pET-CCL19/BSP41  BioVector NTCC质粒载体菌种细胞基因保藏中心

pET-CCL19/BSP41  BioVector NTCC质粒载体菌种细胞基因保藏中心
Construction of pET-CCL19/BSP41 plasmid and expression of CCL19 monomer: The cDNA sequence for mature human CCL19-chemokine was produced by PCR amplification with primers CCL19.5P (5′-GGAATTCCATATGGGAACAAATGATGCTGAAGACTGC-3′ (SEQ ID NO:______)) and CCL19.3P (5′-CGGGATCCACTGCTGCGGCGCTTCAT-3′ (SEQ ID NO:______)), using the plasmid AI-pTyT30 (IncyteGenomics, St. Louis, Mo.) as a template. CCL19.5P contains a Nde I-restrictase adapter sequence, followed by the initiating ATG codon and the nucleotides complementary to the 5′ portion of the CCL19 coding sequence. CCL19.3P contains BamH I-restrictase adaptor sequence and nucleotides complementary to the 3′ portion of the CCL19 coding sequence preceding the TAA stop codon. The amplified DNA fragment was digested with Nde I and BamH I and inserted into the pJA1 plasmid linearized with the same enzymes. In this construct, the nucleotide coding sequence for mature CCL19 was placed downstream of the T7 promoter and upstream of the BSP41 biotinylation site coding sequence followed by a TGA-stop codon. The orientation of the insert and the authenticity of its sequence was verified by nucleotide sequence analysis. The resulting plasmid, pET-CCL19/BSP41, encodes a CCL19 protein fused with 17 amino acids of BSP41 biotinylation site in E. coli and its expression is regulated by the IPTG-inducible T7 promoter.
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