Construction Of Shuttle Vector Containing Calcitonin Gene Related Peptide

Background: Calcitonin gene related peptide was first discovered in 1983 using the technology of DNA genetic recombination and molecular biology.It is structurally related and belong to the calcitonin family of peptides. It is a 37 amino acid peptide that results from alternative splicing of calcitonin gene transcripts. It exists in two forms α- and β- CGRP, which show considerable homology with amylin and adrenomedullin. CGRP is produced in both the central and perhpheral nervous system; innervate blood vessels, digestive canals and several endocrine tissues. Release of CGRP causes increased blood flow in trigeminal and cardiac tissues and CGRP is regarded to be the most potent vasodilatory neuropeptide.Molecular biology has developed rapidly in recent years. People have learned more about the structure and function of viral genome and the mechanism of viral infection. The technology of construction viral vector has been improved. As one of the most widely used viral vector, adenoviral vector can transduce a wide spectrum of cell types and do not require division of the target cell for gene transfer or expression. The adenovinis chromosome remains episomal in the transduce cell, thus avoiding the possibility of insertional mutagenesis.In this study, we first got the mRNAs of CGRP from the dorsal root ganglion (DRG). We designed the primers, so that the upstream primer contained the recognition sequence of Sal I, while the downstream primer contained the recognition sequence of EcoR V. Then, we used the technology of reversetranscription-polymerase chain raction to get the complete DNA sequence of CGRP, which involved 433bps with the additional 12 bps of sites for restriction enzyme Sal I and EcoR V. The PCR product was inserted into the PUC57 plasmid vector after digested. The recombinant plasmids(PUC57-CGRP) were transformed, screened and identified by PCR. Then, the recombinant plasmids and the pShuttle-CMV vectors were both digested and ligated to construct a new recombinant plasmidspShuttle-CMV-CGRP. This provides the preparation for the construction of denovirus vector.Methods: We chose the male mice(25g) which are bred and housed in our institute. The first step is to get the complete DNA sequences of CGRP.After killing the mouse, we searched for the dorsal root ganglion (DRG) and kept it in the liquid nitrogen. We used the Classic Total RNA Isolation Kit to obtain the total RNA of the mouse. Then, with the help of MMLV Single Step RT-PCR Kit, we successfully got the complete DNA sequences of CGRP which has 423bps with the additional 12 bps of sites for restriction enzyme Sal I and EcoR V. The second step is to construct the recombinant plasmids PUC57-CGRP. Both the PCR products and PUC57 plasmids were digested, then the PCR product was inserted into the PUC57 plasmid vector with the help of T4 DNA ligase. The recombinant plasmids(PUC57-CGRP) were transformed, screened and identified by PCR. The last step is to construct the recombinant plasmids pShuttle-CMV-CGRP. Both the PUC57-CGRP and the pShuttle-CMV vectors were digested and ligated to construct a new recombinant plasmids pShuttle-CMV-CGRP.Results:We successfully amplified the complete DNA sequences of CGRP which has 423bps.We successfully constructed the recombinant plasmids PUC57-CGRP.We successfully constructed the recombinant plasmids pShuttle-CMV-CGRP. Conclusions: Molecular biology has developed rapidly in recent years. People have learned more about the strcture and function of viral genome and the mechanism of viral infection. The technology of constructing viral vector has been improved. As one of the most widely used viral vector, adenoviral vector can transducer a wide spectrum of cell types and do not require division of the target cell for gene transfer or expression. The adenovirus chromosome remains episomal in the transduced cell, thus avoid of insertional mutagenesis. The recombinant plasmids pShuttle-CMV-CGRP which we successfully constructed provides the preparation for the construction of adenovirus vector.