The Initial Research Of The Preparation Of Humanized Mouse Anti-human CTnI Antibody

Background:Cardiac troponin I (cTnl) is a basic regulatory protein found as part of a ternary complex responsible for calcium dependent muscle constraction. It is a highly sensitive and specific marker of myocardial injury. There was a significant association between the presence of a positive cTnI assay and the occurrence of a cardiovascular event. Anti-cTnl antibody has been widely used in the diagnosis of myocardium injury. The hybridoma which secret the anti-cTnl antibody effectively were obtained in our laboratory. The anti-cTnl antibody kit has been used to detect the serum cTnI of the patients with myocardial injury.But the use of murine antibodies in patients is problematic, owing to their decreased serum half-lives and induction of human anti-mouse antibody immune responses directed mainly against mouse Ig constant (C) region. So the anti-cTnl antibody must be humanized to be used as the vector to treat the cardiac injury diseases in vivo. The first generation humanized antibody is chimeric antibody which is relatively easy to be prepared. The chimeric antibody is full and could be retained in the body for a long time. The affinity and specificity of the antibody is reserved. No reports about the humanization of anti-cTnl antibody has been foundpresently. Aim:To obtain the murine anti-cTnl Fab fragment and analyse the nucelotide and deduced amino acid sequences; to get the variable region gene and to construct the chimeric antibody cTnl eukaryon expression vectors which be transfected into the SP2/0 cell lines; to detect the chimeric antibody expressed protein by ELISA, RT-PCR and Western Blot and lay the foundation of binding some drug with the chimeric antibody and treating the cardiac injury disease. Methods:1. One step method was used to extract total RNA from hybridoma cells secreting anti-cTnl antibody and Fab fragment gene were amplified by reverse transcription-polymerase chain reaction (RT-PCR) method. The PCR products were inseted into pMD-18 T vector. The recombinant vectors were then transformed into Ecoli DH5ot and sequenced.2. The variable region genes were lined out and the primers with signal peptide gene and reastriction endonuclease sites of EcoR V, Nhe I , Sal I were designed. Total RNA was isolated from hybridoma cells and the variable region genes were amplified by RT-PCR method. The PCR products were inseted into pMD-18 T vector. The recombinant vectors were transformed into Ecoli DH5a and sequenced. The variable region genes were digested and inserted into the pAG4622, pAH4604 whichcontain human k and human IgGl constant regions, respectively. The eukaryon expression vectors pAG4622-LV and pAH4604-HV were constrcucted and cotransfected into myeloma cells SP2/0. The mycophenolic acid at 1.5ug/mL was used to select the surviving cell clones and the positive cell clones were selected by screening ELISA 20 days later. The positive cell clones were further detected by RT-PCR and western blot method. Results:1. Two bands of 723bp,654bp DNA fragments were amplified using IgGl heavy chain primers and Klight chain primers of Fab fragment.2. Two bands of 405bp, 402bp were amplified using IgG heavy chain primers and Klight chain primers which were verified as the variable region genes by sequencing.3. The eukaryon expression vectors pAH4604-HV, pAG4622-LV were constrcucted successfully by enzyme digestion and PCR.4. The recombinant vectors were cotransfected into the SP2/0 cells by lipofectamine or electroporation method and the cell clones were screened by mycophenolic acid. Over 40 positive cell clones were identified by screening ELISA 20 days later. The positive incidence was 30% or so which is consistent with the literature reports.5. Total RNA was isolated from positive cells and two bands of about 350 base pairs were amplified by RT-PCR method which indicated...