Protein and Antimicrobial
Activity of Its Recombinant Protein in vivo and in vitro
LIU Lei,HE Guangzhi,WANG Wenjia(Basic Medical College,Guizhou University of TCM,Guiyang 550025,
China)
ABSTRACT OBJECTIVE:To obtain Ginkgo biloba antimicrobial peptide(GBA)recombinant protein,and to investigate in vivo/
in vitro antimicrobial activity of the protein so as to provide experimental basis for solving bacterial resistance and large-scale
production of new plant-derived antimicrobial agents. METHODS:Based on gene technology,according to GBA gene sequence
(FJ 865399) published by Genebank,recombinant expression vector plasmid pET32a(+)-GBA was constructed. Prokaryotic
expression of recombinant protein was conducted by Escherichia coli,and then the protein was purified and identified by gel
electrophoresis and Western blotting. Drug sensitivity of obtained recombinant protein to E. coli, Staphylococcus aureus,
Pseudomonas aeruginosa,Salmonella typhimurium were investigated by Kirby-Bauer test. The minimum antibacterial concentration
(MIC) and minimum bactericidal concentration (MBC) were determined by broth dilution method. The protective effects of
recombinant protein on S. aureus infection model mice were investigated. RESULTS:Target recombinant protein was expressed
successfully and purified(molecular weight of 32 kDa). The recombinant protein was moderately sensitive to S. aureus and low
sensitive to other three bacterias. MIC and MBC of the recombinant protein to S. aureus were(50.00±5.00)mg/mL and(138.33±
12.58)mg/mL,and MIC was significantly higher than those to other 3 kinds of bacterias(P<0.05). High-dose of recombinant protein
(8.0 g/kg) could significantly reduce the S. aureus-induced mortality of mice (P<0.05),and had similar protective effect as
positive drug penicillin. CONCLUSIONS:Obtained recombinant protein has obvious antimicrobial effects on S. aureus,inhibits E.
coli and P. aeruginosa to certain extent and shows poor inhibitive effect on S. typhimurium. High-dose of recombinant protein
shows significant protective effect for S. aureus infection model mice.
KEYWORDS Ginkgo biloba; Antimicrobial peptide; Vector plasmid; Recombinant protein; Prokaryotic expression;
Antimicrobial effect;in vivo;in vitro;Mice
近年来,由于抗生素的滥用导致许多耐药菌株出 现,这对人类生命健康安全构成了巨大威胁,如何解决
病原菌对抗生素的耐药问题已迫在眉睫,现已成为生
Δ 基金项目:贵州省教育厅创新群体重大研究项目(No.黔教合
KY 字[2016]036);贵州省科技计划课题(No.黔科合中药字[2012] 物、医药领域的研究热点。抗菌肽(Antimicrobial pep-
LKZ7024 号);贵州省科学技术基金项目(No.黔科合 J 字[2012]2075 tides),也称为肽类抗生素或天然抗生素,是一类由20~
号)
60 个氨基酸残基组成,对细菌、真菌等微生物及某些昆
* 讲 师 ,硕 士 。 研 究 方 向 :中 西 医 结 合 基 础 。 电 话 :0851- [1]
虫和植物细胞具有抑制作用的小分子多肽 。从 1972
88308013。E-mail:23583305@qq.com
年瑞典科学家 Boman HG 等首次成功分离天蚕素(Ce-
# 通信作者:教授,硕士生导师,博士。研究方向:中西医结合基
础。电话:0851-88308013。E-mail:392724549@qq.com cropin)开始,到目前为止,已发现并分离获得多种具有
中国药房 2019年第30卷第18期 China Pharmacy 2019 Vol. 30 No. 18 ·2513 ·