稳定表达禽β防御素2的DF-1细胞系的建立及其抗大肠杆菌活性

Establishment of DF-1 Cell Line Stably Expressing Avianβ-defensin 2 and Its Anti-Escherichia coli Activity

旨在建立一株稳定表达禽β防御素2(AvBD2)的细胞系,并检测细胞系分泌产物对耐药菌株的抑制效果。首先,根据同源重组设计方法设计一组含有同源臂的AvBD2基因扩增引物及载体反向扩增引物,将片段连接至pLOV-eGFP真核表达载体,构建真核重组质粒pLOV-eGFP-AvBD2,利用三质粒表达系统将重组质粒与慢病毒包装质粒pSPAX2、外膜质粒pMD2.G共转染293T细胞,收集细胞分泌上清液感染DF-1细胞,嘌呤霉素最适浓度筛选获得阳性克隆细胞系,通过RT-PCR技术和Western Blot方法对该细胞系构建结果加以验证;将细胞系分泌产物与耐药大肠杆菌菌株混合培养后,检测该蛋白抗菌效果,并通过扫描电镜观察菌体的损伤现象。结果表明,已成功构建一种稳定表达AvBD2蛋白的DF-1细胞系,其最适筛选浓度为含有1μg/mL的嘌呤霉素的细胞培养液。该细胞系传递20代后,在mRNA转录水平及蛋白表达水平均验证正确,将细胞系的分泌产物培养耐药大肠杆菌,细菌存活率曲线显示随着培养时间的增加,细胞系上清可明显抑制耐药菌株的增殖,在培养第5小时使供试菌的存活率低于50%。扫描电镜显示供试菌体表面皱缩,存在明显损伤,而对照组菌体光滑饱满。建立了稳定表达AvBD2蛋白的DF-1细胞系,为抗生素替代品的生产制备提供探索方向,同时也为后续评价和研究AvBD2的抗菌作用奠定基础。

This experiment aims to establish a cell line stably expressing avian beta defensin 2(AvBD2),and to detect the inhibitory effect of the secreted product of the cell line on drug-resistant strains.First,according to the homologous recombination design method,a set of AvBD2 gene amplification primers containing homology arms and vector reverse amplification primers were designed,and the fragments were ligated to the pLOV-eGFP eukaryotic expression vector to construct the recombinant plasmid pLOV-eGFP-AvBD2.The recombinant plasmid and the lentiviral packaging plasmid pSPAX2 and the outer membrane plasmid pMD2.G were co-transfected into 293T cells using a three-plasmid expression system,and the cell secretion supernatant was collected to infect DF-1 cells.The optimal concentration of puromycin was screened to obtain positive clones.The results of the cell line construction were verified by RT-PCR technology and Western Blot method;the secreted product of the cell line was mixed with drug-resistant Escherichia coli strains to detect the antibacterial effect,and the damage of the bacteria was observed by scanning electron microscope.The results showed that a DF-1 cell line stably expressing AvBD2 protein had been successfully constructed,and its optimal selection concentration was a cell culture medium containing 1μg/mL puromycin.After 20 generations of the cell line,the mRNA transcription level and protein expression level were verified correctly.The secreted product of the cell line was cultured in Escherichia coli.The bacterial growth curve showed that as the culture time increased,the supernatant of the cell line could significantly inhibit drug-resistant strains.The proliferation of the test bacteria makes the survival rate of the tested bacteria less than 50%at the 5th hour of culture.Scanning electron microscopy showed that the surface of the test bacteria was shrunk and there was obvious damage,while the control group was smooth and full.This study established a DF-1 cell line stably expressing AvBD2,which provided an exploration direction for the production and preparation of antibiotic substitutes,and also laid a foundation for the subsequent evaluation and study of the antibacterial effect of AvBD2.