Research About High Expression And Immunologic Activity Of ScFv Against Clenbuterol

The research is for the purpose of exploring the anti-Clenbuterol single chain antibody activity. Several kinds of single chain antibody which reject easy residual medicine in food are going to be connected and methods which are used to fastly detect the easy residual medicine in food to be established .The experiment will lay the foundation for establishing the method.Primers are designed according to the consensus sequence in varable region of monoclonal antibody against Clenbuterol. By PCR technology single chain antibody gene fragment is cloned from the hybridoma cell, secreting monoclonal antibody of anti-Clenbuterol. After CL ScFv and pMD18-T connected, the gene fragment was sequenced by Shanghai Sangon. The single chain antibody gene fragment contain 742bp, including restriction Enzyme sites, primer, goal gene sequence and linker,of the total 11-719 basic group for CL ScFv sequence,coding 236 amino acids residues. Analyzed on the the Blast database of National Center for Biotechnology Information, the target sequence had the characters of variable domain of reconstructed functional mouse antibody. The sequence of VH include three obvious CDR region and FR region and the sequence of VL include three obvious CDR region and two FR region.The characteristic dissulfide linkage formed between the 21st Cys residue and 98st Cys residue.The secondary structure and physico-chemical property were forcasted on Expasy website. The result is as follow, secondary structure, Sequence length: 235; Alpha helix:14 is 5.96%; Extended strand:102 is 43.40%; Random coil:119 is 50.64%. physico-chemical property, Total number of atoms:3432; Formula: C1096H1670N298O357S11; Molecular weight:25085.7; Theoretical pI:8.28 Estimated half-life:The N-terminal of the sequence considered is M (Met).The estimated half-life is: 30hours (mammalian reticulocytes, in vitro):>20hours (yeast, in vivo):>10hours (Escherichia coli, in vivo). Instability index: The instability index (II) is computed to be 53.56;This classifies the protein as unstable;Aliphatic index: 55.19; Grand average of hydropathicity (GRAVY): -0.355.Prokaryotic expression genetic engineering bacterium CL ScFv-pET-22b-DE3 and eukaryotic expression genetic engineering bacterium CL ScFv-pPIC9-GS115 were constructed and differently induced by IPTG and methanol to express the interest protein. Genetic engineering bacteria CL ScFv-pET22b-DE3 was induced by IPTG with the final concentration 0.75mmol/L. Analyzed with SDS-PAGE, the expressed interest protein to be biggest expression quantity induced for 5h at 37℃. The interest protein occupies the total thallus protein 21.732%. The interest protein was expressed in two forms, dissolbability protein and cytoryctes, majority interest protein expressed as cytoryctes. The interest protein expressed all night at 25℃was dissolbability protein. The interest protein occupies the total thallus protein 11.532%. Induced by methanol, the genetic engineering bacteria CL ScFv-pPIC9 was cultivated for 3-6d at 28℃. Analyzed with SDS-PAGE, the interest protein is 25ku.Complete antigen was obtained with conjugating CL and BSA by diazotization. Protein-L as dis-antibody, the interest protein was detected with indirect ELISA. Results show CL ScFv with high immunologic competence. The immunologic activity of protein expressed in GS115 was obviously higher than the immunologic activity of protein expressed in E.coli DE3.