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目的观察血吸虫感染家兔体内抗日本血吸虫中国大陆株果糖二磷酸醛缩酶(Sj FBA)抗体反应的动态变化,评价检测抗Sj FBA抗体IgG用于血吸虫现症感染的诊断与疗效考核价值。方法采用RT-PCR方法从日本血吸虫中国大陆株cDNA中扩增出编码Sj FBA的开放阅读框基因片段,然后亚克隆到表达质粒pET28a(+)中,构建重组表达质粒Sj FBA-pET28a。将重组表达质粒转化至大肠埃希菌BL21(DE3)中,用异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导表达,经镍螯合胶亲和层析的方法纯化重组Sj FBA蛋白。用日本血吸虫中国大陆株尾蚴感染日本大白兔,6周后用吡喹酮与青蒿琥酯进行治疗,收集感染前和感染后2、4、6周及治疗后2、4、6、8、10、12、14、16周的血样。以重组Sj FBA为包被抗原,建立检测抗Sj FBA抗体IgG的酶联免疫吸附试验方法(rSj FBA-ELISA),用于检测家兔不同感染阶段血清抗Sj FBA抗体反应模式。分别用5、10、15和20条日本血吸虫中国大陆株尾蚴感染小鼠,收集感染后35d的小鼠血清,采用rSj FBA-ELISA检测抗Sj FBA抗体IgG并观察抗Sj FBA抗体水平与感染度的关系。用rSj FBA-ELISA分别检测血吸虫病患者血清、健康人血清、华支睾吸虫病和卫氏并殖吸虫病患者血清,观察该方法用于血吸虫病诊断的敏感性与特异性;使用该方法检测治疗前及治疗后1年的同一血吸虫现症感染患者的配对血清,观察抗Sj FBA抗体IgG检测用于血吸虫病疗效考核的价值。结果血吸虫感染家兔体内抗Sj FBA抗体IgG水平随感染时间的延长而逐渐增强,感染6周达到高峰(A450值为1.364),但治疗后抗Sj FBA的抗体则迅速下降,治疗6-8周后抗体水平可下降至阴性阈值(A450值为0.4),而未治疗组则抗体水平则始终保持在高水平,表明家兔体内抗Sj FBA抗体反应呈典型的短寿抗体反应模式。不同感染度小鼠血清抗Sj FBA抗体水平随着感染度的升高而逐渐升高,呈剂量依赖性关系。检测抗Sj FBA抗体IgG用于血吸虫现症感染诊断的特异性为88.8%,敏感性为82.5%,阴性预测值和阳性预测值分别为78.6%和91.0%;与华支睾吸虫病患者血清的交叉反应率为10%,与卫氏并殖吸虫病患者血清的交叉反应率为30%。治愈1年后患者血清中抗Sj FBA的抗体IgG阴转率达100%。结论日本血吸虫Sj FBA在宿主体内诱导短寿抗体反应,应用rSj FBA-ELISA检测抗Sj FBA抗体IgG的敏感性和特异性均较高,具有血吸虫病诊断与疗效考核价值。
Objective To observe the dynamic changes of anti-Sj FBA antibodies in Schistosoma japonicum infected rabbits infected with Schistosoma japonicum, and to evaluate the diagnostic and therapeutic value of anti-Sj FBA antibody IgG in the diagnosis of schistosoma infection. Methods The open reading frame (ORF) gene fragment encoding Sj FBA was amplified by RT-PCR from Chinese mainland strains of Schistosoma japonicum and subcloned into the expression vector pET28a (+) to construct the recombinant expression plasmid Sj FBA-pET28a. The recombinant plasmid was transformed into Escherichia coli BL21 (DE3), induced with isopropyl-β-D-thiogalactopyranoside (IPTG) and purified by nickel chelating gel affinity chromatography Sj FBA protein. Japanese Schistosoma japonicum cercariae infected Japanese white rabbits, 6 weeks after treatment with praziquantel and artesunate, before and after infection 2, 4, 6 weeks and 2, 4, 6, 8, 10,12,14,16 weeks of blood samples. The recombinant Sj FBA was used as coating antigen to establish a rSj FBA-ELISA for detection of anti-Sj FBA antibodies in different infection stages of rabbits. The mice were infected with 5, 10, 15 and 20 S. japonicum cercariae respectively and the serum of mice was collected 35 days after infection. The anti-Sj FBA antibody IgG was detected by rSj FBA-ELISA and the level of anti-Sj FBA antibody and the degree of infection Relationship. Serum samples from patients with schistosomiasis, healthy human serum, Clonorchis sinensis and Paragonimus westermani were detected by rSj FBA-ELISA. The sensitivity and specificity of this method for the diagnosis of schistosomiasis were observed. Using this method, Paired sera of patients infected with the same schistosomiasis before treatment and 1 year after treatment were used to evaluate the value of anti-Sj FBA antibody IgG detection in the treatment of schistosomiasis. Results The level of anti-Sj FBA antibody in rabbits infected with Schistosoma japonicum increased gradually with the prolongation of infection time and reached the peak at 6 weeks (A450 = 1.364). However, the antibody against Sj FBA rapidly decreased after treatment for 6-8 weeks After the antibody level can be reduced to the negative threshold (A450 value of 0.4), while the untreated group antibody levels remained at high levels, indicating that rabbit anti-Sj FBA antibody reaction showed a typical pattern of short-lived antibody response. The serum anti-Sj FBA antibody levels of mice with different degrees of infection increased gradually with the increase of infection degree in a dose-dependent manner. The detection of anti-Sj FBA antibody IgG was 88.8% for the diagnosis of infection of schistosoma, the sensitivity was 82.5%, the negative predictive value and the positive predictive value were 78.6% and 91.0%, respectively. Compared with the serum of clonorchiasis patients The cross-reactivity rate was 10% and the cross-reactivity with Paragonimus westermani serum was 30%. Anti-Sj FBA antibody IgG negative conversion rate of 100% in patients’ serum after 1 year of cure. Conclusions Sj FBA of Schistosoma japonicum induces short-lived antibody response in the host. Sensitivity and specificity of rSj FBA-ELISA in the detection of anti-Sj FBA antibody IgG are high, which has the value of diagnosis and efficacy evaluation of schistosomiasis.