人黏着斑激酶(FAK)原核表达及多克隆抗体制备与鉴定  

Prokaryotic expression and polyclonal antibodies preparation and identification of human focal adhesion kinase(FAK)

作　　者：张丽[1,2,3] 张娅 季江颖[2,3] 魏梓妤 刘家宇 许涛 汪洪涛 ZHANG Li;ZHANG Ya;JI Jiangying;WEI Ziyu;LIU Jiayu;XU Tao;WANG Hongtao(Center of Molecular Diagnosis,the First Affliated Hospital,Bengbu Medical College,Bengbu 233030,China;Research Center of Laboratory,School of Laboratory Medicine,Bengbu Medical College,Bengbu 233030,China;Anhui Province Key Laboratory of Immunology in Chronic Diseases,Bengbu Medical College,Bengbu 233030,China;Department of Clinical Laboratory,School of Laboratory Medicine,Bengbu Medical College,Bengbu 233030,China;Department of Immunology,School of Laboratory Medicine,Bengbu Medical College,Bengbu 233030,China)

机构地区：[1]蚌埠医学院第一附属医院分子诊断中心,安徽蚌埠233030 [2]蚌埠医学院检验医学院检验医学实验中心,安徽蚌埠233030 [3]蚌埠医学院慢性疾病免疫学基础与临床安徽省重点实验室,安徽蚌埠233030 [4]蚌埠医学院检验医学院临床检验诊断学教研室,安徽蚌埠233030 [5]蚌埠医学院检验医学院免疫学教研室,安徽蚌埠233030

出　　处：《细胞与分子免疫学杂志》2023年第2期175-180,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：安徽省自然科学基金(1908085MH252,2008085QH405);安徽省高校自然科学研究重点项目(KJ2018A0233);蚌埠医学院"512人才培育计划"项目(by51201309);国家级大学生创新创业训练计划项目(201810367001,202110367026);安徽省大学生创新创业训练计划项目(S202010367063)。

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∶512000,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。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 afinity 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 1:512000,and could specifically react with exogenous and endogenous FAK proteins.Conclusion TheFAKproteinis 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.

关 键 词：人黏着斑激酶(FAK) 多克隆抗体 

分 类 号：S852.43[农业科学—基础兽医学] Q511[农业科学—兽医学] R392.11[农业科学—畜牧兽医]