日本血吸虫肌醇单磷酸酶SjIM的克

发布时间：2018-01-01 21:09

本文关键词：日本血吸虫肌醇单磷酸酶SjIM的克隆、表达及动物免疫保护效果评估　出处：《上海师范大学》2012年硕士论文　论文类型：学位论文

【摘要】：血吸虫病（Schistosomiasis japonicum）是一种危害严重的由血吸虫（Schistosoma japonicum）感染引起的人畜共患的寄生虫病。在我国流行的是日本血吸虫病。近年来，我国血吸虫病防治工作已取得了举世瞩目的成就，但单靠药物不能控制血吸虫病的流行，且长期反复的药物治疗会引起血吸虫产生抗药性的危险，因此加强血吸虫病疫苗的研制开发及疫苗候选分子的筛选无疑是血吸虫病防治的重要手段。肌醇单磷酸酶(Inositol monophosphate)是磷脂酰肌醇信号传递途径中的一种重要的酶，在哺乳动物中，IM能够将胰岛素的脂质第二信使PI(3，4,5)P3水解成PI（3，4)P2。然而在寄生虫中IM基因的功能鲜有报道，探讨SjIM的生物学功能可为血吸虫疫苗候选分子的筛选提供了新思路。本研究利用生物信息学和分子生物学技术首次对日本血吸虫肌醇单磷酸酶的基因进行了克隆、表达、并对SjIM重组蛋白诱导的免疫保护效果进行了初步评估。一．日本血吸虫SjIM基因的克隆及在不同发育阶段虫体中mRNA的表达分析。通过BLAST分析比对，获得SjIM基因的全长cDNA序列，该基因核苷酸序列与日本血吸虫其他已知基因无显著同源性，为日本血吸虫的新基因，命名为SjIM。GenBank登录号为FN319326.1，开放阅读框为834bp，编码277个氨基酸，理论分子质量为30680.48Da。设计特异引物进行PCR扩增，克隆这个基因的全长cDNA。以看家基因Tublin为内参，应用荧光定量PCR技术分析了SjIM基因在日本血吸虫7d、14d、21d、28d、35d和42d雌雄虫体内的表达情况，结果显示，SjAIM在日本血吸虫童虫和成虫中均有表达，其中在35d虫体内表达量最高，且雌虫体内表达量高于雄虫。二．重组表达质粒SjIM-pET-28a的构建、表达及免疫保护效果的评估。成功构建了SjIM-pET-28a重组原核表达质粒，，该重组蛋白在E.coli(DE3)中以包涵体形式表达，分子质量约为31kD，Western blotting结果分析表明该融合蛋白具有较好的抗原性。用纯化的SjIM-pET-28a重组蛋白免疫BALB/c小鼠，结果显示与空白对照组相比，SjIM-pET-28a重组蛋白在小鼠中分别诱导了48.76%减虫率和41.29%的肝脏减卵率，差异显著（p0.05）。应用ELISA方法检测小鼠血清中特异性IgG抗体水平变化，结果表明SjIM-pET-28a重组蛋白免疫组小鼠在3次免疫后诱导产生了高水平的特异性IgG抗体，推测重组蛋白SjIM-pET-28a在小鼠中诱导产生的免疫保护效果可能与免疫小鼠血清中高滴度的IgG特异性抗体有关。三．真核质粒SjIM-pVAX1的构建和DNA疫苗的免疫保护效果评估成功构建了重组真核表达质粒SjIM-pVAX1，用重组真核质粒免疫BALB/c小鼠，结果显示与空白对照组相比，真核表达质粒SjIM-pVAX1在BALB/c小鼠中诱导了28.94%的减虫率和39.07%的肝脏减卵率,差异显著（p0.05），重组pVAX1-SjIM免疫小鼠在3次免疫后诱导产生了高滴度的特异性IgG抗体。推测重组真核质粒SjIM-pVAX1在小鼠中诱导产生的免疫保护效果可能与免疫小鼠血清中高滴度的IgG特异性抗体有关。本文首次克隆和表达了日本血吸虫新基因SjIM。用重组表达蛋白SjIM-pET-28a和真核重组质粒SjIM-pVAX1免疫小鼠，均诱导了部分免疫保护效果。本文研究为深入探讨SjIM的生物学功能，筛选新的血吸虫病疫苗候选分子提供了基础。
[Abstract]:Schistosomiasis (Schistosomiasis japonicum) is a kind of serious harm from Schistosoma japonicum (Schistosoma japonicum) infection caused by zoonotic. Epidemic in China is schistosomiasis. In recent years, the work of schistosomiasis control in China has made remarkable achievements, but rely on drugs can not control the prevalence of schistosomiasis, and drugs for a long time repeated treatment will cause schistosomiasis risk of drug resistance, so to strengthen the screening of schistosomiasis vaccine development and vaccine candidate molecules is undoubtedly an important means of schistosomiasis control. Inositol monophosphatase (Inositol monophosphate) is a kind of phosphatidylinositol signaling pathway important enzyme, in mammals, can IM messenger PI second lipid insulin (3,4,5) and P3 PI (3,4) P2. hydrolysis in parasites of the IM gene function of SjIM are seldom reported. The biological function can provide a new idea for the screening of schistosomiasis vaccine candidate molecules. This study uses bioinformatics and molecular biology technology for Schistosoma japonicum inositol monophosphatase gene was cloned, expressed, and protective immunity induced by SjIM recombinant protein was evaluated.
1. Cloning of the SjIM gene of Schistosoma japonicum and the analysis of the expression of mRNA in the different developmental stages of the insect.
Through BLAST analysis, the full-length cDNA sequence of SjIM gene, the gene sequence of Schistosoma japonicum and no significant homology to other known genes and new genes of Schistosoma japonicum, named SjIM.GenBank and the accession number is FN319326.1, an open reading frame of 834bp, encoding 277 amino acids. The theoretical molecular mass for specific primers for 30680.48Da. design PCR amplification of full-length cDNA. clone of the gene to the housekeeping gene Tublin was used as an internal control, application of fluorescence quantitative PCR analysis of SjIM gene in Schistosoma japonicum 7d, 14d, 21d, 28d, 35d and 42d expression, female male in vivo results showed that SjAIM was expressed in schistosomula of Schistosoma japonicum adult worms and, in 35d insect expression was the highest, and the female body was higher than that of males.
Two. Construction of recombinant expression plasmid SjIM-pET-28a, expression and evaluation of immune protection effect.
The successful construction of the Recombinant Prokaryotic expression plasmid SjIM-pET-28a, the recombinant protein in E.coli (DE3) in the form of inclusion body expression, molecular weight of about 31kD, Western blotting analysis showed that the fusion protein has good antigenicity. The purified recombinant SjIM-pET-28a protein immune BALB/c mice. The results showed that compared with the control group, SjIM-pET-28a the recombinant proteins were induced in mice 48.76% worm reduction rate and 41.29% liver egg reduction rate, significant difference (P0.05). Changes in the levels of specific IgG antibodies in serum were detected by ELISA method, the results showed that SjIM-pET-28a recombinant protein immunized mice after the 3 immunization induced specific IgG antibody at a high level, that the immune protective effect of recombinant protein induced by SjIM-pET-28a in mice and in sera of mice immunized with high titer specific antibody of IgG.
Three. Construction of true nuclear particle SjIM-pVAX1 and evaluation of immune protection effect of DNA vaccine
The successful construction of the recombinant eukaryotic expression plasmid SjIM-pVAX1, the recombinant eukaryotic plasmid immunization of BALB/c mice. The results show that compared with the blank control group, eukaryotic expression plasmid SjIM-pVAX1 was induced in BALB/c mice by 28.94% of worm reduction rate and 39.07% liver egg reduction rate, significant difference (P0.05), recombinant pVAX1-SjIM immunized mice in 3 after immunization induced specific IgG antibody with high titer. The immune protective effect of recombinant eukaryotic plasmid SjIM-pVAX1 induced in mice and in sera of mice immunized with high titer specific antibody of IgG.
For the first time, cloning and expression of Schistosoma japonicum SjIM. gene expression in mouse immune protein SjIM-pET-28a and eukaryotic recombinant plasmid SjIM-pVAX1 by recombinant, could induce a certain immune protective effect. This paper studies to further explore the biological function of SjIM, provide the basis for screening new schistosomiasis vaccine candidate molecules.

【学位授予单位】：上海师范大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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