Preparation Of Specific Nanobody Of Dectin-1 And Its Anti-inflammatory Effect In Fungal Keratitis

Objective:To prepare specific nanobody of Dectin-1 and verify its anti-inflammatory effect in fungal keratitis.Methods:1.The helper phage M13K07 was infected with the phage library to obtain the phage released from the host bacteria.After three rounds of panning with recombinant Dectin-1at concentrations of 100,10,and 1 μg/m L,polyclonal phage indirect ELISA method was used to verify the specific phage is enriched.Spreaded the phage obtained in the third round of panning into a dish and picked 95 single colonies.Monoclonal phage indirect ELISA method was used to verify the specificity of the phage carrying the monoclonal gene specific to Dectin-1 nanobody.2.First,the target gene of the specific nanobody of Dectin-1 was cloned by PCR technology.The gene sites of Bam H I and Not I were selected to cut the p ET28 a plasmid.T4 DNA ligase was used to ligate the target gene with the plasmid after digestion.3.Introduced the expression vector plasmid into competent cells,added IPTG at a final concentration of 0.5 mmol/L and cultured at 37°C for 4 hours and 16°C overnight.4.The cells after culture were broken with an ultrasonic cell disruptor,and the supernatant and precipitate were subjected to 12% SDS-PAGE respectively to verify that the expression of the nanobody.5.8 M urea was used to denature the protein in the inclusion body to make it a soluble protein.Purify the soluble protein with a nickel ion column,using 20 mmol/L,50 mmol/L,100 mmol/L,200 mmol/L,500 mmol/L imidazole eluted protein.6.Used the Bradford method to determine the concentration of the protein.Indirect ELISA and immunofluorescence were used to verify the specificity of Dectin-1 nanobody to Dectin-1 recombinant protein and Dectin-1 expressed in HCECs and RAW264.7 cells.7.Mice models of A.fumigatus keratitis were divided into PBS control group and nanobody treatment group.The corneas of each group of mice at different modeling time points are recorded with a slit lamp.Clinical score was used to evaluate the severity of A.fumigatus keratitis.rt-PCR and ELISA were used to verify the effect of Dectin-1nanobody on the m RNA and protein expression levels of IL-1β and IL-6 in HCECs and mice cornea after stimulation by A.fumigatus.Results:1.Three rounds of panning were performed on the phage carrying the nanobody gene.The indirect ELISA results showed that there was no significant difference between the phage OD450 nm after the first round of panning and the negative control.The OD450 nm increased significantly after three rounds of panning.A total of 26 positive clones were selected,and the positive rate was 27%.Four specific phages were selected for ELISA verification experiments.The results showed that the OD450 nm of the four specific phages were all three times higher than the negative control value.The results showed that the specific nanobody gene band appears at about 500 bp.The phage vector is extracted and sent for sequencing to determine the gene sequence of the nanobody.The sequencing results showed that the target gene of Dectin-1 nanobody was 357 bp.The NCBI comparison results showed that the gene was of shark origin.2.The optimal expression condition of nanobody was to induce expression at 16°C overnight after adding a final concentration of 0.5 mmol/L IPTG.The results of 12% SDSPAGE showed that there were obvious protein bands around 17 k Da.Nanobody specific to Dectin-1 was expressed in inclusion bodies.The protein was purified by a nickel ion column.The results of 12% SDS-PAGE showed that 20 mmol/L imidazole can wash out impurities in the protein,and 100 mmol/L imidazole can completely wash out the target protein.After purification,the purified protein was renatured by dialysis and concentrated by ultrafiltration.After 12% SDS-PAGE,there was a single protein band around 17 k Da.3.The indirect ELISA method showed that the OD450 nm of the specific Dectin-1nanobody at a concentration of 5 mg/m L reached about 3.0,and the OD450 nm decreased with the decrease of the nanobody concentration.The OD450 nm of 0.5 mg/m L was still 3times higher than that of the negative control.There was no significant difference between the OD450 nm of the negative nanobody and the blank control.The immunofluorescence showed that the cell membrane and cytoplasm of the HCECs in the Dectin-1 nanobody treatment group showed strong fluorescence,and the fluorescence in the cells stimulated by the A.fumigatus hyphae was enhanced.The strong fluorescence can be seen in the cell membrane and cytoplasm of RAW264.7 cells after stimulation.4.After 3 and 5 days infected by A.fumigatus,compared with the PBS control group,specific nanobody of Dectin-1 treatment can alleviate the severity of fungal keratitis of mice and reduced the clinical score.Compared with PBS control group IL-1β,IL-6 in corneas infected for 3 days after nanobody treatment were significantly decreased.5.Sepecific nanobody of Dectin-1 pretreatment of HCECs stimulated by A.fumigatus can reduce the m RNA and protein expression levels of inflammatory cytokines IL-1β and IL-6.Conclusion:In summary,nanobodies can be successfully expressed through the prokaryotic expression system,and the expression yield is high.Nanobodies specific to Dectin-1 have high affinity to Dectin-1 antigen,which can alleviate the severity of A.fumigatus keratitis in mice,reduce clinical scores,and decrease the expression of inflammatory cytokines.These results indicated that the specific nanobody of Dectin-1 has a great potential to be used as a new anti-inflammatory drug for the treatment of fungal keratitis.