Cloning of anti-human CD4 single chain variable fragment (scFv) with fluorescent tag in pCold I

Single-chain variable fragment (scFv) is a fusion protein of the light and heavy chain variable domains of immunoglobulin, connected with a short linker peptide of 25 amino acids. ScFvs offer several advantages compared to parent monoclonal antibodies like better pharmacokinetic properties and the relative ease of producing in large quantities, atlow cost. ScFvs are powerful tools as new reagents in biological research, diagnostic imaging, tumor therapy and treatmentof neurodegenerative and infectious diseases. CD4 is a surface glycoprotein expressed on helper T cells and other cells.Aim: In this study in other to improve the protein quality, anti-human CD4 scFv in fusion with a fluorescent protein( FP) was cloned in pCold I system. Method: FP-scFv fragment in pAB1 plasmid was amplified by PCR using FPup and scFvdown primers containing EcoRI and HindIII restriction sites respectively and digested with the enzymes. Then the digested vector and insert were ligated using T4 ligase. The competent cells were prepared from E.coli DH5α by using calcium chloride. Then the recombinantplasmid was introduced into competent E.coli DH5α and the bacteria were allowed to grow on medium containingampicilin. The cloning reaction was confirmed using PCR, double digestion and sequencing.Results: The expected band with1506 bp length for FP-scFv on agarose gel was obtained. After performing double digestion on pCold I, the expected band with 4,407bp length was obtained. After PCR and double digestion on the recombinant plasmid, the expected bands were obtained.

کلیدواژه ها:

ScFv ، FP ، pAB1 ، pCold I ، DH5α