Characterization and immunoprotective effect of SjIrV1,a 66 kDa calcium-binding protein from Schistosoma japonicum

钙结合蛋白是日本血吸虫生长发育不可缺少的蛋白,具有非常广泛而重要的功能。在课题组日本血吸虫体被表膜蛋白研究基础上,利用PCr技术克隆了中国大陆株日本血吸虫66 kdA钙结合蛋白(SJIrV1)编码基因的CdnA序列,blAST分析与菲律宾株日本血吸虫SJIrV1 CdnA编码序列一致,荧光定量PCr分析表明该基因在童虫和成虫期不同发育阶段均有表达,其中在35 d和42 d成虫中表达量较高,在42 d雌虫中该基因表达水平远高于42 d雄虫。构建重组表达质粒PET28A(+)-SJIrV1,在大肠杆菌中成功诱导表达,重组蛋白主要以可溶性形式存在,通过高效液相色谱法(rP-HPlC)以及串联质谱法(MS/MS)鉴定所获蛋白为目的蛋白SJIrV1。蛋白质印迹(WESTErn blOTTIng)分析结果显示重组蛋白能被感染日本血吸虫鼠血清和免疫鼠血清所识别,SJIrV1蛋白在虫体各发育阶段中均表达。免疫荧光染色实验观察表明SJIrV1主要分布在日本血吸虫成虫的表膜。应用重组蛋白免疫bAlb/C小鼠后,免疫鼠血清中检测到较高水平的特异性Igg、Igg1和Igg2A抗体。结果表明SJIrV1可能在日本血吸虫的生长发育过程中起着重要作用。Calcium-binding protein is an indispensable protein which performs extensive and important functions in the growth of Schistosoma japonicum.Based on our primary study on tegument surface proteins of S.japonicun,a cDNA encoding a 66 kDa calcium-binding protein of S.japonicum(Chinese strain) was cloned,sequence analysis revealed that it was identical with that of SjIrV1 of Philippines strains S.japonicum.The expression of SjIrV1 were detected by Real-time PCR,using cDNA templates isolated from 7,14,21,28,35 and 42 days worms and the results revealed that the gene was expressed in all investigated stages,and the mRNA level of SjIrV1 is much higher in 42 d female worms than that in 42 d male worms.The cDNA containing the open reading frame of IrV1 was subcloned into a pET28a(+) vector and transformed into competent Escherichia coli BL21 for expression.The recombinant protein was purified using a Ni-NTA purification system,and confirmed by high performance liquid chromatography(RP-HPLC) and tandem mass spectrometry(MS/MS).Western blotting analysis showed that recombinant SjIrV1(rSjIrV1) could be recognized by the S.japonicum infected mouse serum and the mouse serum specific to rSjIrV1,respectively.Immunofluorescence observation exhibited that SjIrV1 was mainly distributed on the tegument of the 35-day adult worms.ELISA test revealed that IgG,IgG1 and IgG2a antibodies are significantly increased in the serum of rSjIrV1 vaccinated mice.The study suggested that rSjIrV1 might play an important role in the development of S.japonicum.国家自然科学基金(No.31172315); 上海科技发展基金(No.12140902700); 中国博士后科学基金(No.2012M510630)资助~

Identification and preliminary analysis of a novel full-length cDNA encoding retinoid X receptor 2 from Schistosoma japonicum

通信作者 E-mail: [email protected][中文文摘]目的克隆编码日本血吸虫视黄酸X受体2(SjRXR2)蛋白的全长cDNA,并对其进行初步研究。方法利用cDNA末端快速扩增技术(RACE)获得SjRXR2蛋白全长编码cDNA。利用生物信息学技术,对基因结构进行初步分析。利用实时荧光定量(Real time)PCR技术对该基因在日本血吸虫不同时期虫体中的转录情况进行分析。应用在线抗体表位预测软件获得SjRXR2配体结合区抗原性较强的一个多肽序列,合成该多肽片段,并免疫小鼠制备抗血清。利用Western blot技术分析该蛋白在日本血吸虫中的表达。结果采用RACE技术成功获得了SjRXR2蛋白全长编码cDNA,总长度为5 960bp,其完整开放阅读框为4 308 bp,编码1 435个氨基酸,预测分子量为159 kDa。生物信息学分析表明该基因编码的蛋白质序列具有核受体家族2的典型结构域特征,且与曼氏血吸虫RXR2有较高的相似性。Real time PCR分析表明,该基因在21、42 d龄日本血吸虫虫体内有较高的转录水平。Western blot分析表明,小鼠SjRXR2多肽免疫血清可特异性识别日本血吸虫虫体150 kDa蛋白。结论成功获得了编码SjRXR2蛋白的全长 cDNA，并制备了针对该蛋白的特异性多克隆抗体，为进一步研究该蛋白的功能奠定了基础。 .国家自然科学基金（31172315）；公益性行业（农业）科研专项（20090303036