小麦热激蛋白WHSP90基因的原核表达及多克隆抗体制备

Expression of Wheat Heat Shock Protein WHSP90 Gene and Preparation of Its Polyclonal Antibodies

为了进一步研究HSP90基因的功能,将WHSP90基因构建到原核表达载体pET-28a(+)中,得到His-WHSP90融合表达载体,并转化到大肠杆菌BL21(DE3)中,发现1 mmol/L IPTG诱导4 h蛋白表达量最大;经过蛋白标记亲和层析柱(HisTrapTMHP)纯化得到的纯化蛋白浓度达到0.4 mg/mL。免疫家兔制备抗体,用间接ELISA法检测免疫后家兔抗血清效价大于125 000,满足后续试验要求的效价值,为在蛋白水平上研究WHSP90基因功能提供了基础。

To investigate the function of the WHSP90 gene, the WHSP90 gene was inserted into the pET- 28a （＋） vector to get fusion vector His-WHSP90, then transformed into E. coli BL21 （DE3） cells. The most high expression quantity was induced by 1 mmol/L IPTG at 4 h. The protein was purified using His- Trap^TMHp, and the concentration of purified protein was 0.4 mg/mL. The purified protein was injected into a rabbit, and the titer of the rabbit＇s anti-serum was measured by indirect ELISA. The rabbit＇s antiserum with high titer （〉125 000） was obtained. The polyclonal antibodies can be used for further investigation on the function of the WHSPgO gene in protein level.