细粒棘球蚴中国大陆株铁蛋白基因的克
发布时间：2018-07-23 19:54
【摘要】： 目的扩增中国大陆株细粒棘球蚴(Echinococcus granulosus Eg)铁蛋白(ferritin)基因,构建重组质粒,并对重组蛋白免疫学特性进行鉴定,为包虫病分子疫苗的研制提供新的候选基因。方法(1)以细粒棘球蚴原头蚴的RNA为模板,根据互联网GenBank检索出的细粒棘球蚴铁蛋白基因序列设计引物,采用RT-PCR技术扩增目的基因;(2)将目的基因亚克隆到pGEM-T载体,转化入大肠杆菌JM109,对重组基因进行DNA测序;(3)利用DNAman、NCBI/BLAST公共数据库对目的基因的同源性进行比较分析。应用DNAstar、Biosun等生物分析软件对该铁蛋白的二级结构、抗原表位、三维结构进行预测;(4)重组表达质粒的构建及鉴定Ⅰ:a.将目的基因亚克隆于表达载体pGEX-6P-1,转化入Bl21,诱导融合蛋白(Eg.ferritin/GST)的表达,经亲和层析法纯化Eg.ferritin/GST;b.用含Eg.ferritin/GST的凝胶条带免疫小鼠的抗血清及细粒棘球蚴免疫家兔的抗血清,通过Western-blot对Eg.ferritin/GST的免疫学特性进行初步研究;(5)重组表达质粒的构建及鉴定Ⅱ:a.将目的基因亚克隆于pET-28a表达载体,转化入BL21(DE3)plysS,诱导表达重组蛋白(Eg.ferritin),经含Ni2+的His-bind树脂柱纯化Eg.ferritin;b.用Eg.ferritin免疫小鼠的抗血清,通过Western-blot及ELISA对Eg.ferritin的免疫学特性进行研究。结果(1)PCR得到562bp的目的基因。(2)该基因与互联网检索的基因序列在390bp内同源性为97.7%,氨基酸序列在130AA内同源性为97.7%。不同生物间的同源性平均达40.79%。生物软件预测:Eg.ferritin的分子量约16.7 kD,非极性氨基酸数目为68个,预测抗原表位的肽段为7N-12E,36H -43V, 55S-62H, 69Q-76R, 82A-89E, 102I-107E, 117A 124S,129L 136T。(3)构建了Eg.ferritin/pGEX-6P-1/BL21基因工程菌株,表达融合蛋白Eg.ferritin/GST,因以包涵体形式存在,无法纯化。western-blot结果:Eg.ferritin/GST能被Eg免疫家兔的抗血清和Eg.ferritin/GST凝胶条带免疫小鼠的抗血清识别,同时小鼠抗血清还可识别天然抗原囊液、原头蚴中约19kD的条带。(4)构建了Eg.ferritin/pET-28a /BL21(DE3)plysS基因工程菌株,表达并纯化出Eg.ferritin。Western-blot检测:Eg.ferritin免疫小鼠的抗血清可识别Eg.ferritin、天然抗原(囊液及原头蚴),其位置大致相同约19kD。ELISA结果表明:免疫组血清中的IgG值明显高于对照组血清(P0.01)。结论(1)成功扩增出中国大陆株细粒棘球蚴铁蛋白基因,构建了基因工程菌株Eg.ferritin/ pGEM-T/JM109。(2)铁蛋白结构功能及抗原表位的预测对本实验的实施及选取有价值的抗原肽段提供了理论依据。(3)成功构建基因工程菌株Eg.ferritin/pGEX-6P-1/BL21,表达分子量约42kD的融合蛋白Egferritin/GST,但无法纯化。(4)成功构建基因工程菌株Eg.ferritin/pET-28a/BL21(DE3) plysS,表达并纯化出分子量约19kD的重组Eg.ferritin,经免疫学鉴定初步说明Eg.ferritin有较好的抗原性及免疫原性,具有作为包虫疫苗候选分子的潜能。
[Abstract]:Objective to amplify the (Echinococcus granulosus Eg) ferritin (ferritin) gene of Echinococcus granulosus strain from mainland China, construct the recombinant plasmid and identify the immunological characteristics of the recombinant protein, so as to provide a new candidate gene for the development of hydatidosis molecular vaccine. Methods (1) using RNA of echinococcus granulosus as template, primers were designed according to the sequence of ferritin gene of Echinococcus granulosus (GenBank), and the target gene was amplified by RT-PCR. (2) the target gene was subcloned into pGEM-T vector. The recombinant gene was transformed into E. coli JM109 and sequenced by DNA. (3) the homology of the target gene was analyzed by using DNAman NCBI / blast public database. The secondary structure, antigen epitope and three-dimensional structure of the ferritin were predicted by bioanalysis software such as DNA starstarsun. (4) Construction and identification of recombinant expression plasmid 鈪，
本文编号：2140474
[Abstract]:Objective to amplify the (Echinococcus granulosus Eg) ferritin (ferritin) gene of Echinococcus granulosus strain from mainland China, construct the recombinant plasmid and identify the immunological characteristics of the recombinant protein, so as to provide a new candidate gene for the development of hydatidosis molecular vaccine. Methods (1) using RNA of echinococcus granulosus as template, primers were designed according to the sequence of ferritin gene of Echinococcus granulosus (GenBank), and the target gene was amplified by RT-PCR. (2) the target gene was subcloned into pGEM-T vector. The recombinant gene was transformed into E. coli JM109 and sequenced by DNA. (3) the homology of the target gene was analyzed by using DNAman NCBI / blast public database. The secondary structure, antigen epitope and three-dimensional structure of the ferritin were predicted by bioanalysis software such as DNA starstarsun. (4) Construction and identification of recombinant expression plasmid 鈪，
本文编号：2140474
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