抗菌肽Cathelicidin-1真核表达及发酵液抑菌活性鉴定  

Eukaryotic expression of Cathelicidin-1 and validation of its antimicrobial activity in the fermentation broth

作　　者：李飞航 武浩恒 李宏[1] 李娟娟 马香[1] 唐燕琼[1] 刘柱[1] LI Feihang;WU Haoheng;LI Hong;LI Juanjuan;MA Xiang;TANG Yanqiong;LIU Zhu(School of Life Science,Hainan University,Haikou,Hainan 570228,China)

机构地区：[1]海南大学生命科学学院,海口570228

出　　处：《热带生物学报》2023年第5期474-480,共7页Journal of Tropical Biology

基　　金：海南省重点研发计划项目(ZDYF2021XDNY181)。

摘　　要：为了获得体外表达的高活性抗菌肽,通过分子克隆技术构建了抗菌肽Cathelicidin-1重组载体,在毕赤酵母(Pichia pastoris)GS115中表达,并鉴定包含该抗菌肽的发酵液的抑菌活性。通过DNA全合成技术合成编码抗菌肽Cathelicidin-1的全长基因,采用PCR技术扩增靶标基因并与真核表达载体pGAPZαA连接,获得重组质粒pGAPZαA-Cathelicidin-1;将重组质粒转化至大肠杆菌DH5α中扩增,提取质粒并经单酶切处理后转化毕赤酵母GS115;使用YPD培养基培养酵母,72 h后收集发酵液,通过Tricine-SDS-PAGE检测抗菌肽表达,并通过抑菌圈与抑菌曲线测定鉴定发酵液的抑菌活性。结果表明,抗菌肽Cathelicidin-1以分泌肽形式表达于发酵液中,并且该发酵液能够显著抑制大肠杆菌(Escherichia coli)的生长,而对金黄色葡萄球菌(Staphylococcus aureus)则无明显抑菌效果。In order to obtain exogenously expressed antimicrobial peptide,cathelicidin-1 expression recombinant vector was constructed using molecular cloning technique,and the secreted cathelicidin-1 was expressed in Pichia pastoris.The antimicrobial activity of the fermentation broth containing Cathelicidin-1 was evaluated.The full-length gene encoding cathelicidin-1 was synthesized by total DNA synthesis and amplified by PCR.The full-length gene was linked to the eukaryotic expression vector pGAPZαA,and the recombinant plasmid pGAPZαA-Cathelicidin-1 was transformed into Escherichia coli DH5αfor multiplication.The multiple plasmids were extracted,single digested,and transformed into the yeast Pichia pastoris GS115.The transformed yeast was cultured in the YPD culture medium,and the fermentation broth was collected after 72 hrs of culture and used to express the antimicrobial peptide Cathelicidin-1 in a secreted form by using Tricine-SDS-PAGE.The antimicrobial activities of the fermentation broth were evaluated by using inhibition zone of the antimicrobial peptide and the microbial growth curve.Measurements of inhibition zone and growth curve showed that the fermentation broth from the yeast expressing Cathelicidin-1 exhibited significant inhibitory activity against E.coli,but did not show any bacteriostatic effect on Staphylococcus aureus.

关 键 词：抗菌肽 真核表达 发酵液 抑菌效果 

分 类 号：Q78[生物学—分子生物学] TQ920.1[轻工技术与工程—发酵工程]