الفهرس 
LITERATURE REVIEWOnly 14 pages are availabe for public view
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Abstract
Summary
Human IGF-1 is a 70 amino-acid peptide with a molecular mass of 7.6 kDa and three conserved cysteine disulfide bridges. hIGF-1 controls proliferation and differentiation in a range of cell types.
The present study, the IGF-1 was cloned and expressed in E. coli system, nanoencapsulation of purified rhIGF-1, and then to study its biological activity. The pBSK (+) vector harboring 228bp IGF-1 gene was transformed into TOP10 chemically competent cells of E. coli.
Successful transformation was confirmed using several techniques including PCR technique which revealed a sharp band with " ~ " 228 bp on 2% agarose gel electrophoresis, minipreparation technique showed a specific band at MW 3145 bp for recombinant vector, and Restriction analysis was performed using NcoI and BamHI restriction enzymes resulting in two fragments; one for a plasmid of 2917 bp and the other for insert of 228 bp comparing to undigested vector which give one band at 3145 bp which were separated on 1% agarose gel electrophoresis. The IGF-1 DNA was excised and eluted from agarose gel using GeneJETTM Gel Extraction Kit; the released insert IGF-1 was ligated into the linearized pET-15b expression vector then transformed into Top10 competent cells. the transformed colonies were picked from agar LB plates carrying the amplified gene were analyzed by PCR using IGF-1 cloning primers using T7 universal PCR primer and reverse IGF-1primer (R) also and lysed by miniprep Kit to prepare plasmid DNA. Electrophoresis evaluated the DNA on 1% agarose gel giving sharp band which represent recombinant vector pET-15b containing IGF-1 gene at expected molecular weight 5.9 kb which referencing to 1kb DNA ladder. The rhIGF-1 has been expressed successfully in Rosetta (DE3) pLysS bacterial cell when induced with 2mM IPTG. SDS-PAGE electrophoresis of bacterial lysate revealed a protein of MW 7.6 kDa, which is the MW of rhIGF-1. In contrast, we did not detect such band in a lysate of non-induced bacterial cells. Confirmation of rhIGF-1 expression in Rosetta (DE3) pLysS bacteria was performed via western blot technique. The Cell lysates were size separated by SDS-PAGE and transferred onto PVDF membrane. The blot was probed with the rabbit anti- hIGF-1 antibody as a primary antibody and anti-rabbit IgG conjugated with alkaline phosphatase as a secondary antibody. One specific band has a molecular weight of 7.6 kDa was recognized in both; the expressed rhIGF-1 protein and the standard rhIGF-1 on the membrane. After preparation of the induced bacterial cells, we obtained 33% of the whole recombinant bacterial pellet was IBs containing rhIGF-1 protein. In order to solubilize rhIGF-1 IBs isolated from bacterial cell lysates, different concentrations of chaotropic agent as GdmCl 6, 7 and 8 M were used. Data showed that the maximum level of soluble form hIGF-1 protein was achieved at 6 M. In this study, the optimum pH buffer for high rhIGF-1 protein refolding was at pH 9 and concentration of 2 M L-arginine gave the highest O.D595 which was 0.561. In order to maximize protein recovery and buffer exchange, the refolded rhIGF-1 protein was concentrated before application to the chromatography, the buffers which were used 20 mM Tris HCl pH 8 and 20 mM Tris HCl pH 8.5. Total protein concentration was 1.8 mg/ml which used as mixture sample for chromatographic separation.
In order to purify the rhIGF-1, we tried to obtain IGF-1 from one chromatographic step without any tag fusion. We used anion exchange chromatographic columns using different experimental conditions. First, we made trial 1 by using DEAE column (weak anion column); all proteins did not bind to the column matrix and get out during wash step. Then we made trial 2 by changing the column to ANX column (strong anion column) which did not work and mixture components get out with a low resolution at wash step. Finally, we change pH for buffers which used in trial 3, this change gave 3 peaks were separated, 2 peaks represent all proteins were come out the column without separation in the washing step and another one peak was separated as a good peak. Therefore, these peaks scouted by 15% SDS-polyacrylamide gel, which gave a single band at peak 3 which represented the successfully purified of rhIGF-1 protein with MW 7.6 kDa referring to rhIGF-1 standard protein. The concentration of purified rhIGF-1 protein was 300µg/ml, which has been measured using NanoDROP 2000C spectrophotometer. Also, identification of the purified recombinant protein was specifically confirmed by ELISA assay. The protein concentration of purified rhIGF-1 was 300 µg/ml using curvilinear equation at O.D 405 nm.
There are several techniques for drug delivery which are used for therapeutic proteins. One of the major strategies is protein nanoencapsulation which increasinge drug solubility and biocompatibility. The chitosan/rhIGF-1 nanocapsule was prepared and its particle size, zeta potential, and the polydispersity index (PDI) were determined by zetasizer instrument. from the acquired results, The particle size distribution of empty chitosan nanoencapsules were 132±6 nm and increased to be 172.2±8 nm after successful upload of hIGF-1 forming chitosan/rhIGF-1 nanocapsules as shown in figures (30, 31), and also the nanometric size was 171.8 nm and dark-colored spherical capsular structure of nanocapsules was confirmed by using TEM analyses. PDI of the loaded chitosan nanocapsule with rhIGF-1 increased from 0.216 ± 0.02 to 0.4 ± 0.04. Zeta potential of empty chitosan nanoparticles is +21.1 ± 4 which is considered strongly cationic, this value increased to be +30.7 ± 2 after loading of chitosan nanoparticles with IGF-1 indicating increase colloidal stability of charged particles.
In the present study, the successful encapsulation with efficiency was calculated to be 99.3 %. The proliferation activity of the purified rhIGF-1 was testified on a vero cells to ensure its safety for further processing and application. Crystal violet assay was used to measure the cell proliferation and survival. Dose-dependent effect on cell proliferation was observed between the concentrations of 12.5-100 μg/ml. A previous study showed marked elevation in cell proliferation of NP cells and the dose-dependent effect was found between the concentrations of 10-100 μg/L of IGF-1. The proliferation activity of our purified rhIGF-1 in international units at cell count 7000 cells generated from the equation obtained from the standard curve of reference rhIGF-1 used as a standard was found to be 2604.17 IU/mg which was stronger than the reference rhIGF-1 that possesses an activity of 1000 IU/ml at the same tested concentration. Cell proliferation ratio of rhIGF-1 protein was two-times higher than nanoencapsulated rhIGF-1 referencing to cell control. We performed in vitro drug release test which show the rhIGF-1 release percent was higher at pH 7.4 at 120 h was 96%, whereas it was lower at pH 5.5 was 93%.We found that the rhIGF-1 release percent at 7.4 was approximately 50% after 24 h incubation, that explain the data obtained from cell proliferation ratio of rhIGF-1 protein was two-times higher than nanoencapsulated rhIGF-1 referencing to cell control which performed after 24 h incubation.
Conclusion
The successful expression of the rhIGF-1 protein was performed using pET based expression system.
Refolding and purification techniques have been optimized which resulting high purity and biological active rhIGF-1. Nanoencapsulation and drug release study of chitosan/ rhIGF-1 nanocapsules have been studied.
To complete the characterization of this molecule in vivo study for stability and activity should be carried out to validate the therapeutic efficacy of the developed rhIGF-1