| Gentamicin(Gent)is an aminoglycoside antibiotic commonly used to treat Gram-negative bacterial infection.It has been extensively used for treatment and growth promotion in livestock management.However,Gent has significant adverse effects such as brain neurotoxicity,loss of hearing or of vestibular function(ototoxicity)and impairment of renal function(nephrotoxicity).The overuse of Gent in animal husbandry could result into accumulation of its residues in animal derived foods.The presence of Gent residues in food has been considered as high risk for consumption due to its adverse effects.In order to monitor and control the antibiotic abuse,the regulatory authorities in EU and China have established the maximum residue limits(MRL)of Gent in food stuffs.Based the previous work,this study explores the feasibility of the chicken scFv in antibiotic residues detection and establishes a rapid ic-ELISA detection method.The research contents are as follows:1.Synthesis and identification of Gent artificial antigenAccording to the characteristic of structure of Gent,Gent was linked to carrier proteins,bovine serum albumin(BSA)and ovalbumin(OVA),through glutaradehyde method.The results of ultraviolet spectrophotometry(UV),SDS-PAGE proved that the artificial antigen Gent-BSA and Gent-OVA were successfully synthesized.2.Selection of anti-Gent chicken-derived scFv antibody and prokaryotic expressionAfter the fourth booster immunization of Gent-BSA,the chicken was euthanized and RNA was extracted from chicken spleen,the cDNA was synthesized by reverse transcriptase.The heavy and light chain variable regions(VH and VL)were amplified by PCR.Then the VH and VL were subjected to overlap PCR with a linker to full length scFv fragments,which were further digested with Sfi I and Not I.The digested scFv fragments were cloned into the pCANTAB5 E vector subsequently and recombinant vector was transformed into E.coli TG1 strain to construct phage display antibody library.After four rounds of bio-panning,the special antibody was effectively enrichment and the size of each library was 3.7×105、6.7×106、3.71×109、1.65×109.Thirty three clones were randomly selected from the fourth library and detected by phage-ELISA.Two phage-scFvs which had strong reactivity in phage-ELISA were selected and the genes of the two scFvs were cloned into pET-30 a vector.The recombinant plasmids were transformed into BL21(DE3)strain.The scFvs antibodies were expressed in E.coli.BL21(DE3)as existed in a form of inclusion body.After denaturation and renaturation,they were used in the subsequent experiments.3.Development of Gent ic-ELISAAccording to the optimal working conditions,a rapid ic-ELISA detection method of Gent was established and the feasibility of this method was evaluated through the cross-reaction and spiked recovery test.The results showed that the liner regression equations were S-1: y=0.74407-0.23033x(R2=0.9849)and S-5: y=0.76066-0.23479x(R2=0.97475).The IC50 were 12.418ng/mL and 14.674ng/mL,respectively.The cross-reactivity with analogues of Gent(kanamycin and amikacin)were both less than 0.04%.The spiked recovery test indicated that the recovery rate in intra-group was from 70.70% to 118.09%,and the recovery rate in inter-group was from 60.91% and 118.04%.All results showed that the method was feasible in residue detection.In the present study,the artificial antigen of Gent was successfully synthesized,chicken anti-Gent phage display antibody library was constructed and high specific scFv antibody was obtained.The ic-ELISA method based anti-Gent scFv antibody specially aimed at the Gent residue detection has been developed,which is contribute to the development of the kit of the Gent residue detection.This study also proved that the chicken scFv can be used in antibiotics residue analysis. |
