日本血吸虫精氨酸酶新基因的鉴定及作为疫苗的保护性研究

Identification of the SjARG Novel Gene and Study on Its Protective Potential as a Vaccine

目的识别和鉴定日本血吸虫(Sj)精氨酸酶(ARG)新基因,并研究纯化的重组SjARG对Sj感染小鼠的保护性。方法通过巢式PCR,从日本血吸虫尾蚴cDNA文库特异扩增ARG基因cDNA序列的5′端,并用生物信息学进行鉴定;将ARG的完整编码序列(CDS)克隆到pET30a(+)载体,表达并纯化表达产物后,以鸟氨酸-茚三酮法鉴定酶活性;用蛋白质印迹法(Westernblotting)比较重组ARG蛋白与Sj天然ARG蛋白的免疫学特性,间接免疫荧光法对ARG在Sj成虫中的分布进行组织定位;用纯化重组ARG蛋白免疫小鼠(PBS和SjGST作对照),Sj尾蚴攻击感染后,检测SjARG作为疫苗的免疫保护力。结果扩增并鉴定SjARG基因的完整CDS长为1095bp;重组SjARG具有酶活性,并具有与Sj天然ARG蛋白相同的免疫学特性;ARG定位于Sj成虫的肠壁和生殖器官。重组SjARG蛋白免疫后的减虫率和减卵率分别为55.8%和48.8%。SjGST蛋白免疫后的减虫率和减卵率分别为28.6%和6.89%。结论获取并鉴定了SjARG新基因;SjARG比SjGST具有更高的疫苗保护性。

Objective To recognize and identify the arginase （ARG） gene of Schistosomajaponicum（Sj）, and to study its protection potential as a vaccine. Methods The 5′-end of the ARG gene from the Sj cercariae cDNA library was amplified by nested-PCR and the sequence was identified by bioinfonnatics. The complete coding sequence（CDS） was cloned into pET30a （＋） vector, and a recombinant SjARG protein （rSjARG） was expressed, purified and used to raise antibodies. ARG＇s activity as an enzyme was tested by ornithine-ninhydrin reaction. Western blotting was used to compare the immunologic characteristics of rSjARG with that of the native one in Sj adult worm. Indirect immunofluorescence assay was used to immunolocalize it. For evaluating the protection potential of rSjARG, mice were immunized by the recombinant protein and challenged by cercariae of japonicum. Results The CDS length of the SjARG novel gene was identified as 1095bp rSjARG showed enzyme activity and the same immunologic characteristics with the native arginase in adult worm. SjARG located in the genital organ and gut of both sexes. The worm reduction rate and egg reduction rate in rSjARG group were 55.8% and 48.8% respectively, higher than that of the rSj26GST group （28.6% and 6.89% respectively）. Conclusion SjARG gene was identified, which shows a higher protection than the Sj26GST.