布鲁氏菌S2疫苗株全基因组测序分析及诊断方法研究

发布时间：2018-01-16 11:17

本文关键词：布鲁氏菌S2疫苗株全基因组测序分析及诊断方法研究　出处：《内蒙古农业大学》2016年博士论文　论文类型：学位论文

【摘要】：布鲁氏菌病(brucellosis)是由布鲁氏杆菌(brucella spp.)引起的一种广泛流行的人畜共患病。近几年来,我国人畜布鲁氏菌病流行越来越严重,不仅影响经济发展,同时也威胁到人的健康,成为广受关注的公共卫生问题。为了有效控制本病的流行,在畜群中开始推广疫苗免疫,但是常用的诊断方法无法辨别布鲁氏菌疫苗免疫和自然感染抗体,使得畜群的“检疫-净化-免疫”防治措施出现了新的问题。而近年来报道的鉴别诊断方法仍然不能很好地鉴别S2疫苗免疫与羊种布鲁氏菌感染,很难做到免疫羊群中的布鲁氏菌病净化。为此,本研究跟踪市场上普遍使用的主流疫苗布鲁氏菌S2疫苗,对S2疫苗全基因组测序及生物信息学分析。找出与牛羊种布鲁氏杆菌相比较,S2疫苗的特有基因,预测出具有免疫原性的抗原基因,利用重组抗原建立了S2疫苗免疫与羊种布鲁氏菌感染的iELISA鉴别诊断方法。研究结果如下：1.本研究采用高通量测序技术,对布鲁氏菌S2疫苗株进行了全基因组测序及生物信息学分析,布鲁氏菌S2疫苗基因组大小约为3,331,982 bp, GC含量约为57.23%,共7个scaffold,13个contig。基因组组分分析发现,S2预测出3,243个编码基因,52个小卫星序列,14个微卫星序列,58个tRNA,12个rRNA。通过GO数据库比对,将布鲁氏菌S2疫苗株基因其划分为生物学途径、分子功能和细胞组件等三大类,参与生物学途径的基因有3,823个,细胞组件基因有1,949个,分子功能基因有2,585个。通过与KEGG数据库进行比对,将其预测出的基因划分为33类,33类基因序列的表达产物参与不同代谢通路。依据COG数据库将其布鲁氏菌预测出编码基因划分为20类。通过KEGG、COG、GO注释发现S2预测基因中大多数基因与氨基酸代谢、糖代谢、膜转运和氨基酸转运有关。S2疫苗全基因组测序预测出的3,243个编码基因与牛羊布鲁氏菌株序列进行比较,找出了20个特有基因。2.从20个特有基因中预测出编码产物可能具有免疫原性的GL_0002181、GL_0002189基因,同时将BP26当作对照,对目的基因进行克隆测序,将双向测序正确的GL_0002181、GL_0002189及BP26基因连接到pET30(+)表达载体中,并转化至BL21(DE3)感受态中。经IPTG诱导后进行SDS-PAGE电泳检测,发现GL_0002181、GL_0002189及BP26重组蛋白大小分别为31KDa.29KDa及32KDa。Western-bloting检测,发现GL_0002181、GL_0002189重组抗原能够与布鲁氏菌S2免疫血清反应,而不与羊种布鲁氏杆菌自然感染羊血清反应。BP26重组抗原与布鲁氏菌S2免疫血清及自然感染血清均呈阳性反应。3.以GL_0002181、GL_0002189及BP26重组蛋白作为抗原建立了布鲁氏菌iELISA诊断方法,3个重组抗原均有较高的敏感性和特异性,重组抗原GL 0002181敏感性为95%、特异性为95.3%,重组抗原GL_0002189敏感性为95%、特异性为95%,重组抗原BP26敏感性为96.7%、特异性为95%。经对180份SAT和RBPT检测呈阳性的血清进行iELISA检测,GL_0002181抗原检出阳性率为35.5%(64/180), GL_0002189抗原检出阳性率为33.8% (61／180),BP26抗原检出阳性率为98.8%(176／180)。经显著性分析,GL_0002181和GL_0002189两种抗原检出的阳性率差异不显著(P0.05),均可用于布鲁氏菌S2疫苗免疫的抗体检测。
[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.

【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.61

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