A novel method to evaluate myocardial injury: cloning of anti-cTnI murine antibody Fab fragment and DNA sequence analysis

Yanyan LI; Di YANG; Jiannan LIU; Zhiping BIAN; Jindan XU; Xiangjian CHEN; Chunrang GU; Jinan ZHANG

ABSTRACT

BACKGROUND:

To apply mouse anti-human cTnI monoclonal antibody as the drug vector in the treatment and diagnosis of myocardial injury, it is important to degrade the immunity of murine antibody and overcome human anti-mouse reaction. Humanization has been applied as an attempt to resolve this problem.

OBJECTIVE:

To clone murine anti-cTnI Fab fragment and analyse the nucleotide and deduced amino acid sequences.

DESIGN:

Single sample study.

SETTING:

An institute of cardiovascular disease under a medical university-affiliated hospitalMATERIALS The study was conducted in the Institute of Cardiovascular Diseases, First Affiliated Hospital of Nanjing Medical University from January 2003 to May 2004. The hybridoma cell line JS200202 which secrets the anti-cTnI monoclonal antibody was provided by Institute of Cardiovascular Disease, First Affiliated Hospital of Nanjing Medical University.

METHODS:

IgG heavy chain primers and κ light chain primers of amplified mouse were designed. Total RNA was extracted from hybridoma cells which secrete cTnI. Reverse transcription polymerase chain reaction(RT-PCR) was amplified. Cloning and subsequent sequence analysis of the Fab fragment was performed. The deduced amino acid sequence was compared and analysed with previously published sequences.MAIN OUTCOME

MEASURES:

Heavy chain Fd segment and κ light chain gene sequence and its subgroups.

RESULTS:

A band of approximate 700 and 800 base pairs were amplified using IgG heavy chain primers and κ light chain primers respectively. Sequence analysis indicated that the deduced amino acid sequences were in consistent with the characterization of the amino acid in the murine IgGl Fab fragment(GenBank accession NO AY484430, AY484431; Protein Bank accession NO AAR83243, AAR83244).

CONCLUSION:

A complete murine anti-cTnI Fab fragment was obtained in this study, which may provide basis for the production of the chimeric anti-cTnI antibody.