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Expression of Recombinant Human Amelogenin in Iranian Lizard Leishmania and Its Biological Function Assay.

ABSTRACT: Amelogenins are the major components of enamel matrix proteins. Enamel matrix derivatives (EMD) can be used in periodontal diseases to regenerate periodontal tissues. The main aim of this study was to evaluate expression of full-length functional recombinant human amelogenin (rhAm) in Iranian lizard Leishmania (I.L.L.) as an alternative eukaryotic expression system.Human cDNA encoding a 175-amino acid amelogenin expression cassette was sub cloned into a pLEXSY vector. The construct was transferred into Leishmania cells by electroporation. The protein production was surveyed in the transcription and the translation levels. The expressed protein was purified and some of its biological properties were investigated in comparison to EMD and negative control.Expression of rhAm was confirmed by RT-PCR and western blot test in Leishmania cells. Purified rhAm significantly inhibited the formation of tartrate-resistant acid phosphatase positive (TRAP(+)) multinuclear cells in calcitriol stimulated mouse marrow cultures. Moreover, it significantly promoted proliferation and DNA synthesis in L929 mouse fibroblast cells.Functional rhAm was successfully expressed in I.L.L. Easy handling and post translation modification were the main advantages of this expression system. It is suggested to investigate molecular properties of this rhAm in the future.

SUBMITTER: Yadegari Z

PROVIDER: S-EPMC4645770 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Expression of Recombinant Human Amelogenin in Iranian Lizard Leishmania and Its Biological Function Assay.

Yadegari Zahra Z Bandehpour Mojgan M Kazemi Bahram B Sharifi-Sarasiabi Khojasteh K

Iranian journal of public health 20150701 7

<h4>Background</h4>Amelogenins are the major components of enamel matrix proteins. Enamel matrix derivatives (EMD) can be used in periodontal diseases to regenerate periodontal tissues. The main aim of this study was to evaluate expression of full-length functional recombinant human amelogenin (rhAm) in Iranian lizard Leishmania (I.L.L.) as an alternative eukaryotic expression system.<h4>Methods</h4>Human cDNA encoding a 175-amino acid amelogenin expression cassette was sub cloned into a pLEXSY ...[more]

PMID: 26576377

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Project description:The recovery of impaired periodontium is still a challenge to the treatment of periodontitis. This study was the first to apply the mesoporous hydroxyapatites/chitosan (mHA/CS) composite scaffold to periodontal regeneration. The aim of our study is to evaluate the biological effects of mesoporous hydroxyapatite/chitosan (mHA/CS) loaded with recombinant human amelogenin (rhAm) on periodontal regeneration. The physicochemical properties of mHA/CS scaffolds were examined by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM), and Brunauer-Emmett-Teller (BET) analysis. Then, the biological effects of the mHA/CS loaded with rhAm were evaluated, including antibacterial effect, controlled-release capacity, osteogenic and cementogenic effects in vitro and in vivo. The antibacterial effect was tested on 1.5 mg/mL CS; 3 mg/mL mHA; 2.25 mg/mL mHA/CS; 4.5 mg/mL mHA/CS and 20 ?g/mL rhAm. Tryptic Soy Broth culture medium was used as a baseline control. Osteogenic effect of rhAm (20 ?g/mL rhAm), mHA/CS (4.5 mg/mL mHA/CS), and mHA/CS-rhAm (4.5 mg/mL mHA/CS and 20 ?g/mL rhAm) on human periodontal ligament cells (hPDLCs) was evaluated in osteogenic media. The hPDLCs treated either with osteogenic media or Dulbecco's modified Eagle's medium (DMEM) alone were used as the baseline control. In the animal model, 4-week-old nude mice (BALB/c) (n = 6) implanted with root slices subcutaneously were used to observe the cementogenic effect in vivo. The root slices were treated with rhAm (20 ?g/mL rhAm), mHA/CS (4.5 mg/mL mHA/CS), and mHA/CS-rhAm (4.5 mg/mL mHA/CS and 20 ?g/mL rhAm). The root slices treated with osteogenic medium alone were used as the baseline control. The analyses showed that the mHA/CS particles were 2 ?m in diameter and had a uniform pore size. The mesoporous structure was 7 nm in diameter and its surface area was 33.95 m2/g. The scaffold exhibited antibacterial effects against Fusobacterium nucleatum and Porphyromonas gingivalis. The mHA/CS scaffold sustainably released rhAm. The mHA/CS loaded with 20 ?g/mL rhAm upregulated ALP activity, the expression levels of osteogenesis-related genes and proteins in vitro. Additionally, it promoted the formation of cementum-like tissue in vivo. Our findings suggest that mHA/CS loaded with 20 ?g/mL rhAm could inhibit the growth of periodontal pathogens and promote the formation of bone and cementum-like tissue.

Dataset Information

Expression of Recombinant Human Amelogenin in Iranian Lizard Leishmania and Its Biological Function Assay.

Publications

Expression of Recombinant Human Amelogenin in Iranian Lizard Leishmania and Its Biological Function Assay.

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