Preparation of human HSP72cDNA recombinant adenovirus and its ex- pression in IEC-6 cell

AIM: To construct the HSP72 recombinant adenovirus and confirm its expression in intestinal epithelial cells (IEC-6), and to provide gene transfection methods for the studies on ischemia after serious trauma. METHODS: Recombinant adenoviral vector, pACCMV-HSP72, containing whole long human HSP72 gene was constructed by homologous recombination, and then used to transfer IEC-6 in vitro. The rate of HSP72 expression in IEC-6 cells was analyzed by flow cytometry. RT-PCR and Western blot were used to detect the expression of HSP72 mRNA and protein respectively. RESULTS:The result of RT-PCR showed the recombinant adenovirus contained the gene of HSP72.The expression rate of HSP72 was high after transferred into IEC-6 cells for 48 h. RT-PCR and Western blot showed the expression of HSP72 mRNA and protein in transferred cells was positive while that of the control group (IEC-6 cells were transferred with vacant adenovirus ) was negative. CONCLUSIONS: The study indicates that pACCMV-HSP72 did generate high level expression of HSP72 in IEC-6 cells. The recombinant adenovirus pAdCMV-HSP72 can be further used in gene therapy against ischemia injury after serious trauma in vivo.

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