【摘要】 目的探讨应用基因免疫法制备凝血酶调节蛋白(throbomodulin,TM)抗体。方法运用基因免疫法,即PCR特异性扩增TM胞外编码区所有碱基片段(包括信号肽,TMLEO),构建pcDNA3.1/TMLEO真核表达质粒。以碱裂解法提取,PEG沉淀法纯化质粒。将纯化后的质粒pcDNA3.1/TMLEO经生理盐水稀释后直接肌肉注射免疫BALB/c小鼠,检测质粒pcDNA3.1/TMLEO在小鼠体内有无表达以及TM抗体的效价和特异性。结果通过RTPCR以及TM含量测定在RNA以及蛋白水平上均说明质粒pcDNA3.1/TMLEO在小鼠体内有表达。以EVC304包板的cellELISA检测抗体滴度,随着免疫次数的增加,小鼠血清抗体滴度明显增加,到第五次达到最高1∶8000,第6～7次均保持在这一水平。小鼠抗血清通过免疫印迹(Westernblot)和免疫组织化学鉴定,特异性好。结论在天然TM提取纯化困难的情况下,通过基因免疫制备抗体是可行的。更多还原

【Abstract】 Objective To produce anti-thrombomodulin antibodies.Methods Using genetic immunization: Eukaryotic expression plasmid pcDNA3.1/TM(LEO),encoding all the extracellular domain of human thrombomodulin and signal peptides but lacking the transmembrance and cytoplasmic domains was constructed, which recombinant thrombomodulin was secreted soluble product. The plasmid was isolated from large-scale bacterial cultures by treatment with alkali and SDS, purified by precipitation with polyethylene Glycol (PEG). Recombinant plasmid was injected into tibial muscle of BALB/c mice. The productions of TM and anti-TM have been detected. Results The positive of RT-PCR and expressed TM identified the function of the recombinant plasmid. The pcDNA3.1/TM(LEO) induced higher titer of anti-TM. The antibody titer peaked between the 5th and 7th injection with a titer of 1∶8 000 detected by cell-ELISA coated with EVC-304. Specificity has been identified by western blot and immunohistochemistry.Conclusion The production of antibody through genetic immunization was a feasible method due to the difficulties in obtaining and purification of natural thrombomodulin.更多还原