【摘要】：目前,對日本血吸蟲的免疫逃避機(jī)制還不十分了解�，F(xiàn)有研究表明,寄生蟲的半胱氨酸蛋白酶抑制劑(Cystatin)具有抑制宿主免疫應(yīng)答的作用,可能是參與免疫逃避的重要分子。本研究應(yīng)用生物信息學(xué)與分子生物學(xué)方法獲得了日本血吸蟲一個(gè)新的半胱氨酸蛋白酶抑制劑基因SjCystatin,通過研究該基因及其重組蛋白的結(jié)構(gòu)與功能,特別是對宿主的免疫調(diào)節(jié)功能,旨在初步闡明日本血吸蟲Cystatin分子在免疫逃避中的作用及其機(jī)制。 首先,在NCBI中搜索到日本血吸蟲半胱氨酸蛋白酶抑制劑基因部分序列,再結(jié)合日本血吸蟲基因組數(shù)據(jù)庫,利用PCR與3'RACE技術(shù),得到了SjCystatin的全長cDNA序列和genomic DNA序列。利用生物信息學(xué)方法分析發(fā)現(xiàn),該基因cDNA開放閱讀框全長306個(gè)堿基,編碼101個(gè)氨基酸,預(yù)測蛋白質(zhì)理論分子量為11.3 kD,等電點(diǎn)為6.95。其基因組DNA全長376個(gè)堿基,由兩個(gè)內(nèi)含子和三個(gè)外顯子組成。通過比較基因組學(xué)分析發(fā)現(xiàn),SjCystatin與脊椎動(dòng)物的stefins家族和cystatins家族的基因組結(jié)構(gòu)類似,推測它們可能是從共同的祖先基因進(jìn)化而來。系統(tǒng)發(fā)育分析顯示該預(yù)測蛋白與Genebank序列號為XP 002572115的曼氏血吸蟲半胱氨酸蛋白酶抑制劑的進(jìn)化距離最近,相似性為77%。生物信息學(xué)分析顯示預(yù)測蛋白質(zhì)沒有信號肽和跨膜區(qū),因此推測該蛋白應(yīng)可能定位在細(xì)胞內(nèi)。通過GeneDoc軟件分析發(fā)現(xiàn)該預(yù)測蛋白存在三個(gè)保守區(qū),分別是N末端的Gly6,Q49VVAG53序列和C末端的L76P77,與脊椎動(dòng)物stefins家族和cystatins家族的成員具有相同的保守區(qū),并且無二硫鍵。通過對以上特征的分析,認(rèn)為SjCystatin應(yīng)屬于stefins家族的新成員。 本研究成功構(gòu)建和高效原核表達(dá)了SjCystatin基因,并經(jīng)鎳離子鰲合親和層析柱法被純化,SDS-PAGE證實(shí)其分子量為12.1 kD；其免疫血清能特異性識別日本血吸蟲成蟲及蟲卵可溶性蛋白中11.3 kD分子；經(jīng)酶活性測定證明其具有抑制蛋白水解酶的生物學(xué)活性,在37℃、pH值為7.4的反應(yīng)條件下,10μg重組SjCystatin每分鐘可抑制0.0517木瓜蛋白酶活力單位。半定量RT-PCR的結(jié)果表明,日本血吸蟲蟲卵、童蟲和成蟲階段均有該基因的表達(dá)。免疫組化和免疫熒光共聚焦實(shí)驗(yàn)顯示SjCystatin基因編碼的蛋白質(zhì)主要定位在日本血吸蟲蟲卵的毛蚴中和成蟲的腸道表皮和體表。免疫保護(hù)性實(shí)驗(yàn)結(jié)果顯示,重組SjCystatin免疫小鼠的減蟲率和減卵率與PBS對照組相比,無顯著性差異,說明重組SjCystatin對小鼠無明顯的保護(hù)性效果。免疫熒光共聚焦檢測證實(shí),重組SjCystatin可以進(jìn)入到小鼠DC細(xì)胞中,并且只進(jìn)入到DC細(xì)胞的胞漿中。MTS法證明重組SjCystatin具有抑制DC細(xì)胞活性和代謝水平的作用。通過流式細(xì)胞術(shù)檢測小鼠DC細(xì)胞表面MHC-Ⅱ類分子的平均熒光強(qiáng)度,間接證實(shí)了重組SjCystatin的確可以降低DC細(xì)胞對抗原的提呈作用。重組SjCystatin體內(nèi)誘導(dǎo)小鼠CD4+ CD25+ Foxp3+ T細(xì)胞增殖的實(shí)驗(yàn)表明,重組SjCystatin可顯著提高感染小鼠體內(nèi)CD4+ CD25+ Foxp3+免疫調(diào)節(jié)性T細(xì)胞的比例。以上結(jié)果表明SjCystatin基因編碼的蛋白分子具有抑制抗原提呈及誘導(dǎo)抑制性免疫反應(yīng)的作用,提示其可能是一個(gè)血吸蟲蟲源性的免疫抑制因子,在血吸蟲免疫逃避中發(fā)揮作用。
[Abstract]:At present, the immune evasion mechanism of Schistosoma japonicum is not very well understood. It has been shown that cystatin has an important role in inhibiting the host immune response and may be an important molecule involved in immune evasion. In this study, a new cysteine protease inhibitor gene named Cystatin was obtained by bioinformatics and molecular biology method, and the structure and function of the gene and its recombinant protein, especially the immune regulation function of the host, were studied. The purpose of this study is to clarify the role and mechanism of cystatin in immune evasion of Schistosoma japonicum. Firstly, the gene partial sequence of cysteine protease inhibitor of Schistosoma japonicum was searched in NCBI and then combined with the genome database of Schistosoma japonicum. The full-length cDNA sequence and genomic DNA of Cystatin were obtained by PCR and 3 'RACE technique. Sequence analysis showed that the cDNA open reading frame had 306 bases full length, 101 amino acids were coded, the molecular weight of predicted protein was 11. 3 kD, and the isoelectric point was 6. 95. Its genomic DNA has a total length of 376 bases consisting of two introns and three exons. Composition. By comparative genomics analysis, Cystatin is found to be similar to the genome structure of the stews family and the cysteins family of vertebrates, suggesting that they may be evolved from common ancestor genes. The phylogenetic analysis showed that the predicted protein was closest to Genebank serial number XP 0025721115, and the similarity was 7. 7%. Bioinformatics analysis showed that the predicted protein had no signal peptide and transmembrane region, so it was presumed that the protein should be located in fine It is found that there are three conserved regions in the predicted protein, which are the N-terminal Gly6, Q49VVVF AG53 sequence and the C-terminal L76P77, respectively, and the members of the vertebrate stefins family and the cysteins family have the same conserved region, and no two. According to the analysis of the above features, it is considered that Cystatin should belong to the Stefins family. Its molecular weight was 12. 1kD, and the immune serum was able to specifically identify the soluble protein of Schistosoma japonicum and egg soluble protein. The enzyme activity assay showed that it had the biological activity of inhibiting proteolytic enzyme. Under the reaction conditions of 37 鈩，

本文編號：2264584