家蝇抗菌肽AMPs17蛋白原核表达条件的优化及其抗真菌活性检测 <sup>*</sup>

doi:10.13523/j.cb.20190404

中国生物工程杂志

2019, Vol. 39

Issue (4): 24-31 DOI: 10.13523/j.cb.20190404

研究报告

家蝇抗菌肽AMPs17蛋白原核表达条件的优化及其抗真菌活性检测 ^*

杨隆兵,国果(

),马慧玲,李妍,赵欣宇,苏佩佩,张勇

贵州医科大学基础医学院贵州医科大学环境污染与疾病监控教育部重点实验室贵阳 550025

Optimization of Prokaryotic Expression Conditions and Antifungal Activity Detection of Antibacterial Peptide AMPs17 Protein in Musca domestica

Long-bing YANG,Guo GUO(

),Hui-ling MA,Yan LI,Xin-yu ZHAO,Pei-pei SU,Yon ZHANG

School of Basic Medical Sciences, Key Laboratory of Environmental Pollution Monitoring and Disease Control, Ministry of Education,Guizhou Medical University,Guiyang 550025, China

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摘要：

目的:优化AMPs17重组蛋白的原核表达条件,分析重组蛋白的抗真菌活性。方法:比较不同的诱导温度(25℃、28℃、 30℃、32℃、34℃)、异丙基硫代-β-D半乳糖苷(IPTG)诱导浓度(0.025mmol/L、 0.05mmol/L、0.1mmol/L、0.3mmol/L、0.5mmol/L、0.8mmol/L、1.0mmol/L)和诱导时间(12h、15h、18h、21h、24h)对AMPs17重组蛋白表达量的影响,筛选AMPs17重组蛋白的最佳表达条件;采用镍离子金属螯合剂亲和层析柱对重组蛋白进行纯化,SDS-PAGE和ImageJ图像分析系统对表达结果进行分析,Western blot对AMPs17重组蛋白进行鉴定,高效液相色谱分析重组蛋白的纯度,微量液体稀释法及菌落计数法检测其抗真菌活性。结果:在诱导温度为32℃、IPTG浓度为0.05mmol/L的条件下诱导培养15h,AMPs17重组蛋白的表达量最高且最为稳定;HPLC色谱仪分析显示AMPs17重组蛋白纯度可达到90%以上;优化后的AMPs17重组蛋白能有效抑制白色念珠菌的生长。结论:优化了家蝇抗菌肽AMPs17的诱导表达条件,获得了高表达、稳定且具有抗真菌活性的蛋白质,为后续抗菌机制及应用研究提供一定的实验基础。

关键词： 家蝇; 家蝇抗菌肽-17; 原核表达; 条件优化; 抗真菌活性

Abstract:

Objective: To optimize the prokaryotic expression conditions of AMPs17 recombinant protein and analyze the antifungal activity of recombinant protein. Methods: Compare different induction temperatures (25℃, 28℃, 30℃, 32℃, 34℃), isopropylthio-β-D galactoside (IPTG) induced concentration (0.025mmol/L, 0.05mmol/L, 0.1mmol/L, 0.3mmol/L, 0.5mmol/L, 0.8mmol/L, 1.0mmol/L) and induction time (12h, 15h, 18h, 21h, 24h) on the expression of AMPs17 recombinant protein, screening the optimal expression conditions of AMPs17 recombinant protein. The recombinant protein was purified by nickel ion metal chelator affinity chromatography column, and the expression results were analyzed by SDS-PAGE electrophoresis and ImageJ image analysis system. The recombinant protein of AMPs17 was identified by Western blot and the purity of the recombinant protein was analyzed by high performance liquid chromatography(HPLC). The antifungal activity was detected by a micro liquid dilution method and a colony counting method. Results: The results showed that the expression of AMPs17 recombinant protein was the highest and the most stable when induced at 32℃ and IPTG concentration of 0.05mmol/L for 15h. The HPLC analysis showed that the purity of AMPs17 recombinant protein reached 90%. In addition, AMPs17 recombinant protein can effectively inhibit the growth of Candida albicans. Conclusion: The induction and expression conditions of antibacterial peptide AMPs17 were optimized, and proteins with high expression, stability and antifungal activity were obtained, which provided certain experimental basis for the follow-up antibacterial mechanism and application research.

Key words: Musca domestica AMPs17 Prokaryotic expression Condition optimization Antifungalactivity

收稿日期: 2018-09-30 出版日期: 2019-05-08

ZTFLH:

Q819

基金资助: * 国家自然科学基金资助项目(81760647);国家自然科学基金资助项目(81560337)

通讯作者: 国果 E-mail: guoguojsc@163.com

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引用本文:

杨隆兵,国果,马慧玲,李妍,赵欣宇,苏佩佩,张勇. 家蝇抗菌肽AMPs17蛋白原核表达条件的优化及其抗真菌活性检测 ^*[J]. 中国生物工程杂志, 2019, 39(4): 24-31.

Long-bing YANG,Guo GUO,Hui-ling MA,Yan LI,Xin-yu ZHAO,Pei-pei SU,Yon ZHANG. Optimization of Prokaryotic Expression Conditions and Antifungal Activity Detection of Antibacterial Peptide AMPs17 Protein in Musca domestica. China Biotechnology, 2019, 39(4): 24-31.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20190404 或 https://manu60.magtech.com.cn/biotech/CN/Y2019/V39/I4/24

图1 AMPs17基因cDNA片段的产物

图2 重组质粒pET-28a(+)- AMPs17双酶切鉴定图

图3 不同诱导温度对AMPs17蛋白表达量的SDS-PAGE分析

图4 不同诱导时间对AMPs17蛋白表达量的SDS-PAGE分析

图5 不同诱导IPTG浓度对AMPs17蛋白表达量的SDS-PAGE分析

图6 AMPs17蛋白的诱导表达及纯化

图7 AMPs17重组蛋白His标签鉴定图

图8 AMPs17蛋白的纯度测定

表1 AMPs17蛋白的抗真菌活性检测

图9 AMPs17蛋白的抗白色念珠菌活性

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