Enzyme using the same technology to build the end of pET15b.docVIP

 PAGE \* MERGEFORMAT 4 Enzyme using the same technology to build the end of pET15b Author: Yao Lingling, Wang Ning, HUANG Yong-Zhang, GUO Ling-Yun [Abstract] Objective: Using the same technology to build the end of enzyme pET15b-PEP-1-CAT recombinant plasmid for the preparation of PEP-1-CAT fusion protein and thus for the prevention and treatment of myocardial ischemia-reperfusion injury lay the foundation. Methods: The two ends were designed and synthesized with Sal Ⅰ and Bgl Ⅱ restriction sites of the CAT primer, with PCR method to pZeoSV2 ()-CAT plasmid as a template-specific amplification of CAT full-length cDNA. The CAT cDNA amplification reactions and dATP, using Taq DNA polymerase has a non-template-dependent terminal transferase activity in the CAT cDNA 3 ‘end of the plus amp;quot;Aamp;quot; and purified, and then apply the TA cloning strategy will be purified CAT cDNA inserted to the intermediate vector pGEM-T Easy Vector, the by PCR, restriction analysis identified recombinant pGEM-T-CAT. Encoding PEP-1 synthetic double-stranded oligonucleotides, respectively, at both ends of the introduction of Nde Ⅰ and Xho Ⅰ restriction site, will be inserted into pET15b recombinant plasmid pET15b-PEP-1, and restriction enzyme digestion. pGEM-T-CAT, and pET15b-PEP-1, respectively by Sal Ⅰ -Bgl Ⅱ and Xho Ⅰ -BamH Ⅰ restriction enzyme digestion, using the same tail enzymes (Sal Ⅰ and Xho Ⅰ , Bgl Ⅱ and BamH Ⅰ ) can produce the same sticky end of the digestion characteristics of the recovered get CATcDNA fragments with pET15b-PEP-1 linked to the recombinant plasmid pET15b-PEP-1-CAT, by PCR and restriction enzyme digestion and sequencing confirmed by nucleotide sequence. Results: pET15b-PEP-1-CAT recombinants by sequencing analysis confirmed that cloned into pET15b for PEP-1 sequence and Design and Synthesis of PEP-1 consensus sequence, CAT sequence in GeneBank accession number for the ‘AY028632’ of the CAT cDNA consensus sequence. Conclusion: pET15b-PEP-1-CAT