线粒体铁转运蛋白2(mitoferrin 2/SLC25A28)的原核表达及兔多克隆抗体制备  

Prokaryotic expression of mitoferrin 2/SLC25A28 and rabbit polyclonal antibody preparation

作　　者：刘磊 聂雅婷 丁兰 谷雨 袁媛 叶菁 LIU Lei;NIE Yating;DING Lan;GU Yu;YUAN Yuan;YE Jing(Department of Pathology,Basic Medical Science Academy,Air Force Medical University,Xi'an 710032,China)

机构地区：[1]空军军医大学基础医学院病理学教研室,陕西西安710032

出　　处：《细胞与分子免疫学杂志》2023年第3期268-274,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(82170589);陕西省自然科学基础研究计划(2022JM-536);肿瘤生物学国家重点实验室自主课题(CBSKL2017Z20)。

摘　　要：目的构建人线粒体铁转运蛋白2(mitoferrin 2/SLC25A28)的原核表达质粒,进行蛋白表达和纯化,制备兔抗多克隆抗体并进行初步鉴定。方法构建pET28a(+)-SLC25A28-His原核表达质粒,转化至BL21(DE3)大肠杆菌,采用异丙基β-D-硫代半乳糖苷(IPTG)诱导蛋白表达。提取蛋白包涵体,并用8 mol/L尿素溶解,His-NTA柱纯化。获得纯化SLC25A28蛋白免疫新西兰大白兔制备SLC25A28多克隆抗体,Western blot法鉴定SLC25A28抗体特异性。结果成功构建pET28a(+)-SLC25A28-His原核表达载体质粒,并在BL21(DE3)大肠杆菌中成功诱导表达SLC25A28蛋白,蛋白纯度约为90%,Western blot法证明制备的抗体能特异性识别小鼠睾丸组织中的SLC25A28蛋白。结论成功对人SLC25A28进行了原核表达,并制备了具有特异性的兔抗人SLC25A28多克隆抗体。Objective To construct the prokaryotic expression plasmid of human mitoferrin 2(SLC25A28),and to express and purify the protein for preparing its rabbit polyclonal antibody.Methods The prokaryotic expression plasmid pET28a(+)-SLC25A28-His was constructed and transferred into E.coli BL21(DE3),and induced with Isopropyl-β-D-thiogalactopyranoside(IPTG).The SLC25A28 protein was extracted in form of inclusion bodies,and was further purified by His-NTA column after dissolved in 8 mol/L urea.The anti-SLC25A28 polyclonal antibody was prepared by immunizing rabbits,and its specificity was determined by Western blot analysis.Results pET28a(+)-SLC25A28-Hiswasconstructedand SLC25A28 protein was successfully expressed in E.coli BL21(DE3)with the purity up to 90%.The Western blot results indicated that anti-SLC25A28 polyclonal antibody was capable to recognize specifically the SLC25A28 protein in testis.Conclusion The human SLC25A28 is successfully expressed in E.Coli,and the rabbit polyclonal antibody specific to SLC25A28 isprepared.

关 键 词：线粒体铁转运蛋白2(mitoferrin 2/SLC25A28) 原核表达 蛋白纯化 多克隆抗体 免疫特异性 

分 类 号：Q512[生物学—生物化学] S852.43[农业科学—基础兽医学]