Expression And Purification Of TAT-NLS-Nkx6.2Fusion Protein In E.Coli And Its Distribution In Brain

Homeodomain transcription factor Nkx6.2plays a critical role embryonically in neuroblastes and postnatally in oligodendrocytes. Our previous study indicated that thyroid hormone deficiency during nervous system development causes altered noble nervous cells with reduced expression of Nkx6.2. However, the effects of down-regulated Nkx6.2in the brain development retardation caused by thyroid hormone deficiency have not been characterized. By administration of exogenous Nkx6.2to the model of Hypothyroidism, this problem may be solved, or partly solved.Moreover. In contrast to conventional gene transfer strategies, the direct introduction of recombinant protein into cells bypasses the risk of insertional mutagenesis and offers an alternative genetic intervention.So in this research, we engineer a TAT-protein transduction domain (TAT-PTD) and nuclear location sequence (NLS) into transcription factor Nkx6.2protein. firstly, we extract the total RNA from the rat brain and reverse transcribed to cDNA. perform polymerase chain reaction (PCR) to clone the exons of rat Nkx6.2coding gene while the coding sequences of TAT and NLS are synthesized chemically in vitro. And then the target sequences are inserted into prokaryotic fusion expression vector pQE-30Xa. Finally the recombinant plasmid pQE-30Xa/TAT-NLS-Nkx6.2(TN-Nkx6.2) is transformed into E.coli Ml5[pREP4] for expression. At the end, a recombinant cell-permeant form of Nkx6.2protein is generated. Labeled with Cy7-N-hydroxysuccinimide ester (Cy7NHS), this protein is i.v. injected into the tail vein of KM mice. Using the in vivo imaging system and immunofluorescence histochemistry. we find that TN-Nkx6.2can pass through the blood-brain barrier(BBB) and biomembrane efficiently and itself has no obviously biotoxicity.