Zamani Ehsan, Zargan Jamil, Honari Hossein, Hajizade Abbas, Mohammadi Ashkan Haji Noor, Alikhani Hani Keshavarz, Heidari Ahmad, Pour Mohammad Hossein
Department of Biological Sciences, Faculty of Sciences, Imam Hossein University, Tehran, Iran.
Department of Biology, Razi University, Kermanshah, Iran.
Iran J Microbiol. 2021 Apr;13(2):235-242. doi: 10.18502/ijm.v13i2.5985.
antimicrobial peptide (AcAMP) is a fungi-derived peptide with a broad spectrum of activity against pathogenic bacteria and fungi. Natural antimicrobial peptides, including AcAMP, have attracted many attentions in the development of new natural antibiotics against pathogenic bacteria, especially multidrug resistant ones.
In the present study, gene was codon-optimized and chemically synthesized in pUC57 cloning vector, subcloned into pET28a (+) expression vector and transferred into competent BL21 (DE3) cells. The expression of AcAMP was induced by addition of Isopropyl β- d-1-thiogalactopyranoside (IPTG) and the expressed peptide was purified by Ni-NTA. BALB/c mice were immunized with the purified peptide and the ability of the immunized mice sera for the detection of the native AcAMP secreted by IRAN 142C was examined through ELISA and Western blotting techniques.
Both ELISA and Western blotting demonstrated the ability of the sera of the immunized mice to detect the native AcAMP.
The results of the present work show that the raised antibody against recombinant AcAMP can be used to detect AcAMP peptide, an issue which paves the way to develop detection kits for the detection of AcAMP-producing organisms, purification of this valuable peptide for further investigations.
抗菌肽（AcAMP）是一种源自真菌的肽，对病原菌和真菌具有广泛的活性。包括AcAMP在内的天然抗菌肽在开发针对病原菌尤其是多重耐药菌的新型天然抗生素方面引起了广泛关注。
在本研究中，对基因进行密码子优化并在pUC57克隆载体中化学合成，亚克隆到pET28a(+)表达载体中并转入感受态BL21(DE3)细胞。通过添加异丙基-β-D-硫代半乳糖苷（IPTG）诱导AcAMP的表达，并用镍-氮三乙酸（Ni-NTA）纯化表达的肽。用纯化的肽免疫BALB/c小鼠，并通过酶联免疫吸附测定（ELISA）和蛋白质印迹技术检测免疫小鼠血清检测IRAN 142C分泌的天然AcAMP的能力。
ELISA和蛋白质印迹均证明免疫小鼠血清能够检测天然AcAMP。
本研究结果表明，针对重组AcAMP产生的抗体可用于检测AcAMP肽，这为开发用于检测产AcAMP生物的检测试剂盒、纯化这种有价值的肽以进行进一步研究铺平了道路。
