本文選題：1型鴨甲肝病毒 + 鴨坦布蘇病毒　； 參考：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：鴨病毒性肝炎(Duck virus hepatitis,DVH)是危害雛鴨的一種急性、高致死性傳染病,主要侵害3周齡以內(nèi)雛鴨,已在國內(nèi)廣泛流行,嚴(yán)重威脅養(yǎng)鴨業(yè)的健康發(fā)展。鴨坦布蘇病毒病(Duck tembusu disease)是一種傳播迅速的急性傳染病,可使肉鴨表現(xiàn)神經(jīng)癥狀、產(chǎn)蛋鴨產(chǎn)蛋量下降等。自2010年在我國爆發(fā)以來,影響范圍廣,已造成巨大經(jīng)濟(jì)損失。目前,鴨甲肝病毒(Duck hepatitis A virus,DHAV)和鴨坦布蘇病毒(D uck tembusu virus,DTMUV)的抗體檢測主要依賴中和試驗(yàn)和酶聯(lián)免疫吸附試驗(yàn),批量檢測樣品時(shí)費(fèi)時(shí)費(fèi)力,因此臨床急需一種高效、快速的檢測方法對多種病原抗體同時(shí)進(jìn)行檢測。VP1基因和E基因分別是鴨甲肝病毒和鴨坦布蘇病毒的主要抗原基因。本試驗(yàn)將1型鴨甲肝病毒的VP1基因和鴨坦布蘇病毒的E基因分別在BL21(DE3)大腸桿菌中表達(dá),用純化的重組蛋白為抗原建立1型鴨甲肝病毒與鴨坦布蘇病毒的液相芯片抗體檢測方法,為DHA-1和DTMU的快速血清學(xué)診斷奠定基礎(chǔ)。本研究主要包括以下三部分內(nèi)容:1.鴨甲肝病毒的分離鑒定及VP1基因序列列分析分析收集2016年山東、河南等地區(qū)發(fā)病鴨場中疑似鴨甲肝病鴨的肝臟組織,共8份,經(jīng)研磨凍融離心處理后接種鴨胚,中和試驗(yàn)進(jìn)一步確診。結(jié)果有2株分離毒能被1型鴨甲肝病毒陽性血清中和,有6株分離毒能被3型鴨甲肝病毒陽性血清中和,初步確診分離毒株中有2株為1型鴨甲肝病毒(DHAV-1),6株為3型鴨甲肝病毒(DHAV-3)。利用設(shè)計(jì)的特異性引物對1型鴨甲肝病毒和3型鴨甲肝病毒分別進(jìn)行RT-PCR擴(kuò)增,將純化的1型VP1基因和3型VP1基因分別克隆至pMD18-T載體中,構(gòu)建pMD18-T-1VP1和pMD18-T-3VP1基因克隆重組質(zhì)粒并測序,利用MegAlign軟件對VP1蛋白的氨基酸同源關(guān)系進(jìn)行比對,結(jié)果顯示:2016年分離的1型鴨甲肝病毒毒株1-JS DH/16株和1-HZYC/16株與經(jīng)典毒株1-R85952/55的同源性分別是93.8%和93.4%,與其他分離毒株的同源性為最低92%~最高94.5%;3型鴨甲肝病毒毒株3-GD/16株、3-HNSQ/16株、3-SDGT/16株、3-SDXD/16、3-SC1/16株和3-SC2/16株與經(jīng)典毒株AP04114/03株的同源性分別為92.1%、92.5%、92.4%、93.5、92.4和92.4%,與其他分離株的同源性為最低90.1%~最高99.7%;利用MEGA 4.0生物學(xué)軟件進(jìn)行遺傳進(jìn)化分析,繪制進(jìn)化樹,可見:2016年分離毒株1-JSDH/16株和1-HZYC/16株處于同一個(gè)分支,與近幾年的流行株1-CS/14株的親緣關(guān)系最近,與經(jīng)典毒株1-R85952/55株親緣關(guān)系相對較遠(yuǎn);3-GD/16株與3-SC1/16株、3-SC2/16株三者親緣關(guān)系最近,處于同一個(gè)小分支上,3-SDGT/16株與3-HNSQ/16株的親緣關(guān)系近,處于同一分支上,3-SDXD/16株單獨(dú)處于一個(gè)分支,上述分離株與經(jīng)典毒株3-AP04023/04株的親緣關(guān)系較遠(yuǎn)。2.pCold-1VP1和pET-28a-E的原核表達(dá)將純化的1VP1基因克隆至pCold-III原核表達(dá)載體中,篩選獲得含1VP1基因正向插入的、有正確讀碼框的重組質(zhì)粒p Cold-1VP1。用Nde I/Hind III進(jìn)行雙酶切鑒定,經(jīng)瓊脂糖凝膠電泳檢測出兩條條帶,分別在4,377bp和714bp左右,與預(yù)期大小相符。提取鴨坦布蘇病毒(DTMUV)的RNA,用特異性引物進(jìn)行RT-PCR擴(kuò)增,得到1,503bp的E基因,將純化后的E基因克隆至pMD18-T載體中,構(gòu)建pMD18-T-E重組質(zhì)粒并保存,然后將其插入pET-28a(+)原核表達(dá)載體,篩選陽性重組質(zhì)粒p ET-28a-E。用EcoR I/Xho I進(jìn)行雙酶切鑒定,經(jīng)瓊脂糖凝膠電泳檢測出兩條條帶,分別在5,285bp和1,503bp左右,與預(yù)期大小相符。對獲得重組表達(dá)載體p Cold-1VP1和pET-28a-E菌液,進(jìn)行IPTG誘導(dǎo)表達(dá),對表達(dá)的重組蛋白進(jìn)行SDS-PAGE和Western blot分析(一抗分別為1型鴨甲肝病毒陽性血清和鴨坦布蘇病毒陽性血清)。結(jié)果發(fā)現(xiàn),DHAV-1VP1和DTMUV-E蛋白在大腸桿菌BL21(DE3)中可穩(wěn)定、高效地表達(dá)。將表達(dá)的重組蛋白純化后均得到單一的蛋白條帶,分別為26.5kDa和54.8k Da,Western blot檢測表明重組蛋白具有良好的免疫原性。3.DHAV-1和DTMUV液相芯片抗體檢測方法的建立根據(jù)液相蛋白芯片技術(shù)原理,以表達(dá)的重組蛋白為抗原,分別與不同編號的微球偶聯(lián),獲得偶聯(lián)復(fù)合體作為捕獲載體,建立了分別檢測鴨血清中DHAV-1、DTMUV抗體的單一液相芯片檢測方法,以及可以同時(shí)檢測兩種抗體的雙重液相芯片檢測方法,結(jié)果表明,建立的液相芯片檢測方法特異性、靈敏性和可重復(fù)性良好,為DHAV-1和DTMUV抗體監(jiān)測提供了高通量、重復(fù)性好、特異性高的抗體檢測體系,有利于鴨甲肝病毒病和鴨坦布蘇病毒病的快速檢測,給生產(chǎn)生活帶來改善,降低經(jīng)濟(jì)損失。
[Abstract]:Duck virus hepatitis (DVH) is a kind of acute, high fatal infectious disease which is harmful to ducklings. It mainly infringes ducklings within 3 weeks of age. It is widely prevalent in China. It is a serious threat to the healthy development of duck industry. The duck tanbsu virus (Duck Tembusu disease) is a rapid and acute infectious disease, which can make the duck show nerve. Symptoms, the drop of egg production of laying ducks, and so on. Since the outbreak of China in 2010, it has a wide range of influence and has caused great economic loss. At present, the antibody detection of duck hepatitis A virus (Duck hepatitis A virus, DHAV) and duck tanburu virus (D uck Tembusu virus, DTMUV) mainly depends on the test of Lai neutralization and enzyme linked immunosorbent assay to detect samples in batch. It is time-consuming and laborious, so it is urgent to detect the.VP1 gene and E gene, the main antigen gene of duck hepatitis A virus and duck tanb virus, respectively. The VP1 gene of duck hepatitis A virus type 1 and the E gene of duck Tanbu Su virus are in BL21 (DE3) Escherichia coli, respectively. Expression, using the purified recombinant protein as antigen to establish a liquid chip antibody detection method for duck hepatitis A virus type 1 and duck Tanbu Su virus, which lays the foundation for rapid serological diagnosis of DHA-1 and DTMU. This study mainly includes the following three parts: isolation and identification of 1. duck hepatitis A virus and sequence analysis and analysis of VP1 gene in Shandong in 2016 The liver tissues of ducks suspected to be duck hepatitis a disease in Henan and other regions of Henan, were inoculated with duck embryos by lapping and thawing centrifugation, and the neutralization test was further confirmed. The results showed that 2 isolates could be neutralized by the positive sera of type 1 duck hepatitis A virus and 6 isolates could be neutralized by the positive sera of type 3 duck hepatitis A virus (HCV), and the isolated strains were preliminarily confirmed. 2 of them were type 1 duck hepatitis A virus (DHAV-1) and 3 type of duck hepatitis A virus (DHAV-3). The specific primers were designed to amplify RT-PCR of duck hepatitis A virus type 1 and type 3 duck hepatitis A virus, respectively. The purified VP1 gene and the 3 VP1 gene were cloned into the pMD18-T carrier, and the cloning and recombination of pMD18-T-1VP1 and pMD18-T-3VP1 genes were constructed. Plasmid and sequencing, using MegAlign software to compare the amino acid homology of VP1 protein, the results showed that the homology of 1-JS DH/16 strain and 1-HZYC/16 strain of the 1 type duck hepatitis A virus strain of 2016 was 93.8% and 93.4% respectively, and the homology of the other isolates was the lowest 94.5% of the lowest 92%~, 3 type of duck liver disease. The homology of virus strain 3-GD/16 strain, 3-HNSQ/16 strain, 3-SDGT/16 strain, 3-SDXD/16,3-SC1/16 strain and 3-SC2/16 strain and the classical strain AP04114/03 strain were 92.1%, 92.5%, 92.4%, 93.5,92.4 and 92.4% respectively. The homology of the other isolates was the lowest 99.7% of the lowest 90.1%~, and the genetic evolution analysis was carried out using the MEGA 4 biological software to draw the evolutionary tree. See: the isolated strain 1-JSDH/16 and 1-HZYC/16 strains were in the same branch in 2016, closely related to the 1-CS/14 strain of the epidemic strain of recent years, and relative to the classic strain 1-R85952/55 strain. The relationship between 3-GD/16 strain and 3-SC1/16 strain and 3-SC2/16 strain three was recently on the same small branch, 3-SDGT/16 strain and 3-HNSQ/16 strain. The relationship is close to the same branch, and the 3-SDXD/16 strain is in a single branch. The relationship between the above isolated strain and the classic strain 3-AP04023/04 strain is far from.2.pCold-1VP1 and pET-28a-E. The purified 1VP1 gene is cloned into the pCold-III prokaryotic expression vector, and the positive insertion of the 1VP1 gene is screened for the correct reading of the 1VP1 gene. The recombinant plasmid P Cold-1VP1. of the code frame was identified by double enzyme cutting with Nde I/Hind III, and two bands were detected by agarose gel electrophoresis, which were in 4377bp and 714bp, respectively, in accordance with the expected size. The RNA of duck tanbsu virus (DTMUV) was extracted. The 1503bp E gene was obtained by specific primers and 1503bp E genes were obtained. The purified gene was cloned. To pMD18-T vector, pMD18-T-E recombinant plasmid was constructed and preserved, and then inserted into pET-28a (+) prokaryotic expression vector, the positive recombinant plasmid P ET-28a-E. was screened by EcoR I/Xho I for double enzyme digestion, and two bands were detected by agarose gel electrophoresis, which were in 5285bp and 1503bp, respectively. The recombinant expression was obtained. The vector p Cold-1VP1 and pET-28a-E bacteria were induced to induce the expression of IPTG, and the expression of recombinant protein was analyzed by SDS-PAGE and Western blot (one anti 1 duck hepatitis A virus positive serum and duck tanbsu virus positive serum respectively). The results showed that DHAV-1VP1 and DTMUV-E protein could be expressed steadily and efficiently in the BL21 (DE3) of Enterobacteriaceae. The recombinant protein was purified with a single protein band, 26.5kDa and 54.8k Da, respectively, and Western blot detection showed that the recombinant protein has a good immunogenic.3.DHAV-1 and DTMUV liquid chip antibody detection method based on the principle of liquid protein chip technology, with the recombinant protein as the antigen, respectively, and different numbered. By coupling the microspheres and obtaining the coupling complex as the capture carrier, a single liquid phase chip detection method for detecting DHAV-1 and DTMUV antibodies in duck serum, and a dual liquid phase chip detection method for detecting two kinds of antibodies at the same time were established. The results showed that the detection method of liquid phase core was specific, sensitive and reproducible. It provides a high throughput, reproducible and specific antibody detection system for DHAV-1 and DTMUV antibody monitoring, which is beneficial to the rapid detection of duck hepatitis A virus disease and duck Tanbu Su virus disease, to the improvement of production and life, and to reduce economic loss.

【學(xué)位授予單位】：山東農(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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