Studies On The Cloning, Recombination And Expression Of Calcium Metabolism Regulation Related Genes Of The Chicken

Three pairs of primers were designed and synthesizd according to the gene sequences of calcitonin(CT), parathormone(PTH) and the D28k calcium-binding protein(D28k-CaBP) published in GenBank. The genes were amplified by RT-PCR technique from New Yangzhou chicken RNA. Five recombinant CT, PTH and D28k-CaBP eukaryotic expression system were constructed and the quality of the recombinant were evaluated by. injecting into the chicken leg muscle with the levels of the serum calcitonin, parathyroid hormone, estradiol, phosphorus and calcium. This research helps to provide both the necessary material for the mechanism investigation study of calcium nourishment regulation in chicken, and the important data for the gene strengthening therapy of the bone calcium metabolism disease in chickens.1. Cloning and sequence analysis of calcium metabolism modulator genesTotal RNA was isolated from fresh ultimobranchial gland, hyperparathyroid gland and the enteric epithelium of small intestines of four 450-days old of the New Yangzhou chickens. The cDNA of CT, PTH and D28k-CaBP were synthesized by the reverse transcription - polymerase chain reaction (RT-PCR) and cloned into pGEM-T easy plasmid. The recombinant plasmids were characterized with PCR, restriction endonuclease digestion and sequencing. The result is as follows:Three cDNA fragments were obtained with expected length of 417bps for CT, 360 bps for PTH and 800 bps for D28k-CaBP from total RNA extracted with the SV Total RNA Isolation System and primers designed in our lab. These three cDNAs were cloned into the pGEM-T by restriction endonulease digestion and ligation followed by PCR detection, sequencing and BLAST. BLAST suggests that the sequence of CT cDNA contains one basedifferent from the data in GenBank; The parathyroid hormone gene cDNA is completely in accordance with the GenBank record. D28k-CaBP cDNA has an one base difference as compared with the nucleotide sequence in GenBank.The amino acid sequences of calcitonin and parathyroid hormone derived from cDNA sequence analysis from our data did not show any differences as compared with the sequences in the publications although one nucleotide difference was detected in calcitonin cDNA sequence. However, amino acid sequence change has been found of D28k-CaBP in the 252 , with the amino acid valine converted to tryptophan due to a mutation of G to T in +760 site in the cDNA.Homologous comparison of the amino acid sequences of the three calcium modulators among animal genus and species showed that the conservative sequence occupies 75% of CT amino acid sequence of the ten species. The amino acid sequence of CT is homologous to eel with 96.9%, followed by salmon with 93.7%. There exists a big difference of amino acid sequence of parathyroid hormone between the chicken mammalian with the homologue of the sequences of the chicken to cattle, human and rat being 42.5%,35.4%,38.6% with 44.9% respectively. The homologues of amino acid sequence of D28k calcium binding protein among species are high, with that of the chicken to toad being 79.3%.2. The construction of the eukaryotic gene expression plasmids of calcium modulator genesThree calcium modulator genes were ligated to pcDNA3 and pCEP4 eukaryotic expression vectors for the construct of the eukaryotic gene expression plasmids of calcium modulator genes.The vector pcDNA3 and pCEP4 and the target gene CT fragment from recombinant were both cut with Bam H I and Hind III and ligated with T4 ligase for the construction of the CT expression plasmids.Parathyroid hormone gene and the vector pcDNA3 were both digested with EcoR I and BamH I and ligated with T4 ligase for the construction of the parathyroid hormone expression plasmids. To prepare the construct of parathyroid hormone gene expression plasmid using the vector pCEP4, parathyroid hormone gene was cut with EcoR I and Bamll I and the vector pCEP4 was cut with Pvu II followed by Klenow treatment and ligation with T4 ligase.Chicken D28k calcium-binding protein gene and the vector pCEP4 were cut by Xho I and BamH I and ligated w...