Preparation Of Single Chain Variable Fragment(scFv) Against Citreoviridin And Establishment Of The Detection Method

Citreoviridin(CIT),a mainly toxic secondary metabolite,has been produced by Penicillium citreoviride in the environment of low temperature and high humidity.Studies have shown that CIT is a noncompetitive inhibitor of the ATP synthase of oxidative phosphorylation of the respiratory chain reaction,which resulted in the failure of the ATP synthesis and the causes of the disease of cardiac beriberi.At the same time,CIT in animals can cause vomiting,convulsions,shock and inhibition of central nervous system symptoms.It has been reported that the contamination levels of CIT in crop in the target areas of China reached 4.9?33.2 ?g/kg.However,some related safety limits of CIT in food products have not been established by relevant administration until now.The related detection methods were limited in the conventional methods such as High performance liquid chromatography(HPLC),Thin-layer chomatography techniques(TLC),UV spectrum,IR spectrum,Fluorescence detection,Liquid chromatography-mass spectrometry(LC-MS)and Gas chromatography-mass spectrometry(GC-MS).However,these methods are usually time-consuming and expensive,and not suitable for the detection of a large number of samples.In addition,detection of CIT based on immunological method was only monoclonal antibody assay.Unfortunately,the application of mAb in detection was limited severely as its long preparation period and the instability of screting antibody in the process of hybridoma cells.Hence,it is essential to develop a efficient and cheap method to detect CIT toxin in real crop samples.In this study,the anti-CIT hybridoma cells were used to isolate the total RNA.First-strand cDNA was synthesized by reverse transcription PCR(RT-PCR).The coding sequences for the variable regions of the heavy chain(VH)and light chain(VL)were amplified from the first-strand cDNA.A specific linker DNA fragment encoding a short flexible peptide(Gly4Ser)3 was used to assemble scFv gene fragments by SOE-PCR.The assembled scFv fragment was cloned into the pBD-his-mbp-linker expression vector.After IPTG induction,the affinity constant of the purified scFv antibody(Kaff=4.3 × 108L/mol)was determined.Subsequently,the original assembled scFv was used to construct the anti-CIT scFv mutant library by error-prone PCR,generating a capacity of 2×108 pfu/mL mutated phage display library.After bio-panning,the selected scFv-5A10 displayed higher affinity and specificity to CIT antigen,with an increased affinity of 13.25-fold(Kaff=5.7×109 L/mol)to the original scFv.Two critical amino acids(P100 and T151)distributed in H-CDR3 and L-FR1 regions that responsible for the affinity of scFv-5A10 to CIT antigen were found and verified by sequencing and oligonucleotide-directed mutagenesis analysis.Indirect competitive ELISA(ic-ELISA)indicated that the linear range to detect CIT was 25?562 ng/mL with IC50 of 120 ng/mL.The limit of detection was 14.7 ng/mL,and the recovery average was(90.612±3.889)%.The study laid a certain foundation for the establishment of the CIT method based on immunological detection and the development of the high quality test kit.