Construction and identification of recombinant lentivirus overexpression and RNA interference vector containing cardiac adriamycin reactive protein gene / 吉林大学学报(医学版)

ABSTRACT

Objective: To construct the recombinant lentiviral vector containing cardiac adriamycin responsive protein (CARP) gene and small-hairpin RNA (shRNA) targeting CARP gene and to pack the lentivirus, and to lay the foundation for further study on the function and mechanism of CARP in adriamycin (ADR) induced cardiomyopathy. Methods: After the rat CARP gene was amplified by PCR and shRNAs targeting CARP were designed and synthesized, they were inserted into the shuttle plasmids GV-358 or GV-248. respectively. After confirmed by sequencing, the recombinant shuttle vectors containing CARP gene or shRNA and auxiliary packaging plasmid Helper 1 0 and Helper 2 0 were co transfected into the IIEK293T cells for virus packaging and amplification; the viral titer was detected by end point dilution. The IIEK293T cells were infected with the recombinant lentiviruses. and the green fluoresence intensity was observed by fluorescence mircroscope. The H9C2 cells were infected with the recombinant lentivirues and divided into control group. CARP overexpression group and shRNA group; Western blotting method was used to detect the CARP expression levels in the cells in various groups. Results: The DNA sequencing results showed that the sequences of CARP overexpression and shRNA vectors were in accordance with the designed sequences. The expression of green fluorescence protein was seen under fluorescence microscope after transfection of the vectors of the IIEK293T cells. After infection of the H9C2 cells, the expression level of CARP protein in CARP overexpression group was 5. 3 times higher than that in control group ( P=0. 01); while it was down-regulated by 53% in CARP shRNA group compared with control group ( P= 0 02). Conclusion: The lentivirus expression vectors carrying CARP or shRNA targeting CARP are successfully constructed and the lentiviruses obtained could significantly interfere the expression of CARP in the II9C2 cells.