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• W2386168102 abstract "[Objective] To construct and identify replication defective adenoviral vectors with human insulin-like growth factor-1 gene of interest for hIGF-1 gene therapy of intervertebral disk degeneration by Cre-LoxP homologous recombination system, in order to lay a basis for hIGF-1 gene thorapy of intervertebral disk degeneration. [Methods] hIGF-1 gene of interest was synthesized by polymerase chain reaction(PCR) and cloned with pMD18-T vector. The cloned hIGF-1 cDNA fragments were se-quenced and similar analysis was done with NCBI B1AST technique in the internet. The pMD18-T -hIGF-1 and empty plasmid pDNR-1r were digested by EcoR I and BamH I, ligated and transformed competence E. coli DH5α. Positive recombinants were identified by EcoR I and BamHI digestion. pInt. AV1. SpaT-hIGF-1 was synthesized by homologous recombination of pDNR-hIGF-1 and pInt. AV1. SpaT with Cre-loxP system and identified by EcoR I digestion. Replication defective aden-ovirus with hIGF-1 gene was synthesized by cotransfection of pInt. AV1. SpaT-hIGF-1 and pInt. B. B plasmids into 293 cells. Cytopathogenic effects were observed through invert microscope. Adenovirus inclusions were observed through transmission electron microscope. Adenovirus DNA was extracted from cell lysis, hIGF-1 gene was identified with PCR technique. hIGF-1 protein expression was indentified by Western blot technique. The second generation adenovirus titers were tested by tissue culture infection dose 50(TCID50) method. [Results] The synthesized hIGF-1 gene of interest had the same sequence as that of Homo sapiens (GI: 19923111) . Both fragments of 5 400 and 1 000 base pairs were seen by restriction endonuclease digestion of pInt. AV1. SpaT-hIGF-1 as expected. Cytopathogenic effects including cell swelling and shedding were observed in most of 293 cells 12-14 days after transfection. hIGF-1 gene existed in the cell lysis. Hexahedron adenovirus inclusions were found in the cytopathic 293 cellular nuclei. hIGF-1 gene existed at recombinant adenovirus. Recombi-nant adenovirus expressed hIGF-1 protein. The second generation adenovirus tilers were 80×106 PFU/mL. [Conclusion] Replication defective adenovirus with hIGF-1 gene of interesl has been constructed successfully." @default.
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• W2386168102 title "Construction and Identification of Adenovirus with Human Insulin-like Growth Factor-1 Gene" @default.
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