日本血吸虫腺嘌呤磷酸核糖转移酶两种同工酶的相关研究

发布时间：2018-04-01 14:28

本文选题：日本血吸虫　切入点：腺嘌呤磷酸核糖转移酶　出处：《华东理工大学》2011年硕士论文

【摘要】：曰本血吸虫腺嘌呤磷酸核糖转移酶(SjAPRT)是血吸虫嘌呤补救途径中的关键酶,它催化腺嘌呤生成腺苷一磷酸(AMP)。已有文献报道在日本血吸虫鉴定出两条编码APRT的基因序列SjAPRT1和SjAPRT2 (GenBank Accesion: AAW24796,AAW25340)。本研究在已有序列的基础上通过RT-PCR获得了日本血吸虫腺嘌呤磷酸核糖转移酶两条基因(SjAPRTs)并利用Real time PCR及Western blotting技术分析其在日本血吸虫各虫期转录水平和蛋白水平差异。将目的基因亚克隆至载体pET28a,转化至大肠埃希菌BL21菌株进行诱导表达,纯化的蛋白免疫小鼠Western blotting分析其免疫反应性,并进行动物实验了解其保护性效果。以纯化的rSjAPRTs作为包被抗原通过ELISA方法评估其免疫诊断价值。RT-PCR扩增结果显示在虫卵、尾蚴、童虫和成虫均检测到SjAPRT1及SjAPRT2基因转录本,Real time PCR结果显示SjAPRTs在各虫期的转录水平存在一定差异,在成虫及虫卵期的表达量均为童虫期的2倍左右。Western blotting分析结果表明在虫卵、童虫和成虫中检测到rSjAPRTl多抗兔血清特异性识别条带,尾蚴中未检测到特异性条带；而在尾蚴和童虫中检测到rSjAPRT2多抗兔血清特异性识别条带,成虫中识别条带较弱。这一结果暗示SjAPRTs的两种同工酶可能在血吸虫不同发育阶段发挥作用,而SjAPRT 1在成虫中发挥主要作用。此外纯化的重组蛋白rSjAPRTs可被日本血吸虫感染兔血清识别,表明其具有一定的免疫反应性。以rSjAPRTl, rSjAPRT2作为包被抗原检测日本血吸虫病人血清的间接ELISA方法结果显示检测疫区病人血清的敏感性分别为60%,63.3%,非疫区正常人血清特异性分别为96.6%,93.3%。动物保护性实验结果显示rSjAPRT1免疫小鼠后可诱导产生13.6%的减虫率和14.4%的肝脏减卵率。
[Abstract]:Schistosoma japonicum adenine phosphate ribonucleosyltransferase (SjAPRT) is a key enzyme in the purine remediation pathway of Schistosoma japonicum. It catalyzes the production of adenosine monophosphate from adenine monophosphate. It has been reported that two APRT coding gene sequences SjAPRT1 and SjAPRT2 GenBank accession: AAW24796 AAW25340 were identified in Schistosoma japonicum. Based on the existing sequences, Schistosoma japonicum adenopurinine was obtained by RT-PCR in this study. Real time PCR and Western blotting techniques were used to analyze the differences of transcription and protein levels in different stages of Schistosoma japonicum. The target gene was subcloned into vector pET28a and transformed to BL21 coli. The strain was induced to express, The immunoreactivity of purified protein immunized mice with Western blotting was analyzed, and the protective effect of purified rSjAPRTs was investigated by animal experiments. The immunological diagnostic value of purified rSjAPRTs was evaluated by ELISA method. The results of RT-PCR amplification showed that the purified rSjAPRTs was found in eggs and cercariae. Real time PCR of SjAPRT1 and SjAPRT2 gene transcripts were detected in both juvenile and adult worms. The results showed that there were some differences in the transcriptional level of SjAPRTs in each stage, and the expression of SjAPRTs in adult and egg stages was about twice as high as that in juvenile stage. Western blotting analysis showed that SjAPRTs was expressed in eggs. Specific identification bands of rSjAPRTl polyantibody rabbit serum were detected in children and adults, but no specific bands were detected in cercariae, but rSjAPRT2 polyantisera were detected in cercariae and juvenile worms. The results suggest that the two isozymes of SjAPRTs may play a role in different stages of development of Schistosoma japonicum. Moreover, the purified recombinant protein rSjAPRTs can be recognized by the sera of rabbits infected with Schistosoma japonicum. The indirect ELISA method using rSjAPRTl and rSjAPRT2 as coated antigen to detect human serum of schistosomiasis japonicum showed that the sensitivity of detecting sera of patients with schistosomiasis japonica was 60% and 63.3% respectively, and that of normal people in non-epidemic area was specific. The results of animal protective experiment showed that rSjAPRT1 could induce 13.6% worm reduction rate and 14.4% liver egg reduction rate after immunizing mice.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【参考文献】