ABSTRACT
To get the mature peptide of human-derived neurotrophin-6 (NT-6), NT-6 gene encoding mature peptide was amplified by PCR, using the NT-6 cDNA that had been cloned as templet. The gene encoding mature peptide of NT-6 gene was cloned into pGEX1-lambdaT plasmid to construct the fusion expression vector. Expression of fusion protein in Escherichia coli was defected after induction by isopropyl beta-D-thiogalactoside(IPTG). The mature peptide of NT-6 was collected with GST fusion protein purifying kit. It was shown that a fragment of 460bp was gained by PCR. With the techniques of double-cleave and electrophoresis, the recombinant vector was identified as pGEX1-NT-6. The recombinant vector pGEX1-NT-6 transformed Escherichia coli expressed fusion protein of 41KD after induction by IPTG. Cleaved by thrombin, the mature peptide of NT-6 was obtained; its molecular weight was about 15KD. The cloning and expression of human-derived NT-6 gene encoding mature protein has provided a basis for further studies on the function and clinical application of NT-6.
Subject(s)
Humans , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Isopropyl Thiogalactoside , Pharmacology , Nerve Growth Factors , Genetics , Peptides , Genetics , Recombinant Fusion Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To clone human brain-derived neurotrophin-6(NT-6) gene and to observe its expression in the procaryotic cell.</p><p><b>METHODS</b>Total RNA was extracted from aborted antenatal cerebral cortex, and cDNA fragment of NT-6 was amplified through reverse transcript-polymerase chain reaction. After being incised and recovered, the NT-6 gene was cloned into pBK-CMV plasmid to construct a NT-6 gene expression vector. Expression of NT-6 gene in Escherichia coli was studied after being induced by isopropyl beta-D-thiogalactoside(IPTG).</p><p><b>RESULTS</b>The NT-6 gene expression vector was constructed and Escherichia coli with recombinant vector expressed specific protein after induction by IPTG.</p><p><b>CONCLUSION</b>The cloning of human brain-derived NT-6 gene provides a basis for further studying the structure, function and clinical application of NT-6.</p>