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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2019, Vol. 39 Issue (4): 24-31    DOI: 10.13523/j.cb.20190404
    
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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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     
Received: 30 September 2018      Published: 08 May 2019
ZTFLH:  Q819  
Corresponding Authors: Guo GUO     E-mail: guoguojsc@163.com
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Long-bing YANG
Guo GUO
Hui-ling MA
Yan LI
Xin-yu ZHAO
Pei-pei SU
Yon ZHANG
Cite this article:

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.

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

Fig.1 Amplification cDNA fragment of AMPs17 gene Lane M: DNA marker;Lane 1: AMPs17 PCR amplification product
Fig.2 Identification of recombinant plasmit pET-28a(+)-AMPs17 by endonuclease digestion Lane M:DNA marker; Lane 1: AMPs17 PCR amplification product; Lane 2: pET-28a(+)- AMPs17 recombinant plasmid double digestion; Lane 3: pET-28a(+)- AMPs17 recombinant plasmid
Fig.3 SDS-PAGE analysis of the expression of AMPs17 protein at different induction temperatures Lane M:DNA marker; Lane 1: AMPs17 PCR amplification product; Lane 2: pET-28a(+)- AMPs17 recombinant plasmid double digestion; Lane 3: pET-28a(+)- AMPs17 recombinant plasmid
Fig.4 SDS-PAGE analysis of the expression of AMPs17 protein at different induction times Lane M: Protein marker; Lanes 2, 4, 6, 8, 9: 24h, 21h, 18h, 15h, 12h induced expression products
Fig.5 SDS-PAGE analysis of the expression of AMPs17 protein at different IPTG concentrations Lane M: Protein marker; Lane 1: Expression product of whole bacterial solution induced by IPTG; Lanes 28: Adding final concentration of IPTG: 0.025mmol/L, 0.05mmol/L, 0.1mmol/L, 0.3mmol/L, 0.5mmol/L, 0.8mmol/L, 1.0mmol/L induced expression products
Fig.6 Induction expression and purification of AMPs17 protein Lane M: Protein marker; Lanes 16: AMPs17 recombinant protein,inclusion body lysate, uninduced expression product,supernatant after cell disruption,cell pellet after precipitation,induced expression product
Fig.7 Identification of AMPs17 protein by his-tag serum
Fig.8 Determination of purity of AMPs17 protein
菌株 MIC(μg/ml) MBC(μg/ml)
AMPs17 FLC AMPs17 FLC
C. albicans
ATCC10231		 20 3.125 40 6.25
Table 1 Detection of antifungal activity of AMPs17 protein
Fig.9 Anti-C. albicans activity of AMPs17 protein
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