真核表达质粒pBK-IL-1ra的构建及表达

Construction and Expression of Eukaryotic Expression Vector pBK-IL-1ra

Objective Construction of eukaryotic vector with high expression of interleukin 1 receptor antagonist (IL 1ra) for IL 1ra gene delivery. Methods (1) Eukaryotic expression vector pBK IL 1ra was constructed by recombinant DNA technique,and identified by restriction mapping enzymes,PCR,and DNA sequence analysis.(2) Expressions of the IL 1ra mRNA and IL 1ra in vitro and in vivo were detected by in situ hybridization,ELISA,and immunohistochemistry. Results (1)The correct construction of pBK IL 1ra was identified by the method above.(2)The higher levels of IL 1ra in the supernate of COS 7 cells (24 h,(P<0.001;48 h,P<0.01)and the over expression of IL 1ra mRNA (P<0.01) and IL 1ra (P<0.05) in the synoviocytes have been detected after 48 h of the transfection with pBK IL 1ra.(3) The over expression of IL 1ra in muscle of the mice was detected by immunohistochemistry after 4 days of pBK IL 1ra injection (P<0.01) Conclusions We have successfully constructed eukaryotic expression vector pBK IL 1ra which could highly expressed IL 1ra in vitro and in vivo, and it offers a novel possibility in the research of IL 1ra gene therapy.