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  

Patterns and diagnostic value of the antibody response to fructose-1,6-bisphosphate aldolase of Schistosoma japonicum

作　　者：王玠[1] 余传信[1] 肖迪[2] 赵飞[2] 殷旭仁[1] 宋丽君[1] 沈双[1] 高玒[1] 张建中[2] 何利华[2] 许永良[1] 杨静[1] 

机构地区：[1]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,江苏无锡214064 [2]中国疾病控制中心传染病预防控制所传染病诊断室,北京昌平102206

出　　处：《中国病原生物学杂志》2015年第3期232-238,共7页Journal of Pathogen Biology

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)

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Objectives To observe the dynamics of the specific antibody response to fructose-1,6-bisphosphate aldolase of Schistosoma japonicum（Sj FBA）in infected rabbits and to determine the value of detecting specific IgG antibodies against Sj FBA in terms of diagnosing and evaluating the effectiveness of treatments for schistosomiasis. Methods RTPCR was used to amplify a DNA fragment containing the open reading frame encoding the Sj FBA protein from cDNA of a Chinese strain of S.japonicum.This fragment was inserted into the expression plasmid pET28a（＋）to construct the recombinant expression plasmid pET28a-Sj FBA.This recombinant plasmid was transformed into E.coli BL21（DE3）competent cells and isopropylβ-D-1-thiogalactopyranoside（IPTG）was used to induce expression of the recombinant Sj FBA protein.The recombinant Sj FBA protein was purified with nickel-nitrilotriacetic acid（Ni-NTA）resin affinity chromatography.Each rabbit was infected with S.japonicumcercariae via skin of the abdomen and given artesunate and praziquantel orally 6weeks after infection.Serum was collected from each rabbit before infection（healthy stage）,4and 6weeks post-infection,and 4,6,8,10,12,14,and 16 weeks post-treatment.rSj FBA-ELISA was established with recombinant Sj FBA to determine the dynamics of the antibody response to recombinant Sj FBA protein in sera from individual rabbits during the healthy stage,schistosomiasis,and post-treatment.Mice were infected with 5,10,15,and 20 cercariae of S.japonicumvia skin of the abdomen and were then tail-bled on day 35post-infection.The relationship between the degree of infection and antibody response to the recombinant Sj FBA protein in mice was observed using rSj FBAELISA.The sensitivity and specificity with which rSj FBA-ELISA diagnosed schistosomiasis were determined by detecting the recombinant protein in sera from healthy individuals and patients with schistosomiasis,clonorchiasis,or paragonimiasis.The therapeutic value of rSj FBA-ELISA was evaluated by examining dynamic chang

关 键 词：日本血吸虫中国大陆株 果糖二磷酸醛缩酶 短寿抗体反应 现症感染 免疫诊断 疗效考核 

分 类 号：R383.24[医药卫生—医学寄生虫学]