应用免疫蛋白质组学方法鉴定日本血吸虫虫卵诊断抗原

目的虫卵可溶性抗原（soluble egg antigen,SEA）是目前日本血吸虫病免疫诊断中最常用的抗原。本研究联合应用双向凝胶电泳（2-DE）、Western blot和MALDI-TOF质谱等技术,鉴定SEA中诊断抗原蛋白质。方法 SEA经2-DE分离蛋白质后进行银染,或应用日本血吸虫感染兔血清Western blot筛选抗原蛋白点,用MALDI-TOF/TOF串联质谱鉴定每一抗原蛋白质分子。结果虫卵可溶性蛋白分子经2-DE分离后转印PVDF膜上,与感染兔血清孵育出现29个特异性阳性反应点,与健康兔血清出现3个非特异的阳性反应点;29个特异性抗阳性反应点中,从银染2-DE胶图上找到21个匹配的抗原蛋白质点;MALDI-TOF/TOF质谱鉴定和NCBI数据库检索,13个点（61.9%）获得匹配蛋白质,4个点（19.0%）获得匹配EST,4个点（19.0%）未能从数据库中找到相匹配的信息。重组表达筛选出的SjCHGC06040和SjP40两个抗原蛋白质,Western blot结果显示reSjCHGC06040、reSjP40均能与感染兔血清抗体发生特异性反应,具有潜在的应用价值。结论 2-DE、MALDI-TOF质谱联合Western blot的免疫蛋白质组学研究方法,用于筛选、鉴定SEA中诊断抗原蛋白质,是切实可行的;但该方法存在一定的局限性,有待进一步改进。

Identification of diagnostic antigens from Schistosoma japonicum eggs with immunoproteomic approach

In order to identify useful candidate antigens for diagnosis of schistosomiasis,two-dimensional electrophoresis（2-DE） technology combined with Western blot and MALDI-TOF/TOF-MS/MS spectrometry were applied to analyse soluble egg antigen（SEA） of Schistosoma japonicum.After being extracted from eggs,soluble proteins were separated by 2-DE.The gels were used for sliver staining or reacted with S.japonicum infected rabbit sera to screen antigenic proteins by Western blot.Each antigenic molecule identified by MALDI-TOF/TOF-MS/MS.There were 29 spots showed on the PVDF membrane that incubated with infected rabbit sera,while 3 non-specific spots were reacted with normal rabbit sera.Twenty-one of these 29 positive spots were precisely matched with a homologous 2-DE gel.After identified by MALDI-TOF/TOF-MS/MS technique and searched in NCBI database,13 molecules were successfully matched with protein database and 4 were EST data,while 4 were failed.Of these proteins,SjP40 and SjCHGC06040 were successfully expressed,and Western blot results showed a specific reaction between the recombinant proteins and infected rabbit sera.Therefore,our data suggests the immunoproteomic approach is a suitable method for screening and identification of diagnostic candidate antigens from eggs of S.japonicum.