【摘要】：1953年,Rowe等在健康人群腺樣組織的細(xì)胞培養(yǎng)過程中發(fā)現(xiàn)了一種病毒,被國際病毒命名委員會定名為腺病毒(Adenovirus,ADV),原因是該病毒喜歡感染上皮細(xì)胞。而禽腺病毒的最早發(fā)現(xiàn)報道是在1987的巴基斯坦安卡拉地區(qū)附近,故又稱之為安卡拉病。禽腺病毒(Fowl adenovirus,FAV)是一種雙鏈DNA病毒,共分三個亞群:I群、II群、III群。Ⅰ群禽腺病毒共有12個血清型(FAV1~12),并且它們具備共同的群抗原,在這之中的Ⅰ群4型禽腺病毒被認(rèn)為是引發(fā)雞安卡拉病的主要病原。FAVⅠ病毒粒子的直徑是70~90nm,無囊膜,結(jié)構(gòu)為球形、二十面體對稱,有252個病毒殼粒,在這之中的二十面體的頂角殼粒是12個五鄰體,另外240個非頂角殼粒為六鄰體(hexon)。Hexon是FAVⅠ最主要的結(jié)構(gòu)蛋白,和五鄰體蛋白以及纖維蛋白共同構(gòu)成FAVⅠ的外殼,hexon蛋白包含了主要的屬和亞屬特異性抗原決定簇,以及次要的種特異性抗原決定簇,是中和抗體的靶目標(biāo)和主要保護(hù)性抗原基因,與致病性有緊密的關(guān)聯(lián),是以在被當(dāng)作診斷抗原方面擁有很好的遠(yuǎn)景。本研究通過對湖北仙桃雞場送檢病雞進(jìn)行一系列觀察,剖檢發(fā)現(xiàn)心包積液、肝臟泛黃腫大等病變特征;病理切片通過H.E染色后鏡檢觀察,可以發(fā)現(xiàn)肝臟組織脂肪變性;PCR結(jié)果與預(yù)期擴(kuò)增片段大小一致;測序分析對比后發(fā)現(xiàn)該毒株序列同源性與I群4型禽腺病毒同源性最高,達(dá)到99.2%,跟其他血清型相似性對比則相對較低;用盲傳3代病毒液接種1日齡雛雞亦能導(dǎo)致發(fā)病,確定本次發(fā)生的禽安卡拉病病原為禽腺病毒I群4型。對臨床上分離的I群4型禽腺病毒hexon基因進(jìn)行PCR擴(kuò)增并克隆到pMD19-T Simple載體上。重組克隆pMD19-T Simple-hexon經(jīng)雙酶切后,將hexon基因插入到pET-28a載體上,構(gòu)建重組質(zhì)粒pET-28a-hexon。將重組表達(dá)質(zhì)粒轉(zhuǎn)化BL21,經(jīng)由含卡那霉素的LB培養(yǎng)基培育篩選,挑取陽性菌送測序。用IPTG誘導(dǎo)表達(dá)鑒定正確的菌液,繼而對菌體進(jìn)行超聲破碎,通過SDS-PAGE分析其可溶性,利用親和層析分離純化目的蛋白,運用western-blot分析重組蛋白的免疫原性,以盲傳3代含禽腺病毒尿囊液滅活后制備免疫原,以0.2mL/只的劑量采用多點注射的方式分三次免疫小鼠,三免過后采血后分離血清,制備鼠多克隆抗體,最后由Western-blot實驗最終檢測重組六鄰體蛋白特異性。結(jié)果成功對六鄰體蛋白進(jìn)行了重組表達(dá),誘導(dǎo)表達(dá)溫度為37℃、IPTG濃度為1mM、誘導(dǎo)時間為4h�？扇苄苑治霰砻髦亟M六鄰體主要以包涵體的形式進(jìn)行表達(dá),利用親和層析分離純化目的蛋白,目的蛋白在10mM、20mM、50mM、100mM、150 mM、250 mM、500 mM等7個濃度的咪唑中均可洗脫。Westeron-blot分析結(jié)果證明,重組六鄰體蛋白可以與禽腺病毒陽性血清產(chǎn)生特異性結(jié)合,能顯示出特異性條帶,說明重組六鄰體蛋白具有很好的反應(yīng)原性。對小鼠三免過后分離的血清進(jìn)行Westeron-blot鑒定,結(jié)果表面成功制備了鼠抗重組六鄰體蛋白多克隆抗體。研究表明,重組六鄰體蛋白以包涵體的形式進(jìn)行表達(dá),具有較好的抗原性,與FAV病毒3免小鼠血清反應(yīng)性良好,為進(jìn)一步制備禽腺病毒新型疫苗及相關(guān)研究奠定了基礎(chǔ)。
[Abstract]:In 1953, Rowe et al. discovered a virus in the cell culture of adenoid tissues of healthy people. It was named Adenovirus (ADV) by the International Committee for the Nomenclature of Viruses because it likes to infect epithelial cells. Cara disease. Avian adenovirus (FAV) is a double-stranded DNA virus, divided into three subgroups: group I, group II, and group III. Avian adenovirus group I has 12 serotypes (FAV1-12) and they share a common group antigen. Among them, group I-4 avian adenovirus is considered to be the main pathogen causing chicken Ankara disease. Hexon is the most important structural protein of FAV I, and the pentahedral protein and fibrin together form the outer shell of FAV I. Proteins contain major genera and subgenera specific antigenic determinants and minor species specific antigenic determinants. They are the target and major protective antigenic genes of neutralizing antibodies. They are closely related to pathogenicity and have a good prospect of being used as diagnostic antigens. After a series of observations, pericardial effusion and hepatic yellowing and enlargement were found, fatty degeneration of liver tissue could be found in pathological sections by H.E staining and microscopic examination; PCR results were consistent with the expected amplified fragment size; sequencing analysis and comparison showed that the sequence homology of the strain was the highest with that of avian adenovirus type I 4, and reached the highest level. By 99.2%, the similarity with other serotypes was relatively low. Blind inoculation with 3-generation virus solution could also cause the disease in 1-day-old chickens. Avian adenovirus group I type 4 was identified as the pathogen of this disease. The hexon gene of Avian adenovirus group I type 4 was amplified by PCR and cloned into pMD19-T Simple vector. The recombinant plasmid pET-28a-hexon was constructed by inserting the hexon gene into the pET-28a vector. The recombinant plasmid was transformed into BL21. The positive bacteria were selected and sequenced through cultivation and screening on the LB medium containing kanamycin. The correct bacterial liquid was identified by IPTG induction, and then the bacterial body was broken by ultrasound. The recombinant plasmid was transformed into SDS-P-hexon. AGE was used to analyze its solubility, affinity chromatography was used to isolate and purify the target protein, Western-blot was used to analyze the immunogenicity of the recombinant protein. Immunogen was prepared after 3 passages of inactivated allantoic fluid containing avian adenovirus. The mice were immunized three times at a dose of 0.2 mL per mouse by multi-point injection. The specificity of the recombinant hexa-neighbor protein was detected by Western-blot. Results The recombinant hexa-neighbor protein was successfully expressed at 37 C, IPTG concentration was 1 mM and induction time was 4 h. Solubility analysis showed that the recombinant hexa-neighbor protein was expressed mainly in the form of inclusion body and purified by affinity chromatography. The results of Westeron-blot analysis showed that the recombinant hexagonal protein could bind specifically to avian adenovirus-positive serum and display specific bands, indicating that the recombinant hexagonal protein had a good reactant. The results showed that the recombinant hexagonal protein was expressed in the form of inclusion body and had good antigenicity. The recombinant hexagonal protein had good reactivity with the serum of mice immunized with FAV-3 and could be used for further preparation of poultry glands. The new vaccine and related research laid the foundation.
【學(xué)位授予單位】：長江大學(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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