丹东地区鸡传染性贫血病毒的分离鉴定及VP3蛋白促凋亡作用的研究

发布时间：2018-03-22 04:15

  本文选题：鸡传染性贫血　切入点：原核表达　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：鸡传染性贫血病(Chicken infectious anemia CIA)俗称蓝翅病,是由鸡贫血病毒(Chicken anemia virus,CAV)引起的以鸡的贫血和免疫抑制为主要特征的传染病。该病1979年在日本被首次发现后,如今已遍布全球所有的养鸡国家,是危害养禽业的重要传染病。鸡是CAV的唯一宿主,雏鸡感染CAV后的典型病理变化是:贫血、骨髓黄化、胸腺萎缩等组织学损伤。本试验通过临床症状观察、病理剖检、基因片段的PCR扩增、鸡胚接种、动物回归试验等对辽宁省丹东地区4个商品肉鸡饲养场疑似CAV感染的鸡群进行了 CAV的分离、鉴定。结果:成功分离出1株CAV,命名为DD-2016;DD-2016基因编码区全长为2297bp,共编码764个氨基酸。利用生物学软件对分离株DD-2016与GenBank中已发表的国内外具有代表性的7株CAV基因进行对比,结果DD-2016与F507715.1South kores同源性最高,为99.17%,与AF313470.1USA同源性最低,为95.59%;该分离株能引起雏鸡发生贫血、皮下出血、胸腺萎缩、骨髓黄化等典型病理学损伤。分离株DD-2016与参考株CUX-1比较结果显示,DD-2016的VP3基因的碱基序列存在两个位点的碱基突变,并导致了相应的第116位、118位这两个位点的氨基酸发生改变。为了阐明这两个位点的氨基酸的变异是否能影响其促凋亡作用,本实验以分离株DD-2016感染5日龄SPF鸡胚,以感染19日龄鸡胚的胚体基因组DNA为模板,扩增VP3基因,并克隆表达到原核载体pET-32a上,然后转染鸡马立克氏肿瘤细胞-MDCC-MSB1细胞,经流式细胞仪检测,研究分离株VP3蛋白的促凋亡作用。结果:成功构建了 DD-2016VP3基因的原核表达载体pET-32a-VP3,表达产物CAV VP3蛋白能显著促进MDCC-MSB1细胞凋亡:正常生长状态下的细胞中活细胞占84.5%、死亡细胞占9.1%、凋亡早期细胞占1.1%,凋亡晚期占5.3%;试验组细胞中活细胞占24%、死亡细胞占1%、凋亡早期细胞占5.2%,凋亡晚期细胞占69.7%。
[Abstract]:Chicken infectious anemia disease, commonly known as blue wing disease, is an infectious disease characterized by anemia and immunosuppression in chickens caused by the chicken anemia virus Chicken anemia virus. The disease was first discovered in Japan in 1979. Chickens are the only hosts of CAV, and the typical pathological changes in chickens infected with CAV are anemia, bone marrow yellowing. Thymus atrophy and other histological injuries. This study was carried out by clinical symptom observation, pathological examination, PCR amplification of gene fragments, chicken embryo inoculation, Animal regression tests were carried out to isolate CAV from chickens suspected to be infected with CAV in four commercial broiler farms in Dandong area of Liaoning Province. Results: one strain of CAV was successfully isolated and named DD-2016DD-2016 gene encoding a total of 764 amino acids, the total length of coding region was 2297bp.Comparison was made between the isolated DD-2016 and 7 domestic and foreign representative CAV genes published in GenBank by using biological software. Results the homology between DD-2016 and F507715.1South kores was the highest (99.17%), and the homology with AF313470.1USA was the lowest (95.59%). The isolated strain could cause anemia, subcutaneous hemorrhage and thymus atrophy in chicks. The results of comparison between DD-2016 and reference strain CUX-1 showed that there were two base mutations in the VP3 gene of DD-2016. In order to elucidate whether the variation of amino acids at these two sites can affect the apoptosis-promoting effect, the isolated strain DD-2016 was used to infect 5-day-old SPF chicken embryos. The VP3 gene was amplified from the genomic DNA of the chicken embryo infected with 19 days old chicken embryo, and cloned into the prokaryotic vector pET-32a, then transfected into the chicken Marek's tumor cell line -MDCC-MSB1, and detected by flow cytometry. Results: the prokaryotic expression vector pET-32a-VP3 of DD-2016VP3 gene was successfully constructed. The expression product of CAV VP3 protein could significantly promote the apoptosis of MDCC-MSB1 cells. Dead cells accounted for 9.1%, early apoptotic cells accounted for 1.1%, late apoptotic cells accounted for 5.3%, living cells accounted for 24%, dead cells accounted for 1%, early apoptotic cells accounted for 5.2%, and late apoptotic cells accounted for 69.7%.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.65
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本文编号：1647006