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目的重组表达抗线粒体抗体二亚型(AMA-M2)的二个靶抗原丙酮酸脱氢酶复合体E2(PDC-E2)、2-氧戊二酸脱氢酶复合体E2(OGDC-E2)及其连接形成的二联体PO-E2融合蛋白,已用于人原发性胆汁性肝硬化(PBC)的早期发现和临床诊断。方法针对PDC-E2,OGDC-E2的cDNA序列设计引物,从正常人的淋巴细胞中提取RNA,通过反转录PCR方法扩增得到相应的基因片段,经测定序列验证后插入表达载体pET28a(+),构建重组表达载体,pET28a(+)-PDC-E2,pET28a(+)-OGDC-E2,pET28a(+)-P0-E2,转化大肠埃希菌BL21(DE3)后诱导表达蛋白质。表达蛋白经SDS-PAGE,Western-blot等鉴定。结果经核苷酸序列测定和酶切鉴定结果表明,成功地构建了三种重组质粒。IPTG诱导表达后,获得三种融合蛋白。经免疫学鉴定,重组抗原片段具有AMA-M2的免疫原性。结论重组表达的PO-E2融合蛋白将有利于原发性胆汁性肝硬化的实验室诊断。
Objective To recombinantly express the two target antigens of pyruvate dehydrogenase complex E2 (PDC-E2) and 2-oxoglutarate dehydrogenase complex E2 (OGDC-E2) against mitochondrial antibody subtype A (AMA-M2) And its connection to form a double-linked PO-E2 fusion protein has been used for early detection and clinical diagnosis of human primary biliary cirrhosis (PBC). Methods Primers were designed according to the cDNA sequence of PDC-E2 and OGDC-E2. RNA was extracted from normal human lymphocytes and amplified by reverse transcription-polymerase chain reaction (PCR). The sequences were inserted into the expression vector pET28a (+ PET28a (+) - PDC-E2, pET28a (+) - OGDC-E2 and pET28a (+) - P0-E2 were constructed and transformed into Escherichia coli BL21 (DE3) to express the recombinant protein. The expressed protein was identified by SDS-PAGE and Western-blot. Results The results of nucleotide sequencing and restriction enzyme digestion showed that three recombinant plasmids were successfully constructed. After IPTG induced expression, three fusion proteins were obtained. Immunologically identified, the recombinant antigen fragment has the immunogenicity of AMA-M2. Conclusion The recombinant fusion protein PO-E2 will be helpful for the laboratory diagnosis of primary biliary cirrhosis.