Construction, Identification Of Padtrack-cmv-rhsg Recombined Shuttle Plasmid Vector And Bioinformation Analysis Of Rhsg.

Objective (1)Based on former constructed recombinant vector pExpress-1- rHSG, to construct and identify the recombined vector pAdtrack-cmv-rHSG carrying rats hyperplasia suppressor gene which can express in eukaryote cells and which will provide the basis for further researching the mechanisms of adenoviral vector and working on cerebral vasospasm genetherapy. (2)From the sequencing report, analyze the rHSG consequence and construction, in order to recognizing the construction and function of this gene. Methods (1) Extraction of the target gene: TO recover including pExpress-1-rHSG vector Jm109 bacteria, isolating pexpress-1- rHSG vector from plasmid in the agent kit, and amplify rHSG cDNA the target gene in PCR instrument. (2) Construction, Identification of shuttle plasmid:①To recovery the DH5αbacteria of the including adenovirus vector pAdtrack-cmv, isolating pAdtrack-cmv plasmid from plasmid in the agent kit.②After the product was extracted, the cDNA after PCR was insert to pGM-T plasmid, to construct and identify the recombinant plasmid pGM-T-rHSG. After the product was extracted,purified and digested with enzyme BglⅡand Eco RV, then it was inserted to the BglⅡand Eco RV sites of pGM-T-rHSG plasmid, to generate the recombinant vector.③pGM-T-rHSG plasmid was transformed into E. coli DH5αin LB Solid agar medium, and purify pGM-T-rHSG plasmid.④BglⅡand Eco RV were inserted to the pGM-T-rHSG plasmid, then pureify and identify the target gene.⑤BglⅡand Eco RV were inserted to the pAdTrack-CMV plasmid to acquire lineable pAdTrack-CMV segment. The segment was connect with target gene by T4DNA ligase to construct recombined pAdtrack-cmv-rHSG shuttle plasmid.⑥The recombined pAdtrack-cmv-rHSG was identified by analysis of different restriction endonuclease digestion and gene sequence analysis. (3)Bioinformatics analysis of rHSG gene: Based on the gene sequence report, the homology analysis,molecular weight,transmembran domain,functional binding sites,isoelectric point and secondary structure by rHSG ere analyzed and predieted by bioinofmatics. Results (1)The rHSG cDNA was isolated successfully from pexpress-1-rHSG plasmid. (2)BglⅡand Eco RV were inserted to the recombinant of pAdtrack-cmv- rHSG shuttle plasmid, then the recombined was analyzed by the means of Gene Sequencing, the sequence of the cloned rHSG cDNA is accordant with GenBank[AF036536]. As is shown in this result, target gene was inserted in the recombined pAdtrack-cmv-rHSG was positive and haploid. (3)Bioinformatics analysis of rHSG gene revealed that There are 4151bp in rHSG. It coded a protein of 757 amino acids and probably a transmembrane protein .Homology analysis showed that it was similar to HSG in some degree. Conclution (1) cDNA of rHSG can be successfully cloned and inserted into Eukaryote-expression plasmid. The newly constructed plasmid may serve as the potential tool to conduct further comprehensive experiments in future on rHSG function and on gene therapy of cerebral vasospasm. (2) The bioinformatics analysis of rHSG would be engaged to the comprehension of molecular and biological functions of this protein.