Studies On The Construction Of Calbindin-D28k Gene's Eukaryotic Expression Plasimd And Its Effect

Calbindin-D28k are a group of intracellular proteins which is adjusted by active vitamin D. Calbindin-D28k and Calbindin-D9k are dubbed vitamin D-dependent calcium-binding proteins, they have high affinity with calcium and play important role in the course of calcium intracellular transport in intestine and kidney. It is important subject of nutrition and physiology that we have profound and careful investigation on Calbindin-D28k. There are tremendous academic value and utilitarian prospect. In the research we attempted to establish eukaryotic expression plasmid chicken of Calbindin-D28k and express in chicken body making use of molecular biology technology, through which to study the calcium nourishment modulated mechanism of avian. Further finding a kind of feasibility to resolve some ailments as a result of calcium deficiency in production by making use of injection. This research is preliminary probing toward Calbindin-D28k function, as a basis of Calbindin-D28k further investigation and exploitation. We enlarge 789 base pairs integrated chicken Calbindin-D28k cDNA from laying hen' intestine RNA through RT-PCR method. Through TA cloning we clone Calbindin-D28k cDNA into vector pGEM-T, which is identified by enzyme digestion and PCR, afterwards we measure and assay the validity of Calbindin-D28k cDNA sequence.In order to verify cloned Calbindin-D28k cDNA encoding condition, we establish eukaryotic expression plasmid pCEP4-CaBP-D28k, which is made by Calbindin-D28k cDNA cloning into vector pCEP4. COS-1 cell carry out transient expression, which is transfectioned by pCEP4-CaBP-D28k. Immunity fiuorescein look over expressing product. Injecting pCEP4-CaBP-D28k into laying hen, serum concentration of CT, PTH, E2, Ca and P were detected to study the expressing of recombinant plasmid in chicken body.The experiment results showed that:1. The whole sequence analysis of chicken demonstrated the Calbindin-D28k cDNA we got had only one nucleotide difference with the published chicken Calbindin-D28k cDNA, which certified we got was chicken Calbindin-D28k cDNA.2. Identified by enzyme digestion and PCR, recombinant eukaryotic expression plasmid chicken Calbindin-D28k cDNA was success constructed.3. Immunity fluorescein method examined a lot expressing product, which certified recombinant plasmid chicken Calbindin-D28k cDNA could be expressed correctly.4. Compared with the controlled group, injection of recombinant plasmid chicken Calbindin-D28k cDNA caused obvious upswing of Ca in serum and serum concentration of CT, but P in serum had a little decline, it made striking decline of PTH and EI.