ABSTRACT
BACKGROUND: Previous studies found that C57BL/6 mouse was susceptible to atherosclerosis, while BALB/c mouse was resistant to atherosclerosis. The stenosis of the culprit vessel and the severity of myocardial infarction were correlated to the levels of OX40L expression. Whether OX40L has differential expression between C57BL/6 and BALB/c mouse was not identified.OBJECTIVE: To observe the differential expression of OX40L mRNA and protein in C57BL/6 and BALB/c mouse.METHODS: Total RNA and protein were extracted by Trizol and RIPA Buffer from heart, brain, kidney, skeletal muscle and spleen tissues of C57BL/6 and BALB/c mice. RT-PCR and Western Blot were used to detect OX40L mRNA and protein expression in heart, brain, kidney, spleen and skeletal muscle of two kinds of mice. The differential expression of OX40L mRNA and protein between C57BL/6 and BALB/c mice was observed.RESULTS AND CONCLUSION: RT-PCR results showed that the mRNA expression level of OX40L in heart of C57BL/6 mice mouse was significantly higher than BALB/c mice (P ＜ 0.05); the mRNA expression level of OX40L in spleen of BALB/c mice was significantly higher than C57BL/6 mice (P ＜ 0.05). There were no significant differences in the brain, kidney and skeletal muscle between these two strains. The results of Western Blot showed that the protein expression level of OX40L in heart, brain and kidney of C57BL/6 mice were significantly higher than BALB/c mice (P ＜ 0.05). There were no significant differences in skeletal muscle and spleen between these two strains. The OX40L mRNA transcription level in heart was higher in C57BL/6 mouse than BALB/c mouse, while the expression in spleen was lower than the latter. The OX40L protein levels in C57BL/6 mouse heart, brainand kidney were higher than BALB/c mouse. The differences of OX40L expression between the two strains of mice indicated that OX40L may promote to C57BL/6 mouse susceptible to atherosclerosis.
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.