日本血吸虫热休克因子基因结构特征及其可变剪切亚型分析研究  

Molecular structure and alternative splicing analysis of heat shock factors of Schistosoma japonicum

作　　者：谢郁[1] 廖海燕[1] 陈淑洁[1] 石玲玉 欧丽妍 滕萍英 夏丹[1] 陈启伟[1] 郑斯楠 周晓红[1] 

机构地区：[1]南方医科大学公共卫生学院病原生物学系暨广东普通高校新发传染病防治重点实验室,广州510515

出　　处：《中国血吸虫病防治杂志》2016年第4期381-387,共7页Chinese Journal of Schistosomiasis Control

基　　金：国家自然科学基金(81071378;30872204);广东省广州市对外科技合作专项(2012J5100026);广东省大学生创新创业训练计划项目(201412121088)

摘　　要：目的克隆、鉴定日本血吸虫热休克因子(Sj-hsf),并分析其基因结构特征和可变剪切亚型。方法以日本血吸虫尾蚴感染新西兰白兔,42 d后剖杀,以灌注法收集雌虫、雄虫及感染兔肝脏,以Trizol法提取总RNA。以RT-PCR扩增日本血吸虫雌虫、雄虫、虫卵总RNA,获得Sj-hsf开放阅读框(ORF)全长片段和Sj-hsf可变剪切区域片段,并进行克隆、酶切及测序鉴定。用DNAMAN 8.0、Inter Pro、Mega 6结合日本血吸虫和曼氏血吸虫基因组数据库以及Gen Bank等网络分析平台,分析Sj-hsf的基因结构特征、功能结构域、可变剪切模式以及不同物种间HSF序列的同源性和进化树。结果自雌虫、雄虫、虫卵扩增获得靶片段,并克隆获得阳性重组质粒,其中含Sj-hsf ORF的p BSj HSFf-F、p BSj HSFf-M、p BSj HSFf-E以及含可变剪切片段的p BSj HSFs-F、p BSj HSFs-M、p BSj HSFs-E,酶切及测序鉴定正确。序列分析显示Sj-hsf具有3种剪切亚型:Sj-hsf-isoform1(2 050 bp)、Sj-hsf-isoform2(2 086 bp)、Sj-hsf-isoform3(2 111 bp),Gen Bank登录号分别为KU954546、KX119143、KX119144,均位于日本血吸虫基因组SJC-S000780,有8个外显子,在雌虫、雄虫和虫卵阶段3种亚型均存在,以Sj-hsf-isoform1表达量高。Sj-HSF-isoform1(671 aa)、Sj-HSF-isoform2(683 aa)具有HSF特征性DBD(DNA Binding Domain)、HR-A/B和HR-C结构域,Sj-HSF-isoform3(282 aa)提前终止,无HR-C结构域。进化树分析表明Sj-HSF三种剪切亚型隶属HSF1,与曼氏血吸虫HSF近源。结论日本血吸虫雌虫、雄虫、虫卵阶段均存在3种剪切亚型的HSF,以Sj-HSF-isoform1表达量高。本研究为进一步阐明Sj-HSF调控Sj-HSPs等应激防御体系的分子机制奠定了基础。Objective To clone and identify the heat shock factors（HSFs）of Schistosoma japonicum and analyze its molecular structure and alternative splicing pattern. Methods The New Zealand rabbits were infected with the cercariae of Schistosoma japonicum and were killed and dissected 42 days postinfection,and the adult worms of S. japonicum and the livers of the rabbits were harvested. Then,the total RNA was extracted by using Trizol reagent. The Sjhsf open reading frame（ORF）and the alternative splicing fragments were amplified by RTPCR from the female,male and egg samples,then cloned and verified by enzyme digestion and sequencing. DNAMAN 8.0,InterPro,Mega 6 combined with the Internet databases were utilized to clarify the gene structure,functional domains,alternative splicing pattern,and the homology and phylogenetic tree of HSFs. Results Sjhsf ORF and the alternative splicing fragments were amplified from the female,male and egg samples of S. japonicum by RTPCR. After cloning,the positive recombinant plasmids pBSjHSFfF,pBSjHSFfM,pBSjHSFfE containing Sjhsf ORF, pBSjHSFsF,pBSjHSFsM,pBSjHSFsE with Sjhsf alternative splicing fragments were identified by enzyme digestion and sequencing. Three alternative splicing Sjhsf isoforms were observed through sequence analysis：Sjhsfisoform1（2 050 bp）,Sjhsf＆amp;nbsp;isoform2（2 086 bp）and Sjhsfisoform3（2 111 bp）;the GenBank accession numbers were KU954546,KX119143 and KX119144,respectively. All the three isoforms located in the same Contig SJC_S000780 of S. japonicum genome and all expressed at female,male and egg stages,but Sjhsfisoform1 with a highlevel expression. SjHSFisoform1（671 aa）and SjHSFisoform2（683 aa）had DBD（DNA binding domain）,HRA/B and HRC domains,while SjHSFisoform3（282 aa）stopped in advance without HRC domain. Phylogenetic tree analysis of HSFs illustrated that SjHSFs belonged to HSF1 family,with a close phylogenetic relationship to SmHSFs. Conclusions There are three alternative splicing isoforms of SjHSF existing in the female,ma

关 键 词：日本血吸虫 热休克反应 热休克因子 可变剪切 热休克蛋白 

分 类 号：R383.24[医药卫生—医学寄生虫学]