论文部分内容阅读
目的克隆、表达细粒棘球绦虫Eg18基因,评价重组蛋白的免疫反应性。方法根据已知Eg18基因序列,利用RT-PCR方法从青海绵羊肝棘球蚴原头节提取的总RNA中扩增目的基因,克隆到原核表达质粒pET-28a(+)中,转化大肠埃希菌BL21(DE),IPTG诱导表达。表达产物经亲和层析纯化,用免疫印迹试验(Western blot)和酶联免疫吸附试验(ELISA)初步评价其与棘球绦虫及其他蠕虫感染血清的反应性。结果 RT-PCR扩增产物编码的蛋白质序列与GenBank中的Eg18和Em18完全相同。重组蛋白与多种蠕虫病人血清中的IgG和IgG4均有交叉反应,但检测IgG4时,与其他蠕虫的交叉反应显著降低。结论 Eg18/Em18是细粒棘球绦虫和多房棘球绦虫的共同抗原,其特异性IgG4是泡型棘球蚴病较特异的诊断标志物。
Objective To clone and express Eg18 gene of Echinococcus granulosus and evaluate the immunoreactivity of the recombinant protein. Methods According to the known Eg18 gene sequence, the target gene was amplified by RT-PCR from the total RNA extracted from the progenitor cells of Echinococcus hyughallus and then cloned into prokaryotic expression vector pET-28a (+). The recombinant plasmid was transformed into E. coli Strain BL21 (DE), induced by IPTG. The expressed product was purified by affinity chromatography. The reactivity of the expressed product with serum of echinococcus and other helminth worms was preliminarily evaluated by Western blot and enzyme-linked immunosorbent assay (ELISA). Results The protein sequence encoded by RT-PCR amplification products was completely identical with Eg18 and Em18 in GenBank. The recombinant protein cross-reacts with both IgG and IgG4 in the serums of various helminth patients, but significant decreases in cross-reactivity with other worms when tested for IgG4. Conclusion Eg18 / Em18 is a common antigen of Echinococcus granulosus and Echinococcus multilocularis, and its specific IgG4 is a specific diagnostic marker for alveolar hydatid disease.