日本血吸虫SjPRMT1基因的克隆、表达及表达产物的免疫保护效果分析

Cloning,Expression and Immunoprotection of the Novel Gene SjPRMT1 of Schistosoma japonicum

【目的】克隆和表达日本血吸虫精氨酸甲基转移酶1(SjPRMT1)编码基因cDNA,分析其在日本血吸虫不同发育阶段虫体的表达情况,评估该重组抗原在小鼠体内诱导的抗血吸虫免疫保护效果。【方法】从实验室构建的7d童虫消减cDNA文库中,获得一个EST序列,经序列测定和生物信息学分析命名为SjPRMT1,以SjcDNA为模板,获得其全长cDNA。荧光实时定量PCR分析该基因在童虫和成虫的表达情况。以pET28a(+)为载体构建重组表达质粒,Western blotting检测重组蛋白的抗原性与免疫原性,利用重组抗原免疫小鼠评估其免疫保护效果。【结果】获得了SjPRMT1编码基因的全长cDNA,开放阅读框为660bp,编码219个氨基酸。荧光实时定量PCR分析该基因在18d童虫高表达,13和23d次之。获得了SjPRMT1重组蛋白,其具有良好的抗原性和免疫原性。在小鼠免疫试验中,与空白对照组比较,免疫组小鼠获得35.07%的减虫率和48.66%的肝脏减卵率。【结论】获得了日本血吸虫童虫期高表达的SjPRMT1基因的全长cDNA,成功构建了pET28a(+)-SjPRMT1重组质粒,该重组抗原在小鼠体内诱导产生了部分免疫保护效果。

[ Objective ] The present study was intend to clone a full length cDNA encoding protein arginine methyltransferase 1 in Schistosoma japonicum, and further access its immunoprotection in mice for schistosomiasis. [ Method ] A full-length cDNA clone encoding a protein arginine methyltransferase was obtained from schistosomula cDNA enriched library, named S.jPRMT1 based on bioinformatics analysis. The expression profiles of SjPRMT1 was determined by real-time PCR using the template cDNAs isolated from 7, 13, 18, 23, 32 and 42 days parasites. The open reading frame was then subcloned into a pET28a（＋） vector and transformed into competent E.coil/BL21. The recombinant protein was purified and then the immune characters of antigen were accessed by Western blotting. [Result] Bioinformatics analysis indicated that the ORF of S.jPRMT1 is 660 base pairs, which is encoding 219 amino acids. Real-time PCR analysis revealed that there is a highest expression in 18 days schistosomula, and slightly lower in 13 days and 23 days parasites, suggesting that S.jPRMT1 has highly expression in schistosomula stage. The recombinant protein showed a good ability to induce antibodies in mice as examined by ELISA. Animal experiment indicated that the administration of recombinant PRMT1 protein in mice resulted in worm burden reduced by 35.07% and egg burden reduced by 48.66%. [Conclusion] A full-length cDNA differently expression in schistosomula was obtained. Results demonstrated that the recombinant PRMT1 protein could induce partial protection against schistosomiasis in mice.