兔抗细胞周期蛋白依赖激酶6(CDK6)抗体的制备及鉴定  

Preparation and identification of rabbit anti-cyclin dependent kinase 6(CDK6)antibodies

作　　者：叶晓鲜 董海艳 王昱 陈争珍 李军伟 韦玉兵 张丽芳 YE Xiaoxian;DONG Haiyan;WANG Yu;CHEN Zhengzhen;LI Junwei;WEI Yubing;ZHANG Lifang(Department of Anatomy,School of Basic Medicine,Wenzhou Medical University Renji College,Wenzhou 325035,China;Institute of Molecular Viruses and Immunology,Wenzhou Medical University Renji College,Wenzhou 325035,China)

机构地区：[1]温州医科大学仁济学院基础医学部解剖教研室,浙江温州325035 [2]温州医科大学仁济学院分子病毒与免疫研究所,浙江温州325035

出　　处：《细胞与分子免疫学杂志》2023年第8期742-747,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：浙江省科技厅公益项目(LGF18H160030)。

摘　　要：目的 制备兔抗重组细胞周期蛋白依赖激酶6(CDK6)抗体并进行鉴定。方法 合成并构建pET21a(+)-CDK6重组质粒,将重组质粒转化至大肠杆菌BL21(DE3),经异丙基硫代-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达重组蛋白,经SDS-PAGE及Western blot法检测蛋白表达;用镍氮川三乙酸(Ni-NTA)亲和层析柱纯化,SDS-PAGE分析;纯化后的CDK6蛋白多部位、多次(隔周)免疫日本大耳白兔,分别于免疫的0、 2、 4、 6周取血,分离血清。采用间接ELISA检测效价,Western blot法、免疫荧光细胞化学染色和免疫组织化学染色法检测其特异性。结果 表达并获得高纯度的CDK6蛋白,制备了兔抗CDK6重组蛋白抗体,其效价达1∶30 000,能特异性识别宫颈癌细胞及宫颈癌组织中表达的CDK6蛋白。结论 成功制备了高效价、特异性强的兔抗CDK6抗体。Objective To prepare and identify rabbit anti-cyclin dependent kinase 6(CDK6)antibody.MethodsThe recombinant pET21a(+)/CDK6 was successfully constructed,then the recombinant plasmid was transformed into E.coli BL21(DE3)competent cells and was induced by isopropyl-β-D-thiogalactopyranoside(IPTG)for protein expression,which was detected by SDS-PAGE and Western blot analysis.The expressed protein was purified by nickel-chelating nitrilotriacetic acid(Ni-NTA)agarose and then analyzed by SDS-PAGE.Japanese white rabbits were immunized with purified CDK6 protein for many times every two weeks.The blood was collected at O,2,4 and 6 weeks after immunization,and serum was separated from blood.The titer was detected by indirect ELISA.Western blot analysis,immunofluorescence assay and immunohistochemistry were employed to determine the specificity.Results High purity CDK6 protein and high specificity of rabbit anti-CDK6 antibody were successfully prepared.The titer of CDK6 rabbit serum antibody reached 1:30000 after immunization,which could specifically recognize the CDK6 protein expressed in cervical cancer cell line and cervical cancer tissues.Conclusion The high titer and specificity of rabbit anti-CDK6 antibody is successfully prepared.

关 键 词：细胞周期蛋白依赖激酶6(CDK6) 重组蛋白 基因表达 血清抗体 

分 类 号：R392.7[医药卫生—免疫学] Q344.13[医药卫生—基础医学] R392-33[生物学—遗传学]