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| PCGF1蛋白的表达、纯化及其单克隆抗体的制备与鉴定 | |
| 引用本文： | 李辉,吴旭东,龚燕华,阴彬.PCGF1蛋白的表达、纯化及其单克隆抗体的制备与鉴定[J].细胞与分子免疫学杂志,2011,27(1):71-73. |
| 作者姓名： | 李辉 吴旭东 龚燕华 阴彬 |
| 作者单位： | 1. 武警医学院组织学与胚胎学教研室,天津,300162 2. 中国医学科学院基础医学研究所,医学分子生物学国家重点实验室,北京,100005 3. 中国医学科学院基础医学研究所,医学分子生物学国家重点实验室,北京,100005;武警医学院生物化学教研室,天津,300162 |
| 基金项目： | 国家自然科学基金面上项目,医学分子生物学田家重点实验室外部课题(2010年) |
| 摘 要： | 目的:原核表达人表观抑制因子Polycomb家族成员PCGF1蛋白片段,并制备其鼠源单克隆抗体(mAb)。方法:应用PCR技术扩增人PCGF1基因编码区部分序列(CDS区第382～567bp的DNA片段)并插入到pET-32a(+)质粒中,在大肠杆菌BL21中进行His-PCGF1-128/189融合蛋白(简写为His-N128/189)的原核表达。利用所表达的融合蛋白中含有的6×His标签进行亲和层析纯化。用所获得的纯化蛋白免疫BALB/c小鼠,利用杂交瘤技术,分别通过ELISA和Western blot法筛选稳定分泌抗体的杂交瘤细胞株,用免疫双向扩散法鉴定mAb的Ig亚类。取1株杂交瘤细胞按照常规方法制备腹水,利用Millipore公司的mAb纯化试剂盒纯化mAb,用Western blot法检测mAb的效价。结果:表达纯化了重组人His-N128/189蛋白;筛选出2株可稳定分泌特异性抗人PCGF1 mAb的杂交瘤细胞株,其免疫球蛋白类型均为IgG1类;通过腹水制备和纯化获得高效价(1∶6000)、高特异性的鼠抗人PCGF1 mAb。结论:原核表达的PCGF1片段融合蛋白可以被纯化,并具有足够的蛋白免疫原性,所制备的相应mAb可特异识别内源性和外源性PCGF1蛋白,可以运用于PCGF1基因功能的研究。 |
| 关 键 词： | PCGF1 单克隆抗体 杂交瘤技术 |
Protein expression,purification of PCGF1 and its monoclonal antibody preparation and identification |
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| LI Hui,WU Xu-dong,GONG Yan-hua,YIN Bin.Protein expression,purification of PCGF1 and its monoclonal antibody preparation and identification[J].Journal of Cellular and Molecular Immunology,2011,27(1):71-73. | |
| Authors: | LI Hui WU Xu-dong GONG Yan-hua YIN Bin |
| Institution: | 1Department of Histology and Embryology,3Department of Biochemistry,Medical College of Chinese People’s Armed Police Force,Tianjin 300162; 2National Laboratory of Medicine Molecular Biology,Institute of Basic Medical Sciences,CAMS,Beijing 100005,China |
| Abstract: | AIM:To express the human recombinant PCGF1 protein and prepare monoclonal antibody (mAb) against it. METHODS: The recombinant expression plasmid pET32a-His-PCGF1-128/189 was made and transformed into E.coli (BL21), and then the recombinant fusion protein His-PCGF1-128/189 was expressed and purified. The BALB/c mice were immuned with purified protein His-PCGF1-128/189 as antigen. The mAbs against PCGF1 were prepared by using standard hybridoma technique. The hybridoma cell lines were obtained by ELISA and We... |
| Keywords: | PCGF1 |
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