日本血吸虫腺嘌呤磷酸核糖转移酶基因的克隆、表达及虫期表达差异分析

Cloning and Expression of Adenine Phosphoribosyltransferase (APRT) and its Differential Expression Analysis during the Developmental Stages of Schistosoma japonicum

目的克隆和表达日本血吸虫腺嘌呤磷酸核糖转移酶基因1(SjAPRT1),并研究该基因在日本血吸虫不同发育阶段的转录和蛋白表达情况。方法根据SjAPRT1基因序列(GenBank登录号为AAW24796)设计引物,RT-PCR扩增目的基因,分析其在日本血吸虫各发育阶段转录本差异。将目的基因亚克隆至载体pET28a(+),转化至大肠埃希菌BL21(DE3)菌株,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,组氨酸亲和层析法纯化表达产物,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析纯化结果。纯化的蛋白SjAPRT1免疫新西兰白兔制备免疫兔血清,蛋白质印迹(West-ern blotting)分析其免疫反应性及其在日本血吸虫不同发育阶段表达的差异。结果在虫卵、尾蚴、童虫和成虫期均检测到SjAPRT1转录本(561 bp),获得了重组SjAPRT1蛋白。Western blotting分析表明,纯化的重组蛋白SjAPRT1可被日本血吸虫感染兔血清识别,虫卵、童虫和成虫粗抗原可被SjAPRT1免疫兔血清识别,在Mr 25 000处有特异性条带。结论日本血吸虫SjAPRT1蛋白在虫卵、童虫和成虫各期均有表达,并具有一定的免疫反应性。

Objective To clone and express adenine phosphoribosyltransferase gene of Schistosoma japonicum,and analyze its stage-specific transcription and expression at different developmental stages of S.japonicum.Methods Specific primers were designed according to the reported EST sequence of SjAPRT1 gene（GenBank Accession No.AAW24796）.RT-PCR was used to investigate the differential transcription of SjAPRT1 gene during the developmental stages.The gene was cloned into pET28a（＋） plasmid.The recombinant plasmid rSjAPRT1/pET28a（＋） was transformed into E.coli BL21（DE3） and induced with IPTG.The recombinant protein was purified with Ni-NTA resin and analyzed by SDS-PAGE.The purified protein was used to immune New Zealand white rabbits to obtain the antiserum.Western blotting was used to investigate the immunogenicity and the differential expression of rSjAPRT1 at different developmental stages.Results RT-PCR result showed that the specific bands were detected in eggs,cercariae,schistosomula,and adult worms（561 bp）.Western blotting analysis showed that the recombinant protein（rSjAPRT1,about Mr 25 000） existed in eggs,schistosomula and adult worms.The recombinant protein was recognized by pooled sera of infected rabbits.Conclusion The recombinant protein（rSjAPRT1） shows specific immunoreactivity,and is detected in the stage of eggs,schistosomula,and adult worms.