cTnI特殊位点抗原的重复表达、免疫及其抗体效价检测

Expression of cTnI Specific Antigen Sites and Antibodies Detection

构建了含人cTnI蛋白稳定区段2个特殊抗原决定簇位点的pMAL-Antigen质粒.利用BamHI/BgLII两限制性内切酶的同尾酶作用进行连接,在pMAL-p2x质粒的MBP(麦芽糖结合蛋白)之后接入了上述5个相同的片段.此质粒在大肠杆菌E.coliBL21(DE3)中经1.0mmol/LIPTG诱导表达出的融合蛋白MBP-Antigen经Amlyose-resin亲和层析柱纯化后免疫BABL/C小鼠,成功获得了能抗人cTnI和抗牛cTnI的抗血清.

Two specific epitope sites of human cardiac Troponin I （hcTnI）, 38aa-52aa and 87aa-97aa, which can stably exist in the blood serum of acute myocardial infarction （AMI） patients, were selected as objective gene sequences and isolated by 5 lysines with low immunogenicity. Two specific epitopes and lysines constituted AA gene sequence. Plasmid pMAL-Antigen consisting of 5AAs after maltose binding protein （MBP） was constructed by two restriction endonucleases, BamHI and BgLII. The pMAL-Antigen plasmid was diverted into E. coli BL21（DE3）, and MBP-Antigen fusion protein was induced by 1.0 mmol/L IPTG in E. coli BL21（DE3）. The MBP-Antigen fusion protein was purified by Amylose-resin. The result showed that protein antigen of MBP-Antigen fusion protein was partially reduced from C end. The purity of MBP-Antigen fusion protein was over 90%. The hyper-purified MBP-Antigen fusion protein was injected into BALB/C mice and antibodies were detected by ELISA. These antibodies could be applied in the diagnosis of AMI and myocardial damage.