發(fā)布時間：2018-07-23 19:54

【摘要】： 目的擴(kuò)增中國大陸株細(xì)粒棘球蚴(Echinococcus granulosus Eg)鐵蛋白(ferritin)基因,構(gòu)建重組質(zhì)粒,并對重組蛋白免疫學(xué)特性進(jìn)行鑒定,為包蟲病分子疫苗的研制提供新的候選基因。方法(1)以細(xì)粒棘球蚴原頭蚴的RNA為模板,根據(jù)互聯(lián)網(wǎng)GenBank檢索出的細(xì)粒棘球蚴鐵蛋白基因序列設(shè)計(jì)引物,采用RT-PCR技術(shù)擴(kuò)增目的基因;(2)將目的基因亞克隆到pGEM-T載體,轉(zhuǎn)化入大腸桿菌JM109,對重組基因進(jìn)行DNA測序;(3)利用DNAman、NCBI/BLAST公共數(shù)據(jù)庫對目的基因的同源性進(jìn)行比較分析。應(yīng)用DNAstar、Biosun等生物分析軟件對該鐵蛋白的二級結(jié)構(gòu)、抗原表位、三維結(jié)構(gòu)進(jìn)行預(yù)測;(4)重組表達(dá)質(zhì)粒的構(gòu)建及鑒定Ⅰ:a.將目的基因亞克隆于表達(dá)載體pGEX-6P-1,轉(zhuǎn)化入Bl21,誘導(dǎo)融合蛋白(Eg.ferritin/GST)的表達(dá),經(jīng)親和層析法純化Eg.ferritin/GST;b.用含Eg.ferritin/GST的凝膠條帶免疫小鼠的抗血清及細(xì)粒棘球蚴免疫家兔的抗血清,通過Western-blot對Eg.ferritin/GST的免疫學(xué)特性進(jìn)行初步研究;(5)重組表達(dá)質(zhì)粒的構(gòu)建及鑒定Ⅱ:a.將目的基因亞克隆于pET-28a表達(dá)載體,轉(zhuǎn)化入BL21(DE3)plysS,誘導(dǎo)表達(dá)重組蛋白(Eg.ferritin),經(jīng)含Ni2+的His-bind樹脂柱純化Eg.ferritin;b.用Eg.ferritin免疫小鼠的抗血清,通過Western-blot及ELISA對Eg.ferritin的免疫學(xué)特性進(jìn)行研究。結(jié)果(1)PCR得到562bp的目的基因。(2)該基因與互聯(lián)網(wǎng)檢索的基因序列在390bp內(nèi)同源性為97.7%,氨基酸序列在130AA內(nèi)同源性為97.7%。不同生物間的同源性平均達(dá)40.79%。生物軟件預(yù)測:Eg.ferritin的分子量約16.7 kD,非極性氨基酸數(shù)目為68個,預(yù)測抗原表位的肽段為7N-12E,36H -43V, 55S-62H, 69Q-76R, 82A-89E, 102I-107E, 117A 124S,129L 136T。(3)構(gòu)建了Eg.ferritin/pGEX-6P-1/BL21基因工程菌株,表達(dá)融合蛋白Eg.ferritin/GST,因以包涵體形式存在,無法純化。western-blot結(jié)果:Eg.ferritin/GST能被Eg免疫家兔的抗血清和Eg.ferritin/GST凝膠條帶免疫小鼠的抗血清識別,同時小鼠抗血清還可識別天然抗原囊液、原頭蚴中約19kD的條帶。(4)構(gòu)建了Eg.ferritin/pET-28a /BL21(DE3)plysS基因工程菌株,表達(dá)并純化出Eg.ferritin。Western-blot檢測:Eg.ferritin免疫小鼠的抗血清可識別Eg.ferritin、天然抗原(囊液及原頭蚴),其位置大致相同約19kD。ELISA結(jié)果表明:免疫組血清中的IgG值明顯高于對照組血清(P0.01)。結(jié)論(1)成功擴(kuò)增出中國大陸株細(xì)粒棘球蚴鐵蛋白基因,構(gòu)建了基因工程菌株Eg.ferritin/ pGEM-T/JM109。(2)鐵蛋白結(jié)構(gòu)功能及抗原表位的預(yù)測對本實(shí)驗(yàn)的實(shí)施及選取有價值的抗原肽段提供了理論依據(jù)。(3)成功構(gòu)建基因工程菌株Eg.ferritin/pGEX-6P-1/BL21,表達(dá)分子量約42kD的融合蛋白Egferritin/GST,但無法純化。(4)成功構(gòu)建基因工程菌株Eg.ferritin/pET-28a/BL21(DE3) plysS,表達(dá)并純化出分子量約19kD的重組Eg.ferritin,經(jīng)免疫學(xué)鑒定初步說明Eg.ferritin有較好的抗原性及免疫原性,具有作為包蟲疫苗候選分子的潛能。
[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，

本文編號：2140474