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.
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)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
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)。
