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目的制备可溶性表达的日本血吸虫烯醇化酶,观察其免疫学特性及免疫诊断价值。方法采作RT-PCR方法反转录合成编码日本血吸虫烯醇化酶的cDNA片段,亚克隆到表达质粒pET28a(+)中构建重组表达质粒。将重组表达质粒转化到大肠埃希菌BL21中,在不同条件下诱导表达可溶性重组烯醇化酶(rSj Enolase),采用镍亲和层析法纯化rSj Enolase。以rSj Enolase免疫小鼠,制备抗血清,采用Western blot法分析rSj Enolase的免疫反应性与特异性。以rSj Enolase为抗原,建立检测抗Sj Enolase抗体IgG的ELISA方法,并以此方法对血吸虫病患者、其他寄生虫病患者及健康人血清进行检测,观察检测抗Sj Enolase抗体IgG用于血吸虫病诊断的敏感性与特异性;对治疗前及治疗后不同时间点的同一患者配对血清进行检测,观察治疗前、后患者血清中抗Sj Enolase抗体IgG水平的动态变化,并观察该方法的疗效考核价值。结果成功克隆编码Sj Enolase基因,并在18℃低温条件下成功表达可溶性rSj Enolase蛋白,采用镍亲和层析进行纯化rSj Enolase蛋白免疫小鼠获得效价大于1︰100 000的抗血清。Western blot显示rSj Enolase蛋白可被血吸虫病患者血清识别,而不与健康人血清反应;抗rSj Enolase鼠血清能识别血吸虫成虫抗原(AWA)和排泄分泌物(ESA)中的天然Sj Enolase分子。rSj Enolase不被曼氏裂头蚴病患者血清,华支睾吸虫病患者及卫氏并殖吸虫病患者血清识别,ELISA显示以rSj Enolase为抗原检测血吸虫感染者血清中的抗Sj Enolase特异性抗体的敏感性为93.71%,特异性为97.92%,与华支睾吸虫患者血清的交叉反应率为8.5%,与弓形虫病患者血清的交叉反应率为0。治疗后3个月的血吸虫病患者血清抗Sj Enolase抗体IgG水平迅速下降,阴转率达58.92%。结论可溶性重组Sj Enolase表达成功。该蛋白具有较高的抗原特异性,以此为抗原检测抗Sj Enolase抗体IgG具有血吸虫病诊断价值。
Objective To prepare soluble expression of Schistosoma japonicum enolase and observe its immunological characteristics and immunodiagnosis value. Methods The cDNA encoding Schistosoma japonicum enolase was reverse transcribed by RT-PCR and subcloned into the expression vector pET28a (+) to construct a recombinant plasmid. The recombinant plasmid was transformed into Escherichia coli BL21, and the soluble recombinant enolase (rSj Enolase) was induced under different conditions. The rSj Enolase was purified by nickel affinity chromatography. The mice were immunized with rSj Enolase to prepare antiserum. The immunoreactivity and specificity of rSj Enolase were analyzed by Western blot. The ELISA method for detecting anti-Sj Enolase antibody IgG was established by using rSj Enolase as an antigen and the serum of schistosomiasis patients and other parasitic diseases and healthy human beings were detected by this method. The anti-Sj Enolase antibody IgG was detected for the diagnosis of schistosomiasis The sensitivity and specificity of anti-Sj Enolase IgG in sera of patients before and after treatment were measured before and after treatment, and the therapeutic value of this method was observed . Results The Sj Enolase gene was cloned successfully and the soluble rSj Enolase protein was successfully expressed at low temperature of 18 ℃. The rSj Enolase protein was immunized with nickel affinity chromatography to obtain antiserum with the titer of more than 1: 100 000. Western blot showed that the rSj Enolase protein was recognized by the serum of schistosomiasis patients but not by healthy human serum. Anti-rSj Enolase murine serum recognized the native Sj Enolase molecule in the adult worm antigen (AWA) and excretion secretion (ESA) of Schistosoma japonicum. rSj Enolase was not recognized by the sera of patients with sparganosis of Mann’s disease, Clonorchis sinensis and Paragonimus westermani, and ELISA showed that rSj Enolase was used as an antigen to detect the sensitivity of anti-Sj Enolase specific antibodies in sera of patients infected with Schistosoma japonicum The positive rate was 93.71% and the specificity was 97.92%. The cross-reactivity rate with the serum of Clonorchis sinensis was 8.5%, and the cross-reactivity with serum of patients with toxoplasmosis was 0. Serum anti-Sj Enolase IgG levels in patients with schistosomiasis 3 months after treatment decreased rapidly, with a negative conversion rate of 58.92%. Conclusion Soluble recombinant Sj Enolase was successfully expressed. The protein has high antigen specificity, as antigens to detect anti-Sj Enolase antibody IgG has the diagnostic value of schistosomiasis.