本文選題：日本血吸蟲　切入點：腺嘌呤磷酸核糖轉(zhuǎn)移酶　出處：《華東理工大學(xué)》2011年碩士論文

【摘要】：曰本血吸蟲腺嘌呤磷酸核糖轉(zhuǎn)移酶(SjAPRT)是血吸蟲嘌呤補救途徑中的關(guān)鍵酶,它催化腺嘌呤生成腺苷一磷酸(AMP)。已有文獻報道在日本血吸蟲鑒定出兩條編碼APRT的基因序列SjAPRT1和SjAPRT2 (GenBank Accesion: AAW24796,AAW25340)。本研究在已有序列的基礎(chǔ)上通過RT-PCR獲得了日本血吸蟲腺嘌呤磷酸核糖轉(zhuǎn)移酶兩條基因(SjAPRTs)并利用Real time PCR及Western blotting技術(shù)分析其在日本血吸蟲各蟲期轉(zhuǎn)錄水平和蛋白水平差異。將目的基因亞克隆至載體pET28a,轉(zhuǎn)化至大腸埃希菌BL21菌株進行誘導(dǎo)表達,純化的蛋白免疫小鼠Western blotting分析其免疫反應(yīng)性,并進行動物實驗了解其保護性效果。以純化的rSjAPRTs作為包被抗原通過ELISA方法評估其免疫診斷價值。RT-PCR擴增結(jié)果顯示在蟲卵、尾蚴、童蟲和成蟲均檢測到SjAPRT1及SjAPRT2基因轉(zhuǎn)錄本,Real time PCR結(jié)果顯示SjAPRTs在各蟲期的轉(zhuǎn)錄水平存在一定差異,在成蟲及蟲卵期的表達量均為童蟲期的2倍左右。Western blotting分析結(jié)果表明在蟲卵、童蟲和成蟲中檢測到rSjAPRTl多抗兔血清特異性識別條帶,尾蚴中未檢測到特異性條帶；而在尾蚴和童蟲中檢測到rSjAPRT2多抗兔血清特異性識別條帶,成蟲中識別條帶較弱。這一結(jié)果暗示SjAPRTs的兩種同工酶可能在血吸蟲不同發(fā)育階段發(fā)揮作用,而SjAPRT 1在成蟲中發(fā)揮主要作用。此外純化的重組蛋白rSjAPRTs可被日本血吸蟲感染兔血清識別,表明其具有一定的免疫反應(yīng)性。以rSjAPRTl, rSjAPRT2作為包被抗原檢測日本血吸蟲病人血清的間接ELISA方法結(jié)果顯示檢測疫區(qū)病人血清的敏感性分別為60%,63.3%,非疫區(qū)正常人血清特異性分別為96.6%,93.3%。動物保護性實驗結(jié)果顯示rSjAPRT1免疫小鼠后可誘導(dǎo)產(chǎn)生13.6%的減蟲率和14.4%的肝臟減卵率。
[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.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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