Expression And Purification Of TAT-NLS-Nkx6.2Fusion Protein In E.Coli And The Effects On The Differentiation Of Neural Stem Cells

Objective:To investigate the homeobox gene Nkx6.2thyroxine deficiency leads to molecular mechanisms in the brain retardation obtained crossing the blood-brain barrier and transmembrane the TN-Nkx6.2restructuring into the nucleusproteins. This paper aims to observe the homeobox gene Nkx6.2morphological study of development and differentiation of neural stem cells. Laid the material foundation to further explore the mechanism of the homeobox gene Nkx6.2in the model that thyroid reduces.Methods:this experiment uses gene cloning technique, gene TAT-NLS-Nkx6.2and plasmid pET-28a were digested, connection using T4ligase, the TAT-NLS-Nkx6.2and Nkx6.2genes were cloned into pET-28a expression vector, the recombinant plasmid pET-28a-TN-Nkx6.2and pET-28a-Nkx6.2, and the recombinant plasmid pET-28a-TN-Nkx6.2and pET-28a-Nkx6.2into E.Coli DH5a, then transformed into Escherichia coli E.Coli (DE3) BL21expression, by enzyme digestion and sequencing of recombinant enzyme digestion and sequencing, the recombinant plasmid was correctly,which was preserved in10%glycerol, streak, taking one colony to anti Kan LB culture medium overnight, according to1:50inoculated in LB liquid anti Kan medium, when the OD value is about0.6, the target protein was induced expression by IPTG, and induced temperature on induction conditions such as IPTG concentration, induction time, optimization,fusion protein was identified by SDS-PAGE and Western Blot.pET-28a vector had fusion tags His-Tag for detection and purification of the target protein and fusion protein was purified by the histidine Ni2+affinity chromatography.Fusion protein TN-Nkx6.2, Nkx6.2, with the different concentration,respectively,with the the neural stem cells,the NF-200, GFAP, Gal-C were detected by immunohistochemistry to observe differentiated cell types.Results:Enzyme digestion and sequencing confirmed that the recombinant plasmids pET-28a-TN-Nkx6.2and pET-28a-Nkx6.2were constructed successfully, according to1:50inoculation access flask,37℃,250r/min medium,about4h, when the bacterial suspension and the OD was0.6, adding IPTG,lmmol/L,at25℃after8h, the yield of fusion protein is the highest in the E.Coli BL21(DE3) in the high expression of TN-Nkx6.2. Results showed that the molecular weight and the expected with the expression of target protein with the histidine by SDS-PAGE electrophoresis analysis, Ni2+affinity chromatography, the purity of fusion proteins is high. Identificated by Western Blot, the purified fusion protein has immunogenicity and good reactivity and fusion proteins are TN-Nkx6.2and Nkx6.2.By fluorescence microscope observation, only the fusion protein TN-Nkx6.2can pass through the cell membrane into neural stem cells and effect of neural stem cell development. Gal-C positive cells was observed by fluorescence microscopy more than75%, contribute to the development of oligodendrocytes direction, in particular in order to increase the experimental group, the most obvious of200μg/L,500μg/L fusion protein TN-Nkx6.2impact development and differentiation of neural stem cells.Conclusion:The success recombinant plasmid pET-28a-TN-Nkx6.2in E. coli BL21(DE3) highly expressed fusion protein TN-Nkx6.2, and it can pass through the cell membrane into neural stem cells. As an endogenous protein, fusion protein TN-Nkx6.2has a certain impact on differentiation of neural stem cells that induces neural stem cells differentiation to oligodendrocytes direction.