| Autor: |
Kahroba, Houman, Galehdari, Hamid, Shafeei, Mohammad, Khodadadi, Ali, Khatami, Saeed Reza, Jalali, Fatemeh |
| Předmět: |
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| Zdroj: |
Zahedan Journal of Research in Medical Sciences; Jul2016, Vol. 18 Issue 7, p1-6, 6p, 1 Color Photograph, 2 Graphs |
| Abstrakt: |
Background: Human leukemia inhibitory factor (hLIF) is a poly functional cytokine with numerous regulatory effects on different cells. Main application of hLIF is maintaining pluripotency of embryonic stem cells. hLIF indicated effective work in implantation rate of fertilized eggs and multiple sclerosis (MS) treatment. Low production of hLIF in eukaryotic cells and prokaryotic host’s problems for human protein production convinced us to develop a simple way to reach high amount of this widely used clinical and research factor. Objectives: In this study we want to purify recombinant human leukemia inhibitory factor in single simple method. Materials and Methods: This is an experimental study, gene expression: human LIF gene was codon optimized for expression in Escherichia coli and attached his-tag tail to make it extractable. After construction and transformation of vector to E. coli, isopropyl -D-1-thiogalactopyranoside (IPTG) used for induction. Single step immobilized metal affinity chromatography (IMAC) used for purification confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and western blotting. Bioactivity of the hLIF were tested by MTT assay with TF-1 cells and CISH gene stimulation in monocyte and TF-1 by real-time PCR. Induction by 0.4 mM of IPTG in 25°C for 3 hours indicated best result for soluble expression. SPSS indicated P [U+02C2] 0.05 that is significant for our work. Results: Cloning, expression, and extraction of bio active rhLIF was successfully achieved according MTT assay and real time PCR after treatment of TF-1 and monocyte cell lines. Conclusions: We developed an effective single step purification method to produce bioactive recombinant hLIF in E. coli. For the first time we used CISH gene stimulating for bioactivity test for qualifying of recombinant hLIF for application. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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