hErmin160蛋白的表达及其多克隆抗体的制备  

Prokaryotic expression and polyclonal antibody generation of hErmin160 protein

作　　者：徐清[1] 赵玮钦[1] 王涛[1] 关路媛[1] 陈南春[1] 张斌[1] 

机构地区：[1]第四军医大学基础部生物化学与分子生物学教研室,陕西西安710033

出　　处：《第四军医大学学报》2008年第20期1892-1895,共4页Journal of the Fourth Military Medical University

基　　金：国家自然科学基金(30770420);全军医药卫生科研基金(06H036)

摘　　要：目的:对hErmin氨基端160个氨基酸(hErmin160)蛋白进行表达、鉴定和纯化并制备其多克隆抗体,为研究hEr-min功能奠定基础.方法:PCR扩增编码hErmin160的核苷酸序列,克隆入原核表达载体pET41-b(+),转化大肠杆菌BL21(DE3)pLysS,诱导表达hErmin160融合蛋白,表达产物用SDS-PAGE和Western Blot进行鉴定,经镍柱纯化后,免疫家兔,制备其多克隆抗体,并对免疫后抗血清进行亲和纯化,用Western-Blot检测抗体纯化的效果.结果:测序证实获得hEr-min氨基端前160个氨基酸的编码序列;SDS-PAGE分析表明,表达产物的相对分子质量为51ku,与理论值相符;灰度扫描分析hErmin160融合蛋白的表达量占菌体蛋白总量的10%,纯化产物纯度达92%;Western Blot确认所表达的蛋白;抗血清和纯化后的多克隆抗体WestermBlot反应均为阳性.结论:利用大肠杆菌融合表达系统,获得了较高纯度的可溶形式hErmin160融合蛋白,并通过亲和纯化成功制备hEr-min160多克隆抗体.AIM： To express, identify, and purify hErminl60 in prokaryotic system and to generate polyclonal antibody of hErmin in order to research its function. METHODS： The coding sequence of N-terminal 160 amino acids of hErmin （hErminl60） was amplified by PCR from pBluescriptR plasmid loading with full length of hErmin cDNA sequence, and then cloned into prokaryotic expression vector pET41-b（ ＋ ）. The plasmid was transformed into E. coli BI21 （ DE3 ） pLysS to express hErminl60 fusion protein in response to IPTG induction. The products were analyzed by SDS-PAGE and Western-Blot. Expressed protein was purified by Ni^2＋ metal-chelating chromatograph and then injected into rabbits to generate polyclonal antibody. Blood serum after injection was purified using affinity chomatography. The polyclonal antibody was identified by Western Blot. RESULTS： DNA sequencing confirmed that the coding sequence of hErminl60 was completely concordant with the original sequence （ BC026345, hErmin＇s GenBank accession number）. SDS-PAGE showed that the relative molecular masses （Mr） of the expressed, purified products were about 51 ku, which was in accordance with the predicted. Grayscale scanning showed that the expressed hErminl60 fusion protein accounted for 10% of the total bacteria protein, and the purity reached 92%. One positive band was detected in Western- Blot analysis. Blood serum after injection and purified polyclonal antibody were also Western Blot positive. CONCLUSION： We acquire high purification soluble hErminl60 fusion protein through the E. coli fusion expression system and purified polyclonal antibody of hErminl60 protein.

关 键 词：hErmin 原核表达 多克隆抗体 

分 类 号：R392.11[医药卫生—免疫学]