真核表达质粒pBK—IL—1ra的构建及表达

目的 构建可在真核细胞内高效表达白细胞介素－１受体拮抗剂（ＩＬ－１ｒａ）的真核表达质粒ｐＢＫ－ＩＬ－１ｒａ,以便用于ＩＬ－１ｒａ基因治疗研究。方法 采用分子生物学技术将ＩＬ－１ｒａ ｃＤＮＡ插入真核表达载体ｐＢＫ－ＣＭＶ中,构建成真核表达质粒ｐＢＫ－ＩＬ－１ｒａ。通过竞争性ＥＬＩＳＡ、原位杂交和免疫组检测其在体外和体内转染真核细胞后ＩＬ－１ｒａ基因和蛋白的表达。结果 （１）酶切、ＰＣＲ和ＤＮＡ测序

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.