日本血吸虫GSK3蛋白编码cDNA的克隆、表达及其重组蛋白的免疫保护研究

本研究利用生物信息学分析了日本血吸虫(Schistosoma japonicum,sj)Glycogen synthase kinase 3(GSK3)蛋白,并对其中一个GSK3蛋白的编码cDNA进行了克隆和原核表达,制备了特异性的多克隆抗体.同时,还初步评估了重组蛋白的免疫保护效果.生物信息学分析表明在日本血吸虫数据库存在两种GSK3蛋白,且其中一个SjGSK3在日本血吸虫不同发育时期均有转录.Western blot结果表明本研究制备的抗体能特异性识别日本血吸虫SjGSK3重组蛋白,表明该重组蛋白具有良好的免疫原性.动物实验表明免疫SjGSK3重组蛋白的动物与佐剂对照组比较分别获得了平均10.6％减虫率和40.5％肝脏减卵率.

CHARACTERIZATION OF A CDNA ENCODING GSK3 PROTEIN OF SCHISTOSOMA JAPONICUM

In the present study,bioinformatics and molecular techniques were applied to characterize glycogen synthase kinase 3(GSK3) proteins in Schistosoma japonicum.The expression profiles of SjGSK3 were determined at the transcript level in different development stages using Real-time RT-PCR.Bioinformatical analysis indicated that S.japonicum had two GSK3 isoforms.The Real-time RT-PCR results showed that one of two SjGSK3 isoforms was transcribed in all stages investigated and expressed at relatively higher levels in eggs and miracidia.Subsequently,the cDNA fragment encoding partial sequence of this GSK isoform was subcloned into a pET32a(+) vector.The recombinant SjGSK3 was expressed and purified for preparation of polyclonal antibodies and evaluation of immunoprotection in BALB/c mice.The polyclonal murine antibodies specifically recognized the recombinant SjGSK3 protein in Western blot.The mice immunized with the recombinant SjGSK3 showed worm reduction by 10.6% and liver egg reduction by 40.5% post challenge.