重组日本血吸虫FKBP12基因的酶活性及其转录水平鉴定

Enzyme Activity and Transcription Level of the Recombinant FKBP12 of Schistosoma japonicum

目的对日本血吸虫(Schistosoma japonicum,Sj)FK506结合蛋白12(FKBP12)进行肽基脯氨酸顺反异构酶(peptidyl-prolyl cis-trans isomerase,PPIase)活性鉴定,并比较它与26 000 Mr的谷胱甘肽转移酶(Sj26GST)基因在成虫、尾蚴和虫卵的转录水平。方法从成虫RNA中RT-PCR扩增SjFKBP12基因,将其克隆入pGEX-4T-1载体中,诱导表达重组融合蛋白,经纯化后进行PPIase活性测定。利用RT-PCR半定量分析SjFKBP12基因与Sj26GST基因的转录水平。结果将SjFKBP12基因成功克隆入pGEX-4T-1载体中后,表达、纯化的重组融合蛋白有PPIase活性。SjFKBP12在尾蚴与虫卵期的转录水平相仿,都高于Sj26GST基因,是成虫期转录水平的1.5倍左右。结论成功鉴定了重组SjFKBP12酶活性,SjFKBP12在尾蚴和虫卵期较高的转录水平,为将其进行疫苗等研究提供了依据。

Objective To analyze the PPIase activity of purified GST-SjFKBP12 recombinant fusion protein and to compare the transcription level of SjFKBP12 with Sj26GST. Method FKBP12 eDNA obtained from adult worm was cloned into pGEX-4T-1 vector and expressed. PPIase activity of the purified recombinant fusion protein was analyzed. Semi-quantitative RT-PCR was used to analyze the transcription level of SjFKBPI2 and Sj26GST gene. Result SjFKBP12 cDNA was successfully cloned into pGEX-4T-1 vector. The purified GST-SjFKBP12 recombinant fusion protein was found to exhibit PPIase activity. Transcription level of SjFKBP12 in cercariae and eggs is 1.5 times higher than adults. Conclusion Functional GST-SjFKBP12 recombinant fusion protein was produced. High level of transcription of FKBP12 was detected in cercaria and eggs. The results may lay the foundation for the future vaccine development.