The Expression Of C-terminal Parathyroid Hormone Peptide And Its Effect On Rat Osteoblastic Cell Proliferation

Parathyroid hormone(PTH),an important peptide hormone which is synthesized and secreted by parathyroid gland, can regulate the metabolization of calcium and phosphorus, also can regulate the transition of bone. There are three kinds of PTH molecules in blood circulation:PTH1-84,PTH37-84 and PTH7-84,the two later ones are called C-terminal parathyroid hormone peptide(C-PTH).Research discovered that PTH1-34 has the same function of receptor-combinding and the formation of bone, the American FDA has approved the parathyroid hormone(1～34) peptide (rh PTH1-34) to treat osteoporosis of post-menopause women.However, when treated with rhPTH1-34,the incidence rate of osteosarcoma in rats ascends distinctly (highly to 52%).Researchers recently discovered that there exists the receptor of C-PTH (CPTHR) in human body. When combinding with the special receptor, C-PTH can promote the biological effect of bioapoptosis.So we speculate that PTH1-34 may induce cancer, but C-PTH can inhibite the effect of PTH1-34.If the inference is true, there would be an important contribution in PTH function research.ObjectiveClone and express the recombinant human PTH1-84,PTH7-84 and PTH37-84 (rhPTH1-84,rhPTH7-84 and rhPTH37-84) with genetic engineering, through observing the effects on rat primary osteoblast cell multiplication of rhPTH1-84,rhPTH7-84,rhPTH37-84 and hPTH 1-34, to reveal the regulative function in Bone cell cycle and bone tumour of every kind of PTH peptide.Methods1.Gene amplification The original PTH E.coli expression vector PBV220/PTH1-84 as a template, high-fidelity DNA polymerase (KOD Fx) catalyst, primer introduced restriction endonuclease SalⅠand HindⅢrecognition sites, PCR amplification of PTH1-84, PTH7.84 and PTH37.84 coding region, agarose gel electrophoresis to check PCR product molecular weight.2.Construction of recombinant plasmid and sequence analysis Recovered PCR product was digested with restriction endonuclease SalⅠand HindⅢand was cloned into prokaryotic expression vector pQE-30Xa to construct pQE-30Xa/PTH1-84,pQE-30Xa/PTH7-84 and pQE-30Xa/PTH37-84.Every kind of prokaryotic expression plasmid was transformed into E.coli Trans-1 competent cells, after cultured, extract plasmid to digest with restriction enzyme, recombinant plasmids were selected for sequencing and expression.3.Expression of recombinant plasmid in prokaryotic cells Every kind of expression plasmid was transformed into E.coli M15 Competent cells, IPTG induced. Collect sonicating broken bacteria, supernatant and precipitate were collected with cryopreservation.4.SDS-PAGE and Western Blot Identification of expression products The supernatant and precipitation of broken bacterial concentration were identified with 15% SDS-PAGE, observe the expression of peptides;Gel electrophoresis of recombinant expression products of constant pressure on the nitrocellulose membrane switch membrane, goat anti-human PTH C-terminal polyclonal antibody for Western Blot identification, X-ray imaging observe the exposure situation.5.Purification and concentration determination of expression peptide Broken bacterial supernatant of a large number of expression products was purified with Ni2 +-IDA metal affinity chromatography, collecting eluate, BCA method for the determination of total protein concentration, purification using the Chemi Genius gel image analysis system purity of the recombinant protein to calculate peptide concentration.6.Effects of PTH fragments of various peptides on rat primary osteoblastic cell proliferation Recover cryopreserved primary rat osteoblasts to transfer and seed in 96 well plate, put the sub-cultured cells with the post-fusion state of different concentrations of rhPTH1-84, rhPTH1-34, rhPTH7-84 and rhPTH37-84 for 24 hours, using MTT assay to detect the absorbance in 490nm,observe cell proliferation.Results1.PCR amplification results PCR product was detected by 1.5% agarose gel electrophoresis, we can find the clear bands about 270 bp,250bp and 160bp which were the expected lengths. 2.Restriction analysis of recombinant plasmid Digest the recombinant plasmid pQE-30Xa/PTH1-84,pQE-30Xa/PTH7.84 and pQE-30Xa/PTH37-84 with SalⅠand HindⅢ,about 3500bp of the pQE-30Xa linear fragment and 270bp,250bp and 160bp fragment containing the recombinant gene were found. The result of DNA sequencing is fully consistent with expectation.3.Product identification through SDS-PAGE and Western Blot Induced products were identified by SDS-PAGE, Coomassie brilliant blue R-250 staining showed the existence of the protein bands of broken bacteria supernatant, the molecular weight was consistent with the expected size. Anti-human PTH carboxyl terminal polyclonal antibody identified by Western blot the significant band, confirmed bands for the C-terminal peptide containing the PTH peptide.4.Detect the concentration determination by BCA method The products were purified with nickel ion affinity chromatography, with BCA protein concentration determination and gel imaging analysis, calculate the final concentrations of rhPTH1-84,rhPTH7-84 and rhPTH37-84 were:0.243mg/ml,0.203mg/ml,0.180mg/ml.5.Effects of the rhPTH peptide fragments on primary rat osteoblast cell growth By MTT assay, The results of various peptides on rat primary osteoblastic cell proliferation rate shows:rhPTH1-84,rhPTH1-34 can promote the primary rat osteoblast proliferation, and rhPTH1-34 in promoting proliferation was significantly stronger than rhPTH1-84;rhPTH7-84 and rhPTH37-84 on rat primary osteoblasts showed inhibition proliferation, and rhPTH37-84 inhibition of proliferation was significantly stronger than rhPTH7-84.Conclusion1.Using PCR method, amplified the PTH1-84,PTH7-84 and PTH37-84 mature peptide gene, fusion expression plasmids were successfully constructed and confirmed DNA sequence is correct by sequencing.2.IPTG induced protein expression in prokaryotic system,Analysised by gel imaging system,the fusion polypeptide with 6 x His were obtained highly expressed in the M15 strain.3.Western Blot identified the fusion expression products rhPTH1-84,rhPTH7-84 and rhPTH37-84 have antigenicity.4.Purified by nickel ion affinity chromatography, the products obtained with high purity peptides and the concentration was ideal.5.The detection by MTT assay confirmed that rhPTH1-84 and rhPTH1-34 had the promotion of primary rat osteoblast proliferation, while rhPTH7-84 and rhPTH37-84 had inhibition of cell proliferation effect.