安徽省2012-2014年J亚群禽白血病病毒的分离及其囊膜蛋白基因序列的分析

发布时间：2018-07-01 21:07

  本文选题：J亚群禽白血病病毒 + gp85基因　； 参考：《安徽农业大学》2015年硕士论文

【摘要】：J亚群禽白血病(Subgroup J of avian leukosis)是由20世纪80年代末分离自肉鸡的一种由J亚群禽白血病病毒(Avian leukosis virus subgroup J,ALV-J)引起的新的禽白血病的亚群,其临床上主要造成患病禽类的生长和免疫抑制。近年来,该病在我国的养禽业普遍流行,给养禽业带来严重的危害。应用已经建立的ALV-J gp85基因特异性PCR检测方法,对2012至2014年间来自安徽省八个市县的33只疑似患病鸡进行PCR检测,检出阳性患病鸡14只;对安徽省3个鸡场患病鸡的598份肛门拭子及360份蛋清样品进行ALV p27抗原ELISA检测,肛门试子及蛋清样品阳性检出率分别为33.40%和10.58%;将PCR及ELISA检测为阳性的样品接种鸡胚成纤维细胞(CEF)分离ALV细胞毒毒株,PCR方法鉴定获取的ALV毒株,共获得ALV-J病毒毒株19株;通过PCR扩增的方法对分离的ALV-J毒株的囊膜蛋白基因gp85及gp37基因进行序列扩增及测定,获得19个gp85基因序列和10个gp37基因序列;在此基础上,选取GenBank中16个国内外不同的ALV-J参考株与分离株分别进行gp85和gp37的基因序列同源性比对、基因进化树的绘制,gp85基因比对结果表明:19个分离株与16个参考株的同源性在85.3%~98.1%之间,分离株之间的同源性为91.9%~99.9%,分离株与英国原型株HPRS-103的同源性在87.6%~98.6%之间,结合gp85基因遗传进化树结果显示18个分离株处于一个大的分支,与原型株亲缘关系较近;氨基酸序列比对分析发现,在氨基酸110#aa~120#aa,140#aa~150#aa,188#aa~193#aa这3个区域之间存在不同程度的缺失和变异;对变异程度较大的AH11-05的gp85基因序列分析得知,AH11-05序列在579bp与589bp之间插入了一段序列-GGGAACCTT-,对ALV-J分离株的gp37基因的序列比对结果整体上与gp85基因比对结果相一致,但gp37的基因序列同源性相对较高,并无整段基因的插入或者缺失。本试验初步掌握ALV-J在安徽省不同地区鸡群中的存在情况,并成功的分离到了19株ALV-J分离株,为进一步深入研究ALV-J囊膜蛋白基因变异提供基础,为我国ALV-J的流行提供相应的预防措施。
[Abstract]:The J subgroup of avian leukemia (Subgroup J of avian leukosis) is a subgroup of avian leukosis caused by the J subgroup of avian leukosis virus (Avian leukosis virus subgroup J) isolated from the end of the 1980s, which is mainly caused by the growth and immunosuppression of the sick poultry. In recent years, the disease has been found in poultry in our country. The ALV-J gp85 gene specific PCR detection method has been used to detect 33 suspected sick chickens from eight cities and counties in Anhui province from 2012 to 2014, and 14 positive sick chickens were detected. 598 anal swabs and 360 egg white samples from the sick chickens in 3 chicken farms in Anhui province were tested. The positive detection rates of ALV p27 antigen ELISA were 33.40% and 10.58%, respectively. The positive samples of PCR and ELISA were inoculated with chicken embryo fibroblasts (CEF) to separate ALV cell viruline strains, PCR method identified ALV strains obtained by PCR method, and 19 strains of ALV-J virus strains were obtained. The isolated ALV- was carried out by PCR amplification method. The J gene gp85 and gp37 gene were amplified and measured, and 19 gp85 gene sequences and 10 gp37 gene sequences were obtained. On this basis, 16 different ALV-J reference strains at home and abroad were selected to compare the homologous sequence of the gene sequence of gp85 and gp37, the plotting of the gene evolution tree and the gp85 gene ratio. The results showed that the homology of 19 isolates and 16 reference strains was between 85.3%~98.1%, the homology of the isolated strains was 91.9%~99.9%, the homology of the isolated strain and the British prototype strain HPRS-103 was between 87.6%~98.6%, and the results of the genetic evolution tree of the gp85 gene showed that 18 isolates were in a large branch, and the relationship with the prototype plant was more related. The analysis of amino acid sequence alignment found that there were different degrees of deletion and variation between the 3 regions of the amino acid 110#aa~120#aa, 140#aa~150#aa, and 188#aa~193#aa, and the sequence analysis of gp85 gene of the AH11-05 with a large variation of degree showed that the AH11-05 sequence inserted a sequence of -GGGAACCTT- in the 579bp and 589bp, and the g of the ALV-J isolates. The sequence alignment of P37 gene was consistent with the results of gp85 gene alignment, but the sequence homology of gp37 was relatively high, and there was no insertion or deletion of the whole gene. This experiment preliminarily grasps the existence of ALV-J in the chickens in different regions of Anhui Province, and successfully separated 19 ALV-J isolates to further study. To provide basis for gene mutation of ALV-J envelope protein, and provide corresponding preventive measures for the prevalence of ALV-J in China.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.65

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