Abstract: Objective Objective To clone and identify the heat shock factors（HSFs）of Schistosoma japonicum and analyze its molec? ular structure and alternative splicing pattern. Methods Methods The New Zealand rabbits were infected with the cercariae of Schistoso? ma japonicum and were killed and dissected 42 days post?infection，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 Sj?hsf open reading frame（ORF）and the alternative splicing fragments were amplified by RT?PCR 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. Re Re? ? sults sults Sj?hsf ORF and the alternative splicing fragments were amplified from the female，male and egg samples of S. japonicum by RT?PCR. After cloning，the positive recombinant plasmids pBSjHSFf?F，pBSjHSFf?M，pBSjHSFf?E containing Sj?hsf ORF， pBSjHSFs?F，pBSjHSFs?M，pBSjHSFs?E with Sj?hsf alternative splicing fragments were identified by enzyme digestion and se? quencing. Three alternative splicing Sj?hsf isoforms were observed through sequence analysis：Sj?hsf?isoform1（2 050 bp） ， Sj?hsf isoform2（2 086 bp）and Sj ? hsf ? isoform3（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 ex? pressed at female，male and egg stages，but Sj?hsf?isoform1 with a high?level expression. Sj?HSF?isoform1（671 aa）and Sj?HSF? isoform2（683 aa）had DBD（DNA binding domain），HR?A/B and HR?C domains，while Sj?HSF?isoform3（282 aa）stopped in advance without HR?C domain. Phylogenetic tree analysis of HSFs illustrated that Sj ?HSFs belonged to HSF1 family，with a close phylogenetic relationship to Sm?HSFs. Conclusions Conclusions There are three alternative splicing isoforms of Sj ?HSF existing in the female，male and egg stages of S. japonicum，but Sj?HSF?isoform1 expresses in a high?level. This study lays the foundation for further study on molecular mechanisms of Sj?HSFs in regulating the heat shock response system.

Key words: Schistosoma japonicum；Heat shock response；Heat shock factor；Alternative splicing；Heat shock proteins

摘要： 目的 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 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、 InterPro、 Mega 6结合日本血吸虫和曼氏血吸虫基因组数据库以及GenBank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 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pBSjHSFf?F、 pBSjHSFf?M、 pBSjHSFf?E 以及含可变剪切片段的pBSjHSFs?F、 pBSjHSFs?M、 pBSjHSFs?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）， GenBank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 Do? main）、 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 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。

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