日本血吸虫烯醇化酶的表达与诊断价值研究

Expression and diagnostic value of enolase fromSchistosoma japonicum

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。

Objectives To prepare a soluble form of Schistosoma japonicum enolase and to explore its immunological features and value in immunological diagnosis of schistosomiasis. Methods A cDNA fragment encoding Schistosoma enolase was synthesized using a reverse transcription polymerase chain reaction （RT-PCR）, and the fragment was subcloned into the expression plasmid pET28a （＋） to construct a recombinant expression plasmid. The recombinant expression plasmid was transformed into E. coli BL21, and expression of soluble recombinant S. japonicum enolase was induced with IPTG under different conditions. Purified recombinant S. japonicum enolase was prepared using nickel affinity chromatography. Mice were immunized with recombinant S. japonicum enolase to prepare antiserum against S. japonicum enolase. The immunologic reactivity and specificity of recombinant S. japonicurn enolase were analyzed using Western blotting. An enzyme linked immunosorbent assay （ELISA） based on recombinant S. japonicum enolase was established. This technique was used to test the sera of patients with schistosomiasis, patients with other parasitic diseases, and healthy individuals. IgG antibodies against S. japonicurn enolase were tested for their sensitivity and specificity at detecting schistosomiasis and their cross-reactivity with other parasitic diseases. IgG antibodies against S. japonicurn enolase were monitored in the same patient at different times before and after treatment. Changes in levels of IgG antibodies against S. japonicum enolase in serum from patients before and after treatment were observed to determine whether those levels were effective at diagnosing schistosomiasis. Results The gene encoding S. japonicum enolase was successfully cloned, and expression of soluble recombinant S. japonicum enolase was induced at a low temperature of 187 C. The pu- rified recombinant protein was prepared using nickel affinity chromatography, and the recombinant protein was used to immunize mice to produce high-titer antiserum （ 1：100,000）. The results of Western blotting indicated that recombi- nant S. japonicum enolase was recognized by serum from patients with schistosomiasis, but was not recognized by serum from healthy individuals or patients with other parasitic diseases. Mouse antiserum against recombinant S. japonicum enolase recognized natural S. japonicum enolase in the adult worm antigens （AWAs） and excreted-secreted antigens （ES- As） of S. japonicum, indicating that recombinant S. japonicum enolase has satisfactory immunogenicity and antibody reactivity. Recombinant S. japonicum enolase was not recognized by serum from patients with sparganosis, clonorchiasis, or paragonimiasis, which suggests that recombinant S. japonicum enolase had a high level of specificity. ELISA results indicated that IgG antibodies against S. japonicum enolase had a sensitivity of 93.71% and a specificity of 97.92% at detecting schistosomiasis. The level of IgG antibodies against S. japonicum enolase in the serum of patients fell rapidly one to three months after treatment; patient serum tested negative for schistosomiasis at a rate of 58. 92% Three months after schistosomiasis treatment, the effectiveness of that treatment can be ascertained based on antibodies against S. japonicum enolase. Conclusions Soluble recombinant S. japonicum enolase was successfully expressed. Detection of IgG antibodies against S. japonicum enolase has value as a means of diagnosing schistosomiasis.