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Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b.

ABSTRACT: Interferon alpha 2b (IFN?-2b) is an important cytokine and used for antiviral and anticancer treatment. The low cost production of IFN?-2b with high biological activity is necessary to provide the interferon therapy to the hepatitis patients in Pakistan. In the present study, human interferon alpha 2b (hIFN?-2b) gene from a healthy person was cloned and overexpressed in E. coli BL21(DE3). The molecular weight of the expressed hIFN?-2b is 19 kDa. The over expressed recombinant hIFN?-2b was checked by ELISA using antibodies raised against commercially available hIFN?-2b. The biocomputational analysis of recombinant hIFN?-2b gene showed the 99.9% nucleotide sequence and 100% deduced amino acid sequence homology with reported sequences of IFN?-2b. The predicted 3D-structure showed mainly five ?-helices, one 310 helix and two disulfide bonds at Cys1-Cys98 and Cys129-Cys138. The amino acid sequence alignment indicated that the disulfide linkage position is conserved in all IFN? family members. On the basis of sequence homology among interferon alpha family, new potent variants of hIFN?-2b with enhance efficacy can be produced. Indigenous production of IFN?-2b from gene of local population will reduce the cost and increase tolerability of interferon therapy.

SUBMITTER: Gull I

PROVIDER: S-EPMC3695685 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b.

Gull Iram I Samra Zahoor Qadir ZQ Aslam Muhammad Shahbaz MS Athar Muhammad Amin MA

SpringerPlus 20130615 1

Interferon alpha 2b (IFNα-2b) is an important cytokine and used for antiviral and anticancer treatment. The low cost production of IFNα-2b with high biological activity is necessary to provide the interferon therapy to the hepatitis patients in Pakistan. In the present study, human interferon alpha 2b (hIFNα-2b) gene from a healthy person was cloned and overexpressed in E. coli BL21(DE3). The molecular weight of the expressed hIFNα-2b is 19 kDa. The over expressed recombinant hIFNα-2b was checke ...[more]

PMID: 23875128

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Project description:BackgroundLike most protein macromolecular drugs, the half-life of rhIFNɑ-2b is short, with a low drug utilization rate, and the preparation and release conditions significantly affect its stability.MethodsA nanoporous ion-responsive targeted drug delivery system (PIRTDDS) was designed to improve drug availability of rhIFNα-2b and target it to the lung passively with sustained release. Chitosan rhIFNα-2b carboxymethyl nanoporous microspheres (CS-rhIFNα-2b-CCPM) were prepared by the column method. Here, an electrostatic self-assembly technique was undertaken to improve and sustain rhIFNα-2b release rate.ResultsThe size distribution of the microspheres was 5~15 μm, and the microspheres contained nanopores 300~400 nm in diameter. The in vitro release results showed that rhIFNα-2b and CCPM were mainly bound by ionic bonds. After self-assembling, the release mechanism was transformed into being membrane diffusion. The accumulative release amount for 24 hrs was 83.89%. Results from circular dichrogram and SDS-PAGE electrophoresis showed that there was no significant change in the secondary structure and purity of rhIFNα-2b. Results from inhibition rate experiments for A549 cell proliferation showed that the antitumor activity of CS-rhIFNα-2b-CCPM for 24 hrs retained 91.98% of the stock solution, which proved that the drug-loaded nanoporous microspheres maintained good drug activity. In vivo pharmacokinetic experimental results showed that the drugs in CS-rhIFNα-2b-CCPM can still be detected in vivo after 24 hrs, equivalent to the stock solution at 6 hrs, which indicated that CS-rhIFNα-2b-CCPM had a certain sustained-release effect in vivo. The results of in vivo tissue distribution showed that CS-rhIFNα-2b-CCPM was mainly concentrated in the lungs of mice (1.85 times the stock solution). The pharmacodynamics results showed that CS-rhIFNα-2b-CCPM had an obvious antitumor effect, and the tumor inhibition efficiency was 29.2%.ConclusionThe results suggested a novel sustained-release formulation with higher drug availability and better lung targeting from CS-rhIFNα-2b-CCPM compared to the reference (the stock solution of rhIFNα-2b), and, thus, should be further studied.

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Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b.

Publications

Heterologous expression, immunochemical and computational analysis of recombinant human interferon alpha 2b.

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