Chicken Parathyroid Hormone Gene Eukaryotic Expression Plasmid Construction And Effect Of Expression Studies

Parathyroid hormone is an important hormone in adjusting the metabolic balance of calcium and phosphor in vertebrate .It generates from parathyroid organ main cells and targets on bone, small intestine and kidney. Gene recombination technique was used in this study. RNA was extracted from 45 weeks new Yang zhou laying parathyroid organ . After then cDNA of PTH gene was amplified by RT-PCR, This segment of cDNA was inserted into vector pGEM-T through TA clone. DNA sequence analysis showed the base sequence has 100% homology with that published in GenBank.The problems of poor shell quality and broiler chicken leg disease become increasingly endangered to poultry production, we attempt to solve them by gene therapy. The segment of PTH gene cDNA was inserted into eukaryotic expression vector pCDNA3 and pCEP4. two eukaryotic expression construction , pCDNA3-PTH and pCEP4-PTH were constructed respectively. Then, they were transformated into bacteria DH5 a after screening and identification, we got the recombinant plasimd was extracted from recombinant and purified plasmid was injected into experimental chickens, 400ug per chicken. Two experimental groups were injected with pCDNA3-PTH and pCEP4-PTH, control group was treated with 400ul glucose(5%). Each group involved three chickens, plasmids were injected twice at two weeks interval. Blood samples were collected four times, and all the samples were stored at -70℃.All blood samples was analysed at the same time. Experiment results show that:(1) Insert the segment of cDNA into vector pGEM-T, sequencing analysis showed that the base sequence has 100% homology with that published in GenBank.(2) The injection of the eukaryotic expression constructions pCDNA3-PTH and pCEP4-PTH, would cause the phosphor decline and the calcium rise in serum of the chickens, there existed interaction between phosphor and calcium in the serum.(3) Serum concentrations of PTH,CT and E2 were detected by RIA. Following result were found serum CT and PTH level rise in the two experiment group on during the whole experiment.E2 level was on the rise significantly, the pCEP4-PTH group show a better level during the whole experiment.