Abstract: 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 1 μg/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.

Key words: AvBD2, Cell line, Stable expression, Lentiviral vector, Antibacterial activity

CLC Number: 

• S852.6