Construction of recombinant plasmid of chicken calbindin-D28k
鸡CaBP-D28k基因重组质粒构建

To construct the eukaryotic expression vector of pCEP4-CaBP-D28k,a pair of primers was designed according to chicken calbindin-D28k(CaBP-D28k) gene sequence published in GenBank.Chicken CaBP-D28k gene was amplified by RT-PCR from chicken intestine total RNA extracts and was cloned into eukaryotic expression vector pCEP4,and transferred into COS-1,The gene expression was detected by indirect immunofluorescent assay(IFA).The result shows that the cloned CaBP-D28k gene,encoding 261 amino acids,was 789 bp in length which was 99.9% identity but only one nucleotide changed from G to T at site 760 compared with published sequence from GenBank database.The homology of CaBP-D28k gene sequence between chicken and other species such as frog,house mouse,human was 79.7%,78.2%,78.6%,respectively;the target segment in recombinant plasmid was characterized and confirmed by restrictive endonuclease assay as well as sequence analysis.The expression of CaBP-D28k could be detected in COS-1.This research provides a novel means to study regulation mechanism of calcium metabolism in chickens through pCEP4-CaBP-D28k.