Preparation Of Gene Engineering Antibody Against Sulfonamides And Aflatoxin B1

With the continuous development of the economy, people's standard of living have improved constantly, people's dietary needs have been changing while the demand for livestock and poultry food have been increasing year by year, veterinary drug residues and toxin residues have been becoming a focus concerned by public as well. In recent years,more and more reports of abuse of the indiscriminate use of veterinary drugs and toxins.The residues of veterinary drugs and toxins are not only harmful to the growth and reproduction of livestock and poultry, but also may cause adverse effects on human health,what's more, may pollut the environment and destroy the balance of ecosystem. To this end, we urgently need to establish a rapid and effective security testing technology as a technical support.In this study, the total RNA was extracted from the hybrid tumor cell lines secreting SAs monoclonal antibodies, then reversely transcribed into cDNA. Taking the cDNA as a template and using Degenerate Primers, SAs-scFv gene fragments were obtained by PCR amplification which were the. variable region of heavy chain (VH) and light chain variable region (VL) . VH/VL were connectted with pMD18-T respectively, then sequencing. At the same time, after purification of the monoclonal antibodies in the ascites of mice, Fab fragment was obtained by hydrolysis by using papaya protease. After SDS-PAGE detection, VH and VL fragments in the Fab fragments were obtained. By Orbitrap mass spectrometry, we. have known their amino acid sequences. The VH and VL variable region sequences were compared and analyzed by the results of mass spectrometry obtaining the variable region sequence of the specific antibody of SAs while the VH (VH7)is 333bp in length, the length of the variable region of VL (VL1/20) 339bp and 339 BP respectively. The VL variable regions were connected with VH variable region (VH7)by Linker with overlapping extension PCR which obtained SAs-scFv gene fragment of 711bp in length. Using restriction endonuclease, SAs-scFv-pMD18-T plasmid was digested and connected with the expression vector pLIP6/GN after the same enzyme cut, chemical transferred into BL21 (DE3) competentcells. The expression conditions were optimized,in 20?, 200 rpm, 12h, while the final concentration of IPTG was 0.05 mmol/L. To induce the expression of strain, we obtained anti sulfonamides scFv alkaline phosphatase fusion protein (SAs-scFv-AP) . SAs-scFv-AP was purified. And the activity of purified SAs-scFv-AP detected by ELISA Assay is 65.45%. And the activity of purified AFB1-scFv-AP detected is 47.47%.