Preparation Of McAbs Against Chicken CARP And Its Expression During Skeletal Muscle Development

CARP, a protein containing an ankyrin repeat domain, two PEST sequence and a nucleus localization signal (NLS), is as a member of the Muscle Ankyrin Repeat Protein family (MARP). It has been reported to be a novel nuclear transcription cofactor. Recently, CARP has been identified as a novel marker of cardiovascular disease. There is a high expression of CARP in fetal, neonatal and adult heart. As a multifunctional factor, the information on the molecular functions of CARP suggests that the gene plays a critical role in angiogenesis and vasculogenesis, cardiac development, transcriptional regulation, myofibrillar assembly, stretch sensing, and in communication between the sarcoplasm and the nucleus. Later, it has been found that CARP is specifically expressed in skeletal muscle tissue of chicken, which is different from that in mammals, suggesting its potential functions in myogenesis. To investigate its expression during myogenesis of chicken, the recombinant chicken CARP protein and anti-chicken CARP McAbs were prepared and its expression at the levels of translation during skeletal muscle development was assayed in this study.The chicken CARP cDNA encoding the N-terminal 110aa was obtained by RT-PCR and cloned into prokaryotic expression vector pET28b, and then recombinant plasmid was transformed into E. coli strain BL21 (DE3). The recombinant bacterias were induced with IPTG, and the expressed CARP recombinant protein was purified with MagneHis? Protein Purification and analyzed by SDS-PAGE. A soluble protein Mr about 18000 was detected, and its purity can reach 88.5% by analysis with the Bandscan software.The BALB/c mice were immunized with the purified recombinant chicken CARP, the spleen cells from the immuned mice were obtained and fused with myeloma cells. The positive clones were screened by indirect ELISA, and cloned by limiting dilutions.The hybridoma cells secreting McAB at high titers were selected, the ascitic fluid from the imice inoculated with hybridomas in abdominal cavity was prepared and the McAbs were purified. Then the total proteins of skeletal muscle from chicken embryogenesis to adult were extracted, the expression level of CARP was detected by Western blot.The subtype of the monoclonal antibody was detected by isotype analysis kit, and the chromosomal amounts of hybridoma cells were acounted. The specificity of the monoclonal antibody was tested by Western blot. Finally, two hybridoma strains were proved to have the capability of stably secreting monoclonal antibody against chicken CARP, designated as 4A8 and 4E6. The subtype of monoclonal antibodies was IgG1 and the light chain wasκ. The chromosomal amounts of them were more than 89. The McAb titers of ascites were 3.2×104, 1.28×105 and that of the culture supernatant of hybridomas were 2.56×103, 2.56×103 respectively. The McAbs could recognize a protein with approximate molecular weight 36 000 in chicken skeletal muscle by Western blot and they had no cross reaction with CARP of mouse. At the level of translation, CARP was expressed only in skeletal muscle after birth in chicken.Two mouse anti-chicken hybridomas and their secreted monoclonal antibodies have been successfully obtained. They provide useful reagent for further study on the biological function of CARP chicken myogenesis, and the results suggest that CARP play an important role during in skeletal muscle development of chicken.