桦褐孔菌酸性蛋白酶的基因克隆及其在菌核中的表达

Cloning of Acid Protease fromInonotus obliquus and Expression Profile in Sclerotium thereof

根据桦褐孔菌(Inonotus obliquus)酸性蛋白酶(Acid protease,AP)基因(IO-AP)序列密码子的偏好性优化选择酶切位点和载体,成功构建pGEX-4T1-IO-AP原核表达载体;用热激法将该载体转化到大肠杆菌(Escherichia coli)BL21(DE3)中,构建桦褐孔菌酸性蛋白酶(IO-AP)重组菌。以此重组菌表达的IO-AP以包涵体形式存在,重组蛋白分子质量约为60kDa。以获得的较高纯度的重组IO-AP作为免疫原,采用传统3-2-2-2免疫方式免疫4只Balb/c小鼠,通过间接ELISA法测定抗血清效价,4免之后,4只小鼠抗血清效价均大于121500,成功制备了IO-AP多克隆抗体。Western Blotting检测表明,IO-AP在桦褐孔菌菌核形成过程中的表达呈现先上升后下降的趋势,以第二个时期(栽培130d,菌核长至1.33g时)AP蛋白表达量最高。

The acid protease gene from Inonotus obliquus （IO-AP） was codon optimized, assembled into the expression vector pGEX-4TI-IO-AP and then expressed in Escherichia coli BL21 （DE3）as inclusion body with a relative molecular weight of 60 kDa. This recombinant acid protease rIO-AP was then used as a high purity antigen to produce polyclonal antibodies of rIO-AP by the traditional 3-2-2-2 immunization method. Four Balb/c mice were immunized and antiserum titers were determined by the indirect ELISA method. After four times of immunization, all four mice contained antiserum titers greater than 121500, suggesting successful production of the rIO-AP polyclonal antibody. The resultant rIO-AP polyclonal antibodies were then used to analyze the expression profile of acid protease in Inonotus obliquus sclerotium by Western blot. The results showed that the expression of IO-AP in sclerotium reached a peak when sclerotia grew to 1.33 g.