日本血吸虫凝溶胶蛋白的克隆、表达及转录时相分析

Cloning, expression and transcriptional profiles of Schistosoma japonicum gelsolin

目的构建日本血吸虫凝溶胶蛋白（Sjgelsolin）的原核表达载体，获得其表达产物并分析其在不同发育阶段的转录水平。方法根据序列AY814387设计引物，体外扩增Sjgelsolin的蛋白质编码序列，将其插入pET28a（＋）质粒，转化至大肠埃希菌BL21（DE3），用IPTG诱导表达。提取日本血吸虫虫卵，尾蚴，7-d童虫，42-d雌、雄成虫的总RNA，用半定量RT—PCR分析sjgelsolin在每个样本中的转录水平。结果成功构建了pET28a（＋）-Sjgelsolin重组质粒，并获得高水平的表达。Sjgelsolin转录子在尾蚴和雌虫成虫中未被检测到，而在7-d童虫和42-d雄虫成虫中被检测到。结论Sjgelsolin在体外获得表达，且其转录是受发育调控的。

Objective To construct prokaryotic expressional vector of gene gelsolin from Schistosoma japonicum （Sjgelsolin）, obtain its expressional product and analyze its transcriptional levels in different life stages. Methods Primers were designed based on the sequence of AY814387, protein-coding sequence of Sjgelsolin was amplified in vitro, inserted into pET28a（＋） plasmid, and transformed into E. coli BL21 （DE3） cells, and then expressed under the induction of IPTG. Total RNAs were purified from egg, cercariae, 7-day- old larval worms, 42-day-old adult female and male worms of Schistosoma japonicum, and the transcriptional levels of gelsolin in each sample were analyzed by semi-quantitative RT-PCR. Results Recombinant plasmid pET＇28a（＋）-Sjgelsolin was successfully constructed and highly expressed. Sjgelsolin transcripts were not detected in cercariae or adult female worms, but were observed in egg, 7-day-old larval and adult male worms. Conclusion Sjgelsolin was succesfully expressed in vitro, and its transcription was developmentally regulated.