發(fā)布時(shí)間：2018-01-01 21:09

  本文關(guān)鍵詞：日本血吸蟲肌醇單磷酸酶SjIM的克隆、表達(dá)及動(dòng)物免疫保護(hù)效果評(píng)估　出處：《上海師范大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 日本血吸蟲 SjIM 克隆表達(dá) 免疫保護(hù) DNA疫苗

【摘要】：血吸蟲�。⊿chistosomiasis japonicum）是一種危害嚴(yán)重的由血吸蟲（Schistosoma japonicum）感染引起的人畜共患的寄生蟲病。在我國(guó)流行的是日本血吸蟲病。近年來，我國(guó)血吸蟲病防治工作已取得了舉世矚目的成就，但單靠藥物不能控制血吸蟲病的流行，且長(zhǎng)期反復(fù)的藥物治療會(huì)引起血吸蟲產(chǎn)生抗藥性的危險(xiǎn)，因此加強(qiáng)血吸蟲病疫苗的研制開發(fā)及疫苗候選分子的篩選無疑是血吸蟲病防治的重要手段。肌醇單磷酸酶(Inositol monophosphate)是磷脂酰肌醇信號(hào)傳遞途徑中的一種重要的酶，在哺乳動(dòng)物中，IM能夠?qū)⒁葝u素的脂質(zhì)第二信使PI(3，4,5)P3水解成PI（3，4)P2。然而在寄生蟲中IM基因的功能鮮有報(bào)道，探討SjIM的生物學(xué)功能可為血吸蟲疫苗候選分子的篩選提供了新思路。本研究利用生物信息學(xué)和分子生物學(xué)技術(shù)首次對(duì)日本血吸蟲肌醇單磷酸酶的基因進(jìn)行了克隆、表達(dá)、并對(duì)SjIM重組蛋白誘導(dǎo)的免疫保護(hù)效果進(jìn)行了初步評(píng)估。 一．日本血吸蟲SjIM基因的克隆及在不同發(fā)育階段蟲體中mRNA的表達(dá)分析。 通過BLAST分析比對(duì)，獲得SjIM基因的全長(zhǎng)cDNA序列，該基因核苷酸序列與日本血吸蟲其他已知基因無顯著同源性，為日本血吸蟲的新基因，命名為SjIM。GenBank登錄號(hào)為FN319326.1，開放閱讀框?yàn)?34bp，編碼277個(gè)氨基酸，理論分子質(zhì)量為30680.48Da。設(shè)計(jì)特異引物進(jìn)行PCR擴(kuò)增，克隆這個(gè)基因的全長(zhǎng)cDNA。以看家基因Tublin為內(nèi)參，應(yīng)用熒光定量PCR技術(shù)分析了SjIM基因在日本血吸蟲7d、14d、21d、28d、35d和42d雌雄蟲體內(nèi)的表達(dá)情況，結(jié)果顯示，SjAIM在日本血吸蟲童蟲和成蟲中均有表達(dá)，其中在35d蟲體內(nèi)表達(dá)量最高，且雌蟲體內(nèi)表達(dá)量高于雄蟲。 二．重組表達(dá)質(zhì)粒SjIM-pET-28a的構(gòu)建、表達(dá)及免疫保護(hù)效果的評(píng)估。 成功構(gòu)建了SjIM-pET-28a重組原核表達(dá)質(zhì)粒，，該重組蛋白在E.coli(DE3)中以包涵體形式表達(dá)，分子質(zhì)量約為31kD，Western blotting結(jié)果分析表明該融合蛋白具有較好的抗原性。用純化的SjIM-pET-28a重組蛋白免疫BALB/c小鼠，結(jié)果顯示與空白對(duì)照組相比，SjIM-pET-28a重組蛋白在小鼠中分別誘導(dǎo)了48.76%減蟲率和41.29%的肝臟減卵率，差異顯著（p0.05）。應(yīng)用ELISA方法檢測(cè)小鼠血清中特異性IgG抗體水平變化，結(jié)果表明SjIM-pET-28a重組蛋白免疫組小鼠在3次免疫后誘導(dǎo)產(chǎn)生了高水平的特異性IgG抗體，推測(cè)重組蛋白SjIM-pET-28a在小鼠中誘導(dǎo)產(chǎn)生的免疫保護(hù)效果可能與免疫小鼠血清中高滴度的IgG特異性抗體有關(guān)。 三．真核質(zhì)粒SjIM-pVAX1的構(gòu)建和DNA疫苗的免疫保護(hù)效果評(píng)估 成功構(gòu)建了重組真核表達(dá)質(zhì)粒SjIM-pVAX1，用重組真核質(zhì)粒免疫BALB/c小鼠，結(jié)果顯示與空白對(duì)照組相比，真核表達(dá)質(zhì)粒SjIM-pVAX1在BALB/c小鼠中誘導(dǎo)了28.94%的減蟲率和39.07%的肝臟減卵率,差異顯著（p0.05），重組pVAX1-SjIM免疫小鼠在3次免疫后誘導(dǎo)產(chǎn)生了高滴度的特異性IgG抗體。推測(cè)重組真核質(zhì)粒SjIM-pVAX1在小鼠中誘導(dǎo)產(chǎn)生的免疫保護(hù)效果可能與免疫小鼠血清中高滴度的IgG特異性抗體有關(guān)。 本文首次克隆和表達(dá)了日本血吸蟲新基因SjIM。用重組表達(dá)蛋白SjIM-pET-28a和真核重組質(zhì)粒SjIM-pVAX1免疫小鼠，均誘導(dǎo)了部分免疫保護(hù)效果。本文研究為深入探討SjIM的生物學(xué)功能，篩選新的血吸蟲病疫苗候選分子提供了基礎(chǔ)。
[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.

【學(xué)位授予單位】：上海師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392

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