本文關(guān)鍵詞：布魯氏菌S2疫苗株全基因組測序分析及診斷方法研究　出處：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 布魯氏菌 布魯氏菌S2疫苗株 全基因組測序 原核表達 iELISA鑒別診斷方法

【摘要】：布魯氏菌病(brucellosis)是由布魯氏桿菌(brucella spp.)引起的一種廣泛流行的人畜共患病。近幾年來,我國人畜布魯氏菌病流行越來越嚴重,不僅影響經(jīng)濟發(fā)展,同時也威脅到人的健康,成為廣受關(guān)注的公共衛(wèi)生問題。為了有效控制本病的流行,在畜群中開始推廣疫苗免疫,但是常用的診斷方法無法辨別布魯氏菌疫苗免疫和自然感染抗體,使得畜群的“檢疫-凈化-免疫”防治措施出現(xiàn)了新的問題。而近年來報道的鑒別診斷方法仍然不能很好地鑒別S2疫苗免疫與羊種布魯氏菌感染,很難做到免疫羊群中的布魯氏菌病凈化。為此,本研究跟蹤市場上普遍使用的主流疫苗布魯氏菌S2疫苗,對S2疫苗全基因組測序及生物信息學(xué)分析。找出與牛羊種布魯氏桿菌相比較,S2疫苗的特有基因,預(yù)測出具有免疫原性的抗原基因,利用重組抗原建立了S2疫苗免疫與羊種布魯氏菌感染的iELISA鑒別診斷方法。研究結(jié)果如下：1.本研究采用高通量測序技術(shù),對布魯氏菌S2疫苗株進行了全基因組測序及生物信息學(xué)分析,布魯氏菌S2疫苗基因組大小約為3,331,982 bp, GC含量約為57.23%,共7個scaffold,13個contig�；蚪M組分分析發(fā)現(xiàn),S2預(yù)測出3,243個編碼基因,52個小衛(wèi)星序列,14個微衛(wèi)星序列,58個tRNA,12個rRNA。通過GO數(shù)據(jù)庫比對,將布魯氏菌S2疫苗株基因其劃分為生物學(xué)途徑、分子功能和細胞組件等三大類,參與生物學(xué)途徑的基因有3,823個,細胞組件基因有1,949個,分子功能基因有2,585個。通過與KEGG數(shù)據(jù)庫進行比對,將其預(yù)測出的基因劃分為33類,33類基因序列的表達產(chǎn)物參與不同代謝通路。依據(jù)COG數(shù)據(jù)庫將其布魯氏菌預(yù)測出編碼基因劃分為20類。通過KEGG、COG、GO注釋發(fā)現(xiàn)S2預(yù)測基因中大多數(shù)基因與氨基酸代謝、糖代謝、膜轉(zhuǎn)運和氨基酸轉(zhuǎn)運有關(guān)。S2疫苗全基因組測序預(yù)測出的3,243個編碼基因與牛羊布魯氏菌株序列進行比較,找出了20個特有基因。2.從20個特有基因中預(yù)測出編碼產(chǎn)物可能具有免疫原性的GL_0002181、GL_0002189基因,同時將BP26當(dāng)作對照,對目的基因進行克隆測序,將雙向測序正確的GL_0002181、GL_0002189及BP26基因連接到pET30(+)表達載體中,并轉(zhuǎn)化至BL21(DE3)感受態(tài)中。經(jīng)IPTG誘導(dǎo)后進行SDS-PAGE電泳檢測,發(fā)現(xiàn)GL_0002181、GL_0002189及BP26重組蛋白大小分別為31KDa.29KDa及32KDa。Western-bloting檢測,發(fā)現(xiàn)GL_0002181、GL_0002189重組抗原能夠與布魯氏菌S2免疫血清反應(yīng),而不與羊種布魯氏桿菌自然感染羊血清反應(yīng)。BP26重組抗原與布魯氏菌S2免疫血清及自然感染血清均呈陽性反應(yīng)。3.以GL_0002181、GL_0002189及BP26重組蛋白作為抗原建立了布魯氏菌iELISA診斷方法,3個重組抗原均有較高的敏感性和特異性,重組抗原GL 0002181敏感性為95%、特異性為95.3%,重組抗原GL_0002189敏感性為95%、特異性為95%,重組抗原BP26敏感性為96.7%、特異性為95%。經(jīng)對180份SAT和RBPT檢測呈陽性的血清進行iELISA檢測,GL_0002181抗原檢出陽性率為35.5%(64/180), GL_0002189抗原檢出陽性率為33.8% (61／180),BP26抗原檢出陽性率為98.8%(176／180)。經(jīng)顯著性分析,GL_0002181和GL_0002189兩種抗原檢出的陽性率差異不顯著(P0.05),均可用于布魯氏菌S2疫苗免疫的抗體檢測。
[Abstract]:Brucellosis (brucellosis) by Brucella (Brucella spp.) is a widespread zoonosis originated from. In recent years, China's livestock brucellosis epidemic is more and more serious, not only affects economic development, but also a threat to human health, has become a public health problem in order to wide public concern. The effective control of the epidemic, began to promote vaccination in the herd, but commonly used diagnostic methods cannot identify Brucella vaccine immune antibody and natural infection, the herd "Quarantine - purification - immune prevention of the emergence of new problems. The differential diagnosis methods reported in recent years is still not very good identification of S2 vaccine immunization with Brucella melitensis infection, difficult to achieve immune in the sheep brucellosis purification. Therefore, the widespread use of this study tracking the market mainstream S2 vaccine of Brucella vaccine, S2 vaccine Analysis of whole genome sequencing and bioinformatics. Find out compared with cattle and sheep brucellosis, specific gene S2 vaccine antigen gene, predict immunogenic, established S2 vaccine and Brucella melitensis infection iELISA differential diagnosis method using recombinant antigen. The results are as follows: 1. this study used high-throughput sequencing technology of Brucella vaccine strain S2 of whole genome sequencing and bioinformatics, S2 of Brucella vaccine genome size is about 3331982 BP, the content of GC is about 57.23%, a total of 7 scaffold, 13 contig. for group analysis, S2 predicted 3243 genes encoding, 52 small satellite sequences. 14 microsatellite sequences, 58 tRNA, 12 rRNA. by GO database, the Brucella vaccine strain S2 gene into biological pathways, molecular function and cellular components in three categories, participate in biological This way of 3823 genes, 1949 genes and cell component, molecular function and 2585 genes. By comparison with the KEGG database, the predicted genes are divided into 33 categories, 33 kinds of expression product of gene sequences in different metabolic pathways. On the basis of the COG database the cloth Lu's bacteria predicted encoding the gene is divided into 20 categories. Through KEGG, COG, GO comments found that the majority of gene and amino acid metabolism, S2 gene prediction in glucose metabolism, membrane transport and amino acid transport related.S2 vaccine genome sequencing predicted 3243 genes encoding sequences were compared with cattle and sheep Brucella strains, identified 20 specific gene.2. encoding product prediction may have the immunogenicity of GL_0002181, from 20 unique gene GL_0002189 gene, and BP26 as the control, cloning and sequencing of target gene, GL_0002181 bidirectional sequencing, GL_00021 Connect the 89 and BP26 gene into pET30 (+) expression vector, and transformed into competent BL21 (DE3). After induced by IPTG SDS-PAGE electrophoresis, GL_0002181, GL_0002189 and BP26 recombinant protein size were 31KDa.29KDa and 32KDa.Western-bloting detection showed that GL_0002181, GL_0002189 can react with recombinant antigen of Brucella S2 immune serum. But not with the sheep brucellosis infected sheep serum reaction of recombinant.BP26 antigen with Brucella S2 immune serum and natural infection serum showed positive reaction of.3. with GL_0002181, GL_0002189 and BP26 recombinant protein as antigen to establish a iELISA method for diagnosis of Brucella, 3 recombinant antigen has high sensitivity and specificity, sensitivity of 0002181 GL recombinant antigen 95%, the specificity was 95.3%, recombinant antigen GL_0002189 sensitivity was 95%, specificity was 95%, recombinant antigen BP26 sensitivity was 96.7%,. The specific 95%. of 180 SAT and RBPT positive serum were detected by iELISA. The positive rate of GL_0002181 antigen was 35.5% (64/180), the positive rate of GL_0002189 antigen was 33.8% (61 / 180), the positive rate of BP26 antigen was 98.8% (176 / 180). By the analysis of significant differences in the positive rate GL_0002181 and GL_0002189 were detected in two antigen was not significant (P0.05), can be used for the detection of antibody to Brucella S2 vaccine.

【學(xué)位授予單位】：內(nèi)蒙古農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S852.61

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