摘要
【目的】为建立能够稳定表达禽β-防御素9(AvBD9)的DF-1细胞系,并检测其表达产物对耐药大肠杆菌的抗菌活性。【方法】将AvBD9基因克隆至慢病毒载体pLOV-eGFP中,将重组质粒与psPAX2和pMD2.G共转染293T细胞,包装成含AvBD9基因的重组慢病毒,并将其感染DF-1细胞,通过嘌呤霉素筛选和纯化,获得稳定表达AvBD9蛋白的DF-1细胞系。利用RT-PCR和Western blot验证AvBD9基因在DF-1中的转录和表达;将细胞培养上清液与耐药大肠杆菌混合培养,检测其抗菌效果,并用扫描电镜观察其对菌体的损伤情况。【结果】筛选出了稳定表达AvBD9的DF-1细胞系,且AvBD9在转录水平和蛋白水平均有表达;细胞培养上清使耐药大肠杆菌的存活率低于55%,电镜观察显示菌体皱缩,损伤严重。【结论】成功建立能够稳定表达AvBD9的DF-1细胞系,为抗生素替代品的生产制备提供了借鉴思路,为进一步开展AvBD9的抗菌研究提供依据。
[Objective]This study was designed to establish a DF-1 cell line stably expressing avianβ-defensin 9(AvBD9)by infecting DF-1 cells with recombinant lentivirus containing the AvBD9 mature peptide gene,and to detect the antibacterial activity of its expression products against antibiotic-resistant Escherichia coli.[Method]The AvBD9 mature peptide gene was cloned into the lentiviral expression plasmid pLOV-eGFP,and the constructed recombinant plasmid pLOV-eGFP-AvBD9 was co-transfected into 293T cells with packaging plasmid psPAX2 and outer membrane plasmid pMD2.G.DF-1 cells were infected with recombinant lentivirus containing the AvBD9 mature peptide gene,and a DF-1 cell line stably expressing the AvBD9 protein was obtained by puromycin pressure screening and purification.Transcription and expression of AvBD9 gene in this cell line were verified by RT-PCR and Western blot.The cell culture supernatant was mixed with antibiotic-resistant Escherichia coli to detect its antibacterial effect,and its damage to the cells was observed by scanning electron microscopy.[Result]The DF-1 cell line stably expressing AvBD9 was screened and the target gene was expressed at the transcriptional level and protein level.The cell culture supernatant made the survival rate of drug-resistant Escherichia coli less than 55%.Electron microscopic observation showed that the bacteria collapsed and the damage was serious.[Conclusion]This experiment successfully established a DF-1 cell line capable of stably expressing AvBD9 protein,which provided a reference for the production and preparation of antibiotic substitutes,and laid a foundation for further antibacterial research of AvBD9.
作者
熊修琦
耿姝
李芳果
殷冬冬
薛媚
邵颖
涂健
宋祥军
祁克宗
XIONG Xiu-qi;GENG Shu;LI Fang-guo;YIN Dong-dong;XUE Mei;SHAO Ying;TU Jian;SONG Xiang-jun;QI Ke-zong(Anhui Province Key Laboratory of Veterinary Pathobiology and Disease Control,Anhui Agricultural University,Hefei 230036,China)
出处
《江西农业大学学报》
CAS
CSCD
北大核心
2020年第3期530-536,共7页
Acta Agriculturae Universitatis Jiangxiensis
基金
国家自然科学基金资助项目(31772707)
安徽高校协同创新项目(GXXT-2019-035)。
