| Much attention has been pay to the hazard of sulfadiazine (SD) residues in foods and ecological environment, for these residues can cause serious problems including human health and huge economic losses. It is important to develop a rapid and accurate method for detection of SD. In this dissertation, the artificial antibody against SD was prepared by the molecular imprinting technology. Sulfadiazine residues in the pork were detected by the molecular imprinted column coupling with high performance liquid chromatography method. Single-chain variable fragment against SD was prepared by the modern molecular biotechnology and the gene engineering technology. The phage library which the gene of single-chain variable fragment against SD mutated was constructed. The main research results are as follows:1. Such association between the functional monomer and the template molecule has been confirmed by the Ultra Violet absorption spectra and nuclear magnetic resonance spectroscopy (’HNMR). It was clearly observed that the decreased absorption peak of the mixtures and the redshift effect were attributed to an association between the monomer and template molecules via hydrogen bonding. In the mixture solution, the amino group (-NH2) and the carbanyl group (C=O) in a molecule of Acrylic acid (AM) can interact with the functional group (amino group and carbanyl group) of the template molecule, leading to the formation of the precursor through hydrogen bonding.2. The Molecularly imprinted polymers (MIP) for SD was synthesized using bulk polymerization. The characters of MIP were described by the scanning electron microscopy (SEM), the Fourier transform infrared spectrometry, Brunauer-Emmett-Teller (BET), and the adsorption kinetic experiment. It was noted that the surface of the MIP exhibited more porous structure, the BET surface area of the MIP was182.2m2g-1, and the adsorption amount of the MIP for SD reached7.8mg/g. The phenomenons showed that the MIP could be successfully synthesized using bulk polymerization.3. The molecularly imprinted solid-phase extraction (MISPE) column was selected as an extraction device. To obtain the optimum extraction efficiency, several parameters related to the molecular imprinted column, including column solvent, extraction flow-rate, eluent of the sample matrix and eluent volume, were investigated. The recovery of SD was99%under the optimum conditions which the acetonitrile with2.5%of the volume fraction was selected for the column solvent, the flow-rate of column solvent was2mL/min, the mixture of acetonitrile and water (2/3, v/v) solution was the optimum eluent, and the3mL of eluent was selected for the eluent volume.4. To test the SD response linearity, a series of standard solutions of SD in the concentration range50-4000μg kg-1were analyzed, using blank sample extraction solvent. The relationship between peak area (y) and sample concentration (x) was linear for SD according to the equation y=0.063x+0.0294,(R2=0.9998),(n=3). The limit of detection (LOD) and the limit of quantification (LOQ) are10.0μg kg-1and33.3μg kg-1respectively. It can be seen that the recovery of SD in the spiked pork samples treated by the MISPE column are more than75.6%, and the relative standard deviation (RSD) values of them are found to be less than6.1%. The proposed method has been successfully applied to monitoring sulfadiazine residues in pork.5. The gene of scFv against SD was prepared by the reverse transcription-polymerase chain reaction (RT-PCR) technology and the splicing by overlap extension PCR (SOE-PCR) technology. The phy-chemical properties of scFv against SD were analyzed through the bioinformatics methods. The heavy chain and light chain of single-chain variable fragment against sulfadiazine hold four FR and three CDR in the antibody from Murine, respectively. It was not found that the sequence of single-chain variable fragment against sulfadiazine was not the same sequence in the NCBI DNA Blast. The homologous rate was83.7%between the gene of heavy chain and IGHV1-14*01. The homologous rate was91.6%between the gene of light chain and IGKV6-20*01. It is known that the molecular weight is26582.4, the molecular formula is C1174H1779N313O376S9, the isoelectric point is7.6, the hydrophilic index is-0.5484, and the instability index is49.38by ExPasy software. The scFv against SD belongs to instability protein. 6. The prokaryotic expression vector pET22b-SD with the gene of the scFv against SD was prepared by the molecular biotechnology and the gene engineering technology. The expressed conditions were optimized by the single factor experiment mothed. The scFv against SD were expressed as inclusion bodies in E.coli BL21(DE3). The results showed the optimum induced conditions that the induced temperature was30℃, the concentration of IPTG was300μM, the pH was7, the coefficient of charge was30%, and the expression time was8h. Western blotting and Enzyme-linked immunosorbent assay (ELISA) confirmed that the scFv against SD expressed in the E.coli BL21(DE3) had bioactivity.7. The gene of scFv against SD was cloned to the secreting type expression vector pPIC9K. The expression vector pPIC9K-SD was transformed to Pichia pas tor is G115. The Muts type of Pichia past or is GS115recombinants were screened by both MM and MD plates. Multi-copy Pichia pastor is GS115were screened by the YPD agar plates containing G418. The scFv against SD in the Pichia pas tor is GS115was successfully secreting expressed under the methanol induction.8. The gene of the scFv against SD was random mutated by the error-prone PCR technology. The mutation rate was1.3%under the optimal conditions that the concentration of Mg2+was7mmol/L, the concentration of dATP and dGTP was0.2mmol/L respectively, the concentrations of dCTP and dTTP was1mmol/L respectively, and the concentration of Mn2+was0.3mmol/L. A/T mutation rate was higher than G/C mutation rate. The storage capacity of phage library which the gene of the scFv against SD was mutated was1.26x106. |
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