AD7c-NTP与麦芽糖结合蛋白的融合表达、纯化及其抗血清制备

Expression, Purification and Antiserum Preparation of AD7c-NTP Fused with Maltose Binding Protein

人工合成了编码AD7c-NTP的基因,并将其中的6种密码子GGA AGG AGA ATA CTA CCC分别突变成大肠杆菌偏爱密码子GGT CGT CGT ATC CTG CCG,PCR扩增后克隆到表达载体pMAL-c2上,构建表达质粒pMAL-AD7c,重组质粒导人E coli BL21(DE3)构建工程菌,IPTG诱导实现了MBP-ADTc-NTP融合蛋白在工程菌中的高效表达,细胞裂解液经Amylose-resin亲和层析、DEAE-Sepharose离子交换层析以及Su—perdex 200凝胶过滤层析,得到了电泳纯的融合蛋白。纯化的融合蛋白免疫家兔制备了兔抗MBP-AD7c-NTP 融合蛋白的抗血清,利用该抗血清及点杂交技术,初步分析了10例AD疑似患者的尿样.结果表明5例疑似患者的尿样呈阳性,另5例呈阴性。

In order to get enough AD7C-NTP for structural research and clinical use, cDNA encoding AD7C-NTP was synthesized, in which 6 rare codons for E. coli GGA AGG AGA ATA CTA CCC were mutated to GGT CGT CGT ATC CTG CCG, respectively. The synthesized DNA was amplified by PCR and cloned into pMAL-c2, the recombinant plasmid was transformed into E. coli BL21 （DE3） and expression of AD7c-NTP fused with maltose binding protein （MBP-AD7c-NTP） was achieved by IPTG induction. The recombinant MBP-AD7c-NTP was purified through amylose-resin affinity chromatography, ion exchange chromatography on DEAE-Sepharose column, and gel filtration chromatography on Superdex 200 column. The fusion protein was homogeneity as,judged by SDS-PAGE. A specific antiserum was raised using the purified fusion protein, and 10 urine samples of Alzheimer＇s disease-like individuals were tested by dot blotting. The result showed that 5 samples were positive.