日本血吸虫果糖二磷酸醛缩酶抗体反应模式及诊断价值的研究
发布时间：2019-04-26 21:44
【摘要】：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。
[Abstract]:Aim to observe the dynamic changes of anti-Schistosoma japonicum Chinese strain fructose diphosphate aldolase (Sj FBA) antibody reaction in rabbits infected with Schistosoma japonicum, and to evaluate the value of detecting anti-Sj FBA antibody IgG in the diagnosis and therapeutic evaluation of schistosome infection. Methods the open reading frame gene encoding Sj FBA was amplified by RT-PCR from Schistosoma japonicum Chinese strain cDNA and subcloned into the expression plasmid pET28a () to construct the recombinant expression plasmid Sj FBA-pET28a.. The recombinant expression plasmid was transformed into Escherichia coli BL21 (DE3) and induced by isopropyl-尾-D-thiogalactoside (IPTG). The recombinant Sj FBA protein was purified by Ni chelating gel affinity chromatography. Rabbits were infected with cercariae of Schistosoma japonicum in mainland China. Six weeks later, praziquantel and artesunate were treated with praziquantel and artesunate. Blood samples were collected before infection and 2,4,6,8,10,12,14,16 weeks after infection. An enzyme-linked immunosorbent assay (rSj FBA-ELISA) for detection of anti-Sj FBA antibody IgG was established by using recombinant Sj FBA as coated antigen, which was used to detect the reaction pattern of anti-Sj FBA antibody in sera of rabbits at different stages of infection. Mice were infected with 5, 10, 15 and 20 cercariae of Schistosoma japonicum (Schistosoma japonicum) Chinese mainland strain respectively. The sera of mice were collected 35 days after infection. Anti-Sj FBA antibody IgG was detected by rSj FBA-ELISA and the relationship between the level of anti-Sj FBA antibody and the degree of infection was observed. The sera of schistosomiasis patients, healthy people, clonorchiasis sinensis and paragonimiasis westerns were detected by rSj FBA-ELISA, and the sensitivity and specificity of this method for diagnosis of schistosomiasis were observed. This method was used to detect the matched sera of the same schistosomiasis infected patients before and 1 year after treatment, and to observe the value of anti-Sj FBA antibody IgG detection in evaluating the curative effect of schistosomiasis. Results the level of anti-Sj FBA antibody IgG in rabbits infected with Schistosoma japonicum increased gradually with the prolongation of infection time and reached its peak at 6 weeks after infection (A450 = 1.364). However, the level of anti-Sj FBA antibody decreased rapidly after treatment. The antibody level decreased to the negative threshold value (A450 = 0.4) after 6 weeks of treatment, while the antibody level remained high in the untreated group, indicating that the response of anti-Sj FBA antibody in rabbits showed a typical response pattern of short-lived antibody. The level of anti-Sj FBA antibody in serum of mice with different degree of infection increased gradually with the increase of infection degree, in a dose-dependent manner. The specificity, sensitivity, negative predictive value and positive predictive value of anti-Sj FBA antibody IgG for diagnosis of schistosome infection were 88.8%, 82.5%, 78.6% and 91.0%, respectively. The cross-reaction rate with clonorchiasis and paragonimiasis was 10% and 30%, respectively. One year after cure, the negative conversion rate of anti-Sj FBA antibody IgG was 100%. Conclusion Schistosoma japonicum Sj FBA can induce short-lived antibody response in the host. The sensitivity and specificity of detecting anti-Sj FBA antibody IgG by rSj FBA-ELISA are high. It is valuable to diagnose schistosomiasis and evaluate the therapeutic effect of Schistosoma japonicum.
【作者单位】： 江苏省寄生虫病分子生物学重点实验室卫生部重要寄生虫病预防与控制技术重点实验室江苏省血吸虫病防治研究所;中国疾病控制中心传染病预防控制所传染病诊断室;
【基金】：国家传染病重大专项(No.2012ZX1004-220) 国家自然科学基金项目(No.81201316,30972581) 江苏省自然科学基金项目(No.BK2012544) 江苏省卫生厅血地寄科技项目(No.X201429,X201402)
【分类号】：R383.24
[Abstract]:Aim to observe the dynamic changes of anti-Schistosoma japonicum Chinese strain fructose diphosphate aldolase (Sj FBA) antibody reaction in rabbits infected with Schistosoma japonicum, and to evaluate the value of detecting anti-Sj FBA antibody IgG in the diagnosis and therapeutic evaluation of schistosome infection. Methods the open reading frame gene encoding Sj FBA was amplified by RT-PCR from Schistosoma japonicum Chinese strain cDNA and subcloned into the expression plasmid pET28a () to construct the recombinant expression plasmid Sj FBA-pET28a.. The recombinant expression plasmid was transformed into Escherichia coli BL21 (DE3) and induced by isopropyl-尾-D-thiogalactoside (IPTG). The recombinant Sj FBA protein was purified by Ni chelating gel affinity chromatography. Rabbits were infected with cercariae of Schistosoma japonicum in mainland China. Six weeks later, praziquantel and artesunate were treated with praziquantel and artesunate. Blood samples were collected before infection and 2,4,6,8,10,12,14,16 weeks after infection. An enzyme-linked immunosorbent assay (rSj FBA-ELISA) for detection of anti-Sj FBA antibody IgG was established by using recombinant Sj FBA as coated antigen, which was used to detect the reaction pattern of anti-Sj FBA antibody in sera of rabbits at different stages of infection. Mice were infected with 5, 10, 15 and 20 cercariae of Schistosoma japonicum (Schistosoma japonicum) Chinese mainland strain respectively. The sera of mice were collected 35 days after infection. Anti-Sj FBA antibody IgG was detected by rSj FBA-ELISA and the relationship between the level of anti-Sj FBA antibody and the degree of infection was observed. The sera of schistosomiasis patients, healthy people, clonorchiasis sinensis and paragonimiasis westerns were detected by rSj FBA-ELISA, and the sensitivity and specificity of this method for diagnosis of schistosomiasis were observed. This method was used to detect the matched sera of the same schistosomiasis infected patients before and 1 year after treatment, and to observe the value of anti-Sj FBA antibody IgG detection in evaluating the curative effect of schistosomiasis. Results the level of anti-Sj FBA antibody IgG in rabbits infected with Schistosoma japonicum increased gradually with the prolongation of infection time and reached its peak at 6 weeks after infection (A450 = 1.364). However, the level of anti-Sj FBA antibody decreased rapidly after treatment. The antibody level decreased to the negative threshold value (A450 = 0.4) after 6 weeks of treatment, while the antibody level remained high in the untreated group, indicating that the response of anti-Sj FBA antibody in rabbits showed a typical response pattern of short-lived antibody. The level of anti-Sj FBA antibody in serum of mice with different degree of infection increased gradually with the increase of infection degree, in a dose-dependent manner. The specificity, sensitivity, negative predictive value and positive predictive value of anti-Sj FBA antibody IgG for diagnosis of schistosome infection were 88.8%, 82.5%, 78.6% and 91.0%, respectively. The cross-reaction rate with clonorchiasis and paragonimiasis was 10% and 30%, respectively. One year after cure, the negative conversion rate of anti-Sj FBA antibody IgG was 100%. Conclusion Schistosoma japonicum Sj FBA can induce short-lived antibody response in the host. The sensitivity and specificity of detecting anti-Sj FBA antibody IgG by rSj FBA-ELISA are high. It is valuable to diagnose schistosomiasis and evaluate the therapeutic effect of Schistosoma japonicum.
【作者单位】： 江苏省寄生虫病分子生物学重点实验室卫生部重要寄生虫病预防与控制技术重点实验室江苏省血吸虫病防治研究所;中国疾病控制中心传染病预防控制所传染病诊断室;
【基金】：国家传染病重大专项(No.2012ZX1004-220) 国家自然科学基金项目(No.81201316,30972581) 江苏省自然科学基金项目(No.BK2012544) 江苏省卫生厅血地寄科技项目(No.X201429,X201402)
【分类号】：R383.24
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相关期刊论文 前4条
1 雷正龙;郑浩;张利娟;朱蓉;郭家钢;李石柱;王立英;陈朝;周晓农;;2010年全国血吸虫病疫情通报[J];中国血吸虫病防治杂志;2011年06期
2 王s，
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