發(fā)布時間：2018-03-22 04:15

  本文選題：雞傳染性貧血　切入點：原核表達(dá)　出處：《沈陽農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：雞傳染性貧血病(Chicken infectious anemia CIA)俗稱藍(lán)翅病,是由雞貧血病毒(Chicken anemia virus,CAV)引起的以雞的貧血和免疫抑制為主要特征的傳染病。該病1979年在日本被首次發(fā)現(xiàn)后,如今已遍布全球所有的養(yǎng)雞國家,是危害養(yǎng)禽業(yè)的重要傳染病。雞是CAV的唯一宿主,雛雞感染CAV后的典型病理變化是:貧血、骨髓黃化、胸腺萎縮等組織學(xué)損傷。本試驗通過臨床癥狀觀察、病理剖檢、基因片段的PCR擴增、雞胚接種、動物回歸試驗等對遼寧省丹東地區(qū)4個商品肉雞飼養(yǎng)場疑似CAV感染的雞群進(jìn)行了 CAV的分離、鑒定。結(jié)果:成功分離出1株CAV,命名為DD-2016;DD-2016基因編碼區(qū)全長為2297bp,共編碼764個氨基酸。利用生物學(xué)軟件對分離株DD-2016與GenBank中已發(fā)表的國內(nèi)外具有代表性的7株CAV基因進(jìn)行對比,結(jié)果DD-2016與F507715.1South kores同源性最高,為99.17%,與AF313470.1USA同源性最低,為95.59%;該分離株能引起雛雞發(fā)生貧血、皮下出血、胸腺萎縮、骨髓黃化等典型病理學(xué)損傷。分離株DD-2016與參考株CUX-1比較結(jié)果顯示,DD-2016的VP3基因的堿基序列存在兩個位點的堿基突變,并導(dǎo)致了相應(yīng)的第116位、118位這兩個位點的氨基酸發(fā)生改變。為了闡明這兩個位點的氨基酸的變異是否能影響其促凋亡作用,本實驗以分離株DD-2016感染5日齡SPF雞胚,以感染19日齡雞胚的胚體基因組DNA為模板,擴增VP3基因,并克隆表達(dá)到原核載體pET-32a上,然后轉(zhuǎn)染雞馬立克氏腫瘤細(xì)胞-MDCC-MSB1細(xì)胞,經(jīng)流式細(xì)胞儀檢測,研究分離株VP3蛋白的促凋亡作用。結(jié)果:成功構(gòu)建了 DD-2016VP3基因的原核表達(dá)載體pET-32a-VP3,表達(dá)產(chǎn)物CAV VP3蛋白能顯著促進(jìn)MDCC-MSB1細(xì)胞凋亡:正常生長狀態(tài)下的細(xì)胞中活細(xì)胞占84.5%、死亡細(xì)胞占9.1%、凋亡早期細(xì)胞占1.1%,凋亡晚期占5.3%;試驗組細(xì)胞中活細(xì)胞占24%、死亡細(xì)胞占1%、凋亡早期細(xì)胞占5.2%,凋亡晚期細(xì)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%.
【學(xué)位授予單位】：沈陽農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.65
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本文編號：1647006