旋毛虫新生幼虫可溶性抗原的免疫蛋白组学分析  

Immunoproteomic analysis on the soluble antigens of Trichinella spiralis newborn larvae

作　　者：郝会囡 程永康 张茹 韩璐璐 宋艳艳 龙绍蓉 刘若丹 张玺 王中全 崔晶 HAO Huinan;CHENG Yongkang;ZHANG Ru;HAN Lulu;SONG Yanyan;LONG Shaorong;LIU Ruodan;ZHANG Xi;WANG Zhongquan;CUI Jing(Department of Pathogens,Basic Medical College,Zhengzhou University,Zhengzhou 450001,Henan,China)

机构地区：[1]郑州大学基础医学院病原生物学系,河南郑州450001

出　　处：《中国寄生虫学与寄生虫病杂志》2023年第2期176-182,191,共8页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(82172300,82272367)。

摘　　要：目的 鉴定能被旋毛虫感染血清识别的新生幼虫蛋白,为抗新生幼虫疫苗筛选候选抗原。方法 从感染旋毛虫的BALB/c小鼠肠道收集成虫,培养后收集新生幼虫,制备新生幼虫可溶性性抗原,通过蛋白质免疫印迹(Western blotting)筛选出能被旋毛虫感染小鼠血清识别的新生幼虫可溶性抗原蛋白带进行液相色谱串联质谱(LC-MS/MS)鉴定,并与Uniprot数据库中的旋毛虫数据进行比对,利用生物信息学在线网站分析所鉴定的旋毛虫蛋白的理化性质,使用WEGO在线软件对匹配的蛋白进行基因本体(GO)分类。收集旋毛虫肌幼虫、肠道感染性幼虫(感染后6 h)、成虫(感染后2 d)和新生幼虫等不同发育期的虫体,提取虫体总RNA,反转录为cDNA。qPCR扩增旋毛虫C型凝集素(CTL)、钙网蛋白(CRT)、锌指蛋白(ZFP)和丙酮酸激酶(PK)基因,以甘油醛-3-磷酸脱氢酶(GAPDH)基因为内参,以肌幼虫的相对转录水平为对照,比较4个旋毛虫基因在不同发育期的相对转录水平。不同发育期相对转录水平的比较采用单因素方差分析。结果 Western blotting结果显示,新生幼虫可溶性抗原的11条蛋白条带中有4条被旋毛虫感染血清识别,相对分子质量分别为Mr99 700、 79 600、 68 900和46 000;LC-MS/MS鉴定出353种旋毛虫蛋白,其中166种蛋白(47.0%)为Mr40 000~70 000, 182种蛋白(51.8%)的等电点为5~6, 31种蛋白有信号肽,58种蛋白有跨膜结构域。GO分析结果显示,353种旋毛虫蛋白中,285种蛋白有功能注释,其中177种蛋白(62.1%)具有催化活性,192种蛋白(67.4%)具有结合活性,158种蛋白(55.4%)参与代谢过程,149种蛋白(52.3%)参与细胞转化过程。qPCR结果显示,以旋毛虫肌幼虫的相对转录水平为对照,肠道感染性幼虫、成虫和新生幼虫的CTL基因相对转录水平分别为140.99%、90.99%和65.71%(F=1 875.105,P?0.01), CRT基因分别为79.33%、 41.59%和58.58%(F=2 192.665, P?0.01), ZFP基因分别为64.93%、105.36%和126.7Objective To identify the soluble antigens of Trichinella spiralis newborn larvae recognizable by the serum of mice infected with T.spiralis for screening candidate antigens of anti-newborn larvae vaccines.Methods The adult worms were collected from the intestine of T.spiralis-infected BALB/c mice and cultured to collect the newborn larvae,of which the soluble antigens were extracted for screening out the antigen band recognized by sera from T.spiralis-infected mice using Western blotting.The recognized antigen on the blotting band was identified by liquid chromatograph tandem mass spectrometer(LC-MS/MS),and aligned with data of T.spiralis in the Uniprot database.The physicochemical properties of identified proteins were analyzed using the bioinformatics online website.The InterProscan software was used to perform proteins sequences searches against InterPro member databases to identify signatures,and the matched terms were further subjected to gene ontology(GO)categorizing using WEGO online software.The worms at different developmental stages including muscle larvae,infectious intestinal larvae(6 h post-infection),adult worms(2 d post-infection)and newborn larvae were collected to extract total RNA,which was then reversely transcribed into cDNA.The relactive transcription levels of the C-type lectin(CTL),calreticulin(CRT),zinc finger protein(ZFP)and pyruvate kinase(PK)at four developmental stages were analyzed by qPCR using glyceraldehyde-3-phosphate dehydrogenase(GAPDH)gene as the internal reference and compared with their transcription level at muscle larval stage using one-way ANOVA.Results Western blotting showed that among the 11 protein bands of the soluble antigens of newborn larvae,4 bands[the relative molecular mass(Mr)were 99700,79600,68900 and 46000]were recognized by T.spiralis-infected mice sera.A total of 353 T.spiralis proteins were identified by LC-MS/MS,of which 166 proteins(47.0%)had Mr 40000-70000,182 proteins(51.8%)had isoelectric point of 5-6,31 proteins had signal peptides and 58 proteins h

关 键 词：旋毛虫 新生幼虫 免疫蛋白组学 LC-MS/MS GO注释 

分 类 号：R383.15[医药卫生—医学寄生虫学]