Construction of retroviral vector inserted SV40 large T antigen gene and its expression in rat hepatocytes / 中国普通外科杂志

ABSTRACT

Objective To construct the retroviral vector inserted SV40 large T antigen gene and transfect it into rat hepatocytes, analyze the status of SV40 large T antigen gene expression in rat hepatocytes, and to establish important basis for clinical hepatocytes transplanation. Methods Retroviral vector inserted SV40 large T antigen gene was(constructed) by DNA recombinant techniques in vitro, then the combinant vector was determined with enzyme(digestion) and sequencing and was transfected into the PA317 cell lines by liposome mediation and screened(anti-G4)18 positive clones. The viral titer was determined with the NIH3T3. After transfected into separated and purified rat primary hepatocytes, the SV40 large T antigen gene expression was detected by PCR and immunohistochemical (methods). Results (1)SV40 large T antigen gene fragment was inserted into retroviral vector in sense orientation. (2)The titer of pseudovirion packed by PA317 cell lines was 1.3?10~6CFU/ml. (3)SV40LT antigen gene was(integrated) into rat primary hepatocytes and its expression in transfected cells at 24 hour was higher than 96 hour (P