| Objective: To study the construction of the lentiviral vector encoding human B-type natriuretic peptide gene, and to achieve efficient , stable expression of human B-type natriuretic peptide gene in skeletal myoblasts. Method: Primary skeletal myoblasts were isolated from Sprague-Dawley rats and cultured by enzymatic digestion. We tested them by Desmin immunohistochemistry stains and knew their viability by Trypan blue. Human B-type natriuretic peptide (hBNP) cDNA was subcloned into expression vector pLenti6/V5-D-TOPO to construct recombinant pLenti6/V5-hBNP. In order to obtain the virus particles, the pLenti6/V5-hBNP and the contructed pLenti6/V5-EGFP were transduced into 293FT cells. The murine primary myoblasts were infected with recombinant virus by lipofectamin 2000. Through counting by the Fluorescence Microscope, we identified the transfection efficacy. After transfected and during selected, the concentration of human B-type natriuretic peptide (hBNP) in cell culture medium was detected by enzyme-link immunosorbent assay (ELISA). Result: PCR, double-enzyme cutting and DNA sequence showed hBNP cDNA was correctly inserted into pLenti6/V5-D-TOPO vector. Desmin immunohistochemical stains showed that 94 % myoblasts'cytolymph was positive reaction and cell nucleas and cytolymph were stained brown or brownish, and Trypan blue showed the viability was up to 94 %. Bright green fluorescence of the transfected cells could be observed under the fluorescent microscope after 12 h transfection with pLenti6/V5-EGFP recombinant virus, the transfection rates reached 60%. The level of human brain peptide in the pLenti6/V5-hBNP groups and pLenti6/V5-EGFP groups was distinct(P<0.01). After 4 weeks, the express of human brain peptide was still stable. Conclusion: Recombinant pLenti6/V5-hBNP has been constructed and expressed ectopic in the skeletal myoblasts, meanwhile this gene expression mediated by lentivirus vector may contribute to cardiovascular fields.
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