抗菌肽Cec4a的重组表达和抗菌活性研究<sup>*</sup>

doi:10.13523/j.cb.2105046

中国生物工程杂志

2021, Vol. 41

Issue (10): 12-18 DOI: 10.13523/j.cb.2105046

研究报告

抗菌肽Cec4a的重组表达和抗菌活性研究^*

陈素芳¹,夏明印¹,曾丽艳¹,安晓琴¹,田敏芳¹,彭建^1,^2,^**(

)

1 贵州医科大学生物与工程学院/基础医学院贵阳 550025
2 环境污染与疾病监控教育部重点实验室贵阳 550025

Recombinant Expression and Detection of Antimicrobial Activity of Cec4a

CHEN Su-fang¹,XIA Ming-yin¹,ZENG Li-yan¹,AN Xiao-qin¹,TIAN Min-fang¹,PENG Jian^1,^2,^**(

)

1 School of Biology and Medical Engineering/Basic Medical College,Guizhou Medical University,Guiyang 550025,China
2 Key Laboratory of Environmental Pollution and Disease Control of Ministry of Education, Guiyang 550025,China

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摘要：

目的:构建Cec4a的原核重组表达体系,通过诱导表达、酶切纯化获得重组蛋白,并检测产物的抗菌活性。方法:基于Cec4a的序列设计引物,克隆Cec4a基因的DNA片段。利用原核表达载体(pCold-SUMO)构建重组原核表达质粒,并将其转化到大肠杆菌C41(DE3)等感受态细胞,使用IPTG进行诱导表达。通过Ni-NTA亲和层析柱纯化,获得含有His-SUMO标签的重组Cec4a融合蛋白。在SUMO蛋白酶酶切后,再次使用Ni-NTA亲和层析纯化,得到目的蛋白,最后用鲍曼不动杆菌(ATCC19606)作为指示菌检测表达产物的抗菌活性。结果:成功构建pCold-SUMO-Cec4a原核表达质粒,测序分析其序列与预期结果一致。Cec4a融合蛋白表达量为42.8mg/L,纯化后的Cec4a重组蛋白对鲍曼不动杆菌的MIC为4 μg/mL。结论:通过原核表达,并经Ni-NTA亲和层析纯化,获得了具有抗菌活性的重组蛋白Cec4a,为研究Cec4a的生物活性、抗菌机制及应用奠定了基础。

关键词： Cec4a; SUMO; 鲍曼不动杆菌; 重组表达

Abstract:

Objective: To construct the recombinant expression system of Cec4a, and to obtain the recombinant protein by induced expression and detect the antibacterial activity of the product. Methods: Based on the primers designed according to the sequence of Cec4a, the mature peptide part of Cec4a gene was amplified by PCR. Recombinant prokaryotic expression plasmid was constructed using prokaryotic expression vector (pCold-SUMO) and transformed into E. coli C41 (DE3) competent cells, which were induced by IPTG. His-SUMO labeled recombinant Cec4a fusion protein was purified by Ni-NTA affinity chromatography. The target protein was purified by Ni-NTA affinity chromatography after SUMO protease digestion. Acinetobacter baumannii (ATCC19606) was used to detect the antibacterial activity of the product. Results: pCold-SUMO-Cec4a prokaryotic expression plasmid was successfully constructed, and the sequencing analysis was consistent with the expected results. The expression level of Cec4a fusion protein was 42.8mg/L, and the MIC of purified Cec4a recombinant protein against Acinetobacter baumannii was 4 μg/mL. Conclusion: The recombinant Cec4a protein with antibacterial activity was successfully constructed and purified by Ni-NTA affinity chromatography. It lays a foundation for further study on the biological activity, the relationship between the structure and function of Cec4a.

Key words: Cec4a SUMO Acinetobacter baumannii Recombinant expression

收稿日期: 2021-05-25 出版日期: 2021-11-08

ZTFLH:

Q819

基金资助: * 国家自然科学基金(82002180);贵州省科技厅科技支撑计划(黔科合支撑[2019]2823号);贵州省高等学校教学内容和课程体系改革项目(2019046);大学生创新训练项目(20195200150);大学生创新训练项目(20195200931)

通讯作者: 彭建 E-mail: pjf66666@126.com

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引用本文:

陈素芳,夏明印,曾丽艳,安晓琴,田敏芳,彭建. 抗菌肽Cec4a的重组表达和抗菌活性研究^*[J]. 中国生物工程杂志, 2021, 41(10): 12-18.

CHEN Su-fang,XIA Ming-yin,ZENG Li-yan,AN Xiao-qin,TIAN Min-fang,PENG Jian. Recombinant Expression and Detection of Antimicrobial Activity of Cec4a. China Biotechnology, 2021, 41(10): 12-18.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2105046 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I10/12

表1 密码子优化

图1 Cec4a片段的密码子优化

图2 Cec4a片段的PCR扩增

图3 融合蛋白的SDS-PAGE分析

图4 融合蛋白纯化后SDS-PAGE分析

图5 目的蛋白酶切

图6 目的蛋白纯化

表2 盐和血清对抗鲍曼不动杆菌肽活性的影响,MIC(μg/mL)

图7 透射电镜显示抗菌肽Cec4a能在1~3 h导致鲍曼不动杆菌裂解

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