【摘要】 目的 克隆、表达与纯化黏着斑激酶(FAK)基因C端黏着斑定位序列(第798～1041位氨基酸),制备兔抗FAK多克隆抗体并鉴定。方法 PCR法体外扩增FAK基因C端序列(2671～3402 bp),克隆至pCZN1,构建pCZN1-FAK重组表达载体，将重组表达载体转化到大肠杆菌表达菌株BL21(DE3)感受态细胞，用异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达；利用镍-次氮基三乙酸(Ni-NTA)金属鳌合亲和层析树脂进行蛋白纯化，并免疫新西兰大白兔制备多克隆抗体；通过间接ELISA和Western blot法进行效价和特异性鉴定。结果 成功构建pCZN1-FAK重组表达载体，FAK蛋白主要以包涵体形式表达。目的蛋白纯化后，制备的兔抗FAK多克隆抗体效价达1∶512 000,且与外源和内源FAK蛋白发生特异性反应。结论 成功克隆、表达与纯化FAK蛋白，制备兔抗FAK多克隆抗体，可用于内源FAK蛋白特异性检测。更多还原

【Abstract】 Objective To Clone, express, and purify the focal adhesion kinase(FAK) gene C-terminal focal adhesion location sequence(aa798-aa1041), and to prepare and identify the rabbit anti-FAK polyclonal antibodies. Methods The C-terminal(2671 bp-3402 bp) gene of the FAK gene was amplified by PCR in vitro and cloned into pCZN1 vector to construct a pCZN1-FAK recombinant expression vector. The recombinant expression vector was transformed into E. coli expression strain BL21(DE3) competent cells, and then induced by isopropy-β-D-thiogalactoside(IPTG). The protein was purified by affinity chromatography resin Ni-NTA and immunized with New Zealand white Rabbit to prepare polyclonal antibodies. The antibody titer was detected by indirect ELISA and the specificity was identified by Western blot analysis. Results The pCZN1-FAK recombinant expression vector was successfully constructed. The FAK protein was mainly expressed in the form of inclusion bodies. After purification of the target protein, the prepared rabbit anti-FAK polyclonal antibody showed a titer of 1∶512 000, and could specifically react with exogenous and endogenous FAK proteins. Conclusion The FAK protein is successfully cloned, expressed and purified, and a rabbit anti-FAK polyclonal antibody is prepared, which can be used for the specific detection of endogenous FAK protein.更多还原