本文選題：J亞群禽白血病病毒 + gp85基因　； 參考：《安徽農(nóng)業(yè)大學》2015年碩士論文

【摘要】：J亞群禽白血病(Subgroup J of avian leukosis)是由20世紀80年代末分離自肉雞的一種由J亞群禽白血病病毒(Avian leukosis virus subgroup J,ALV-J)引起的新的禽白血病的亞群,其臨床上主要造成患病禽類的生長和免疫抑制。近年來,該病在我國的養(yǎng)禽業(yè)普遍流行,給養(yǎng)禽業(yè)帶來嚴重的危害。應用已經(jīng)建立的ALV-J gp85基因特異性PCR檢測方法,對2012至2014年間來自安徽省八個市縣的33只疑似患病雞進行PCR檢測,檢出陽性患病雞14只;對安徽省3個雞場患病雞的598份肛門拭子及360份蛋清樣品進行ALV p27抗原ELISA檢測,肛門試子及蛋清樣品陽性檢出率分別為33.40%和10.58%;將PCR及ELISA檢測為陽性的樣品接種雞胚成纖維細胞(CEF)分離ALV細胞毒毒株,PCR方法鑒定獲取的ALV毒株,共獲得ALV-J病毒毒株19株;通過PCR擴增的方法對分離的ALV-J毒株的囊膜蛋白基因gp85及gp37基因進行序列擴增及測定,獲得19個gp85基因序列和10個gp37基因序列;在此基礎上,選取GenBank中16個國內(nèi)外不同的ALV-J參考株與分離株分別進行gp85和gp37的基因序列同源性比對、基因進化樹的繪制,gp85基因比對結(jié)果表明:19個分離株與16個參考株的同源性在85.3%~98.1%之間,分離株之間的同源性為91.9%~99.9%,分離株與英國原型株HPRS-103的同源性在87.6%~98.6%之間,結(jié)合gp85基因遺傳進化樹結(jié)果顯示18個分離株處于一個大的分支,與原型株親緣關系較近;氨基酸序列比對分析發(fā)現(xiàn),在氨基酸110#aa~120#aa,140#aa~150#aa,188#aa~193#aa這3個區(qū)域之間存在不同程度的缺失和變異;對變異程度較大的AH11-05的gp85基因序列分析得知,AH11-05序列在579bp與589bp之間插入了一段序列-GGGAACCTT-,對ALV-J分離株的gp37基因的序列比對結(jié)果整體上與gp85基因比對結(jié)果相一致,但gp37的基因序列同源性相對較高,并無整段基因的插入或者缺失。本試驗初步掌握ALV-J在安徽省不同地區(qū)雞群中的存在情況,并成功的分離到了19株ALV-J分離株,為進一步深入研究ALV-J囊膜蛋白基因變異提供基礎,為我國ALV-J的流行提供相應的預防措施。
[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.
【學位授予單位】：安徽農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S852.65

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