旋毛虫和伪旋毛虫肌幼虫时期排泄分泌产物iTRAQ法蛋白质组学分析

iTRAQ-based proteomics of excretory-secretory products of Trichinella spiralis and Trichinella pseudospiralis at the muscle larva stage

目的探究旋毛虫和伪旋毛虫肌幼虫时期排泄分泌产物(ESP)中的差异蛋白,分析两者免疫抑制差异的原因和参与包囊形成的潜在功能蛋白。方法收集旋毛虫和伪旋毛虫ESP,采用二喹啉甲酸检测法测定旋毛虫、伪旋毛虫ESP蛋白浓度,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)检测蛋白质量,对经过超滤管酶解法酶解后的ESP多肽段采用同位素相对定量和绝对定量标记技术(iTRAQ)标记,混合标记后的样品进行液相分离高pH反相色谱和液相串联质谱检测。检测完成后,搜索UniProt数据库中的旋毛虫(Trichinella spiralis)和伪旋毛虫(Trichinella pseiudospiralis)数据库,在可信蛋白(得分80以上)的基础上筛选同源蛋白,比较同源蛋白的差异表达情况。利用QuickGO对差异蛋白进行标准化描述。选取9个差异明显的重要蛋白进行实时荧光定量PCR验证,采用△△Ct法验证基因表达水平。采用SPSS 19.0软件进行统计学分析结果经iTRAQ标记鉴定出旋毛虫可信蛋白492个,伪旋毛虫535个,可用于定量分析的旋毛虫蛋白193个,伪旋毛虫蛋白164个。旋毛虫/伪旋毛虫同源比对结果显示,表达蛋白上调162个,下调31个。GO分析结果显示,分子功能主要富集在离子结合功能、肽酶活性、氧化还原酶活性和核酸酶活性,分别为45、18、12和8个。在生物过程中,涉及最多的是小分子代谢过程(15个),碳水化合物代谢过程(12个),生物合成过程(12个),DNA代谢过程过程(8个)。差异蛋白涉及的细胞组成成分主要为细胞质和含蛋白质的复合物。KECG分析显示,与硫胺素代谢,鞘糖脂生物合成,苯丙氨酸、酪氨酸和色氨酸生物合成相关的蛋白大部分上调。plancitoxin-1(E5SXW8)、胰蛋白酶(E5SPA7)、胱抑素(E5S387)、丝氨酸蛋白酶抑制剂(E5SJH4)、5’-核苷酸酶(E5S554)、烯醇酶(E5SQX1)、L-天冬酰胺酶(E5SBA6)蛋白表达和基因转录水平在旋毛虫肌幼虫时期均高于伪旋毛虫(P<0.05);热休克蛋白β-1 (E5RZQ6)、组蛋白H2B (E5S7K8)蛋白表达和基因表达水平在旋毛虫肌幼虫时期低于伪旋毛虫(P<0.05)。结论旋毛虫和伪旋毛虫肌幼虫时期ESP差异蛋白生物信息学分析提示丝氨酸蛋白酶、胱抑素、plancitoxin-1和14-3-3蛋白等可食能与包嚢形成和免疫调节相关。

Objective To analyze differential proteins between excretory-secretory products(ESPs) of Trichinella spiralis and Trichinella pseudosptralis muscle larvae,and find out the causes for different immunosuppression between the two species and the potential functional proteins involved in the formation of cysts.Methods ESPs were collected from T.spiralis and T.pseudo spiralis at the muscle larval stage.The bicinchoninic acid(BCA) detection method was used to determine the ESP protein concentrations.The quality of ESP proteins were assessed by sodium lauryl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE),then the proteins were digested by filter-aided sample preparation(FASP),and labelled with iso baric tags for relative and absolute quantitation(iTRAQ)reagents.The samples with mixed labeling underwent reversed-phase high performance liquid chromatography(RPHPLC) and liquid chromatography-tandem mass spectrometry(LC-MS/MS).Then the uniprot databases for T.spiralis uniport and T.pseudospiralis were searched,to screen for homologous proteins based on the trusted proteins(scores>80) and compare their differential expression.Standardized description of differential proteins was done with Quick GO.Nine proteins with significant difference were selected for qRT-PCR verification using the △△Ct method.Statistical analysis was performed using the SPSS 19.0 software.Results Based on the iTRAQ analysis,we identified 492 credible proteins for T.spiralis,of which 193 were suitable for quantification,and 535 credible proteins for T.pseudo spiralis,of which 164 could be quantified.T.spiralis/T.pse udosptrails homology comparison revealed that 162 proteins with up-regulation and 31 with down-regulation.The GO analysis results showed that the molecular functions were mainly involved in ion-binding function(45 proteins),peptidase activity(18),oxidoreductase activity(12),and nuclease activity(8).As regards biological processes,the small molecule metabolism processes involved the largest number of proteins(15),followed by the carbohydrate metabolism processes(12),the biosynthesis processes(12),and DNA metabolism processes(8).Of various cell components,the cytoplasm and protein-containing complexes had highest enrichment of differential proteins.KEGG analysis showed that most of the proteins related to thiamine metabolism,sphingolipid biosynthesis,and phenylalanine,tyrosine and tryptophan biosynthesis were up-regulated.In addition,at the muscle larva stage,E5 SXW8(plancitoxin-1),E5 SPA7(putative trypsin),E5 S387(cystatin),E5 SJH4(squash family serine protease inhibitor),E5 S554(5’-nucleotidase),E5 SQX1(enolase) and E5 SBA6(L-asparaginase)had higher protein and transcription levels in T.spiralis than in T.pseudospiralis(P <0.05),while E5 RZQ6(heat shock protein beta-1) and E5 S7 K8(histone H2 B) had a reversed pattern(P <0.05).Conclusion The bioinformatics analysis on differential excretory-secretory products between T.spiralis and T.pseudospiralis muscle larva stage suggests that serine protease,cystatin,plancitoxin-1 and 14-3-3 protein may involve in the formation of cysts and immuno modulation.