【摘要】：J亞群禽白血病病毒(Avian Leukosis Virus Subgroup J,ALV-J)自1988年從肉雞中分離出來(lái),在世界范圍內(nèi)廣泛流傳。以垂直和水平兩種傳播方式傳播,誘發(fā)腫瘤、引起生長(zhǎng)緩慢、消瘦、死亡為特征。在種雞上還可降低產(chǎn)蛋率、受精率、孵化率。同時(shí)患病雞骨髓、胸腺、法氏囊發(fā)育受阻導(dǎo)致機(jī)體發(fā)生免疫抑制,不能產(chǎn)生足夠的抗體應(yīng)對(duì)疾病,導(dǎo)致疫苗免疫失效,易于患病,給養(yǎng)禽業(yè)帶來(lái)巨大的經(jīng)濟(jì)損失。近年來(lái),該病在我國(guó)養(yǎng)雞業(yè)中呈現(xiàn)流行趨勢(shì),優(yōu)質(zhì)地方土雞、三黃雞、商品蛋雞中都有發(fā)病情況報(bào)道。目前臨床上尚無(wú)有效的藥物和疫苗能夠用于此病的治療,鑒于上述原因只有通過(guò)淘汰ALV-J陽(yáng)性雞,建立無(wú)ALV-J病原的凈化雞群,才是解決當(dāng)前問(wèn)題的唯一手段。因此,建立雙抗夾心ELISA檢測(cè)出血清中的病毒,及時(shí)淘汰帶毒雞對(duì)防制該病流行、開(kāi)展種群凈化具有重要意義。根據(jù)大多數(shù)病毒和細(xì)菌都是通過(guò)消化道和呼吸道的黏膜侵入動(dòng)物機(jī)體的特性,利用重組活載體疫苗,采用口服免疫的方式把表達(dá)的抗原呈遞到黏膜組織,以此來(lái)誘導(dǎo)全身的免疫應(yīng)答來(lái)抵抗外界的細(xì)菌、病毒的入侵。乳酸桿菌作為一種自然界分布極為普遍,并廣泛分布于動(dòng)物機(jī)體內(nèi)的具有益生保健作用的菌類,近年來(lái)乳酸菌口服疫苗已成為研制的熱點(diǎn)。重組的乳酸菌通過(guò)天然的形式口服介導(dǎo)黏膜免疫,方法簡(jiǎn)便、安全,不僅在局部黏膜產(chǎn)生免疫應(yīng)答,還可介導(dǎo)全身性的免疫應(yīng)答。當(dāng)把重組的含外源基因表達(dá)載體的乳酸桿菌作為活載體菌株時(shí),能將乳酸桿菌的生物學(xué)特性和外源性抗原基因的免疫原性相結(jié)合,同時(shí)具有表達(dá)的外源蛋白不易受內(nèi)毒素的影響、無(wú)包涵體并易于表達(dá)到細(xì)胞外的特點(diǎn)。重組的菌株易于保存擴(kuò)繁,也符合新型基因工程口服疫苗的特點(diǎn)。本研究使用的方法是通過(guò)重組GP85囊膜糖蛋白的干酪乳桿菌口服免疫小白鼠,誘導(dǎo)小白鼠產(chǎn)生特異性抗體,免疫接種后每周檢測(cè)抗體水平。6W后經(jīng)瓊脂雙擴(kuò)散法、Western-blot、直接ELISA檢測(cè)合格的鼠抗ALV-J多抗,經(jīng)提存后對(duì)此抗體進(jìn)行酶標(biāo),作為酶標(biāo)抗體。并利用本實(shí)驗(yàn)室保存的鼠單抗作為捕獲抗體包被ELISA板,鼠多抗作為酶標(biāo)二抗,建立雙抗夾心ELISA方法。并對(duì)雙抗夾心ELISA方法工作條件進(jìn)行優(yōu)化,來(lái)檢測(cè)經(jīng)DF1傳代培養(yǎng)后禽血清中ALV-J。結(jié)果表明,封閉液濃度為5%的脫脂奶粉;單抗包被量為1μg/mL;酶標(biāo)二抗的稀釋度為1:1600;二抗作用時(shí)間為1h;底物顯色時(shí)間為15min。經(jīng)特異性試驗(yàn)、重復(fù)性試驗(yàn)、靈敏性實(shí)驗(yàn)和臨界值確定結(jié)果顯示,該方法具有良好的特性,與新城疫病毒、馬立克病毒、傳染性法氏囊病毒、傳染性支氣管炎病毒均無(wú)交叉反應(yīng),并通過(guò)對(duì)臨床大量樣品的檢測(cè),得到較為科學(xué)的陽(yáng)性率,可以為臨床檢測(cè)ALV-J提供依據(jù)。
[Abstract]:Avian Leukosis virus (Avian Leukosis virus subgroup ALV-J) was isolated from broilers in 1988 and has been widely spread all over the world. It is characterized by vertical and horizontal transmission, inducing tumor, causing slow growth, wasting and death. The laying rate, fertilization rate and hatching rate can also be reduced on the broiler. At the same time, the development of bone marrow, thymus and bursa of diseased chicken lead to immunosuppression, which can not produce enough antibody to deal with the disease, which lead to vaccine failure, easy to fall ill, and bring huge economic loss to poultry industry. In recent years, the disease is popular in the chicken industry of our country. There are reports of the disease in the high quality local chicken, Sanhuang chicken and commercial laying hens. At present, there are no effective drugs and vaccines for the treatment of this disease. Only by eliminating ALV-J positive chickens and establishing purified chickens without ALV-J pathogen is the only way to solve the current problem. Therefore, it is of great significance to establish a double antibody sandwich Elisa to detect the virus in serum and to eliminate the virus in time to control the epidemic of the disease and to carry out population purification. Based on the fact that most viruses and bacteria invade the animal body through the mucous membrane of the digestive tract and the respiratory tract, the expressed antigen is presented to the mucosal tissue by oral immunization using the recombinant live vector vaccine. In order to induce a systemic immune response to resist the invasion of external bacteria, viruses. Lactobacillus is a kind of probiotic bacteria which is widely distributed in nature and widely distributed in animal organism. In recent years oral lactic acid bacteria vaccine has become a hot spot. Recombinant lactobacillus mediated mucosal immunity by oral administration in natural form. The method is simple and safe. It not only produces immune response in local mucosa, but also mediates systemic immune response. When the recombinant Lactobacillus containing exogenous gene expression vector was used as a living vector, the biological characteristics of Lactobacillus could be combined with the immunogenicity of exogenous antigen gene. At the same time, the extraneous proteins were easy to be affected by endotoxin, without inclusion bodies and easy to express out of cells. The recombinant strain is easy to preserve and propagate, and conforms to the characteristics of new genetic engineering oral vaccine. The method used in this study was to induce specific antibody production in mice by oral immunization with Lactobacillus casei, a recombinant GP85 envelope glycoprotein. The antibody level was detected weekly after immunization. The antibody level was detected by Western-blot method with Agar double diffusion method. The qualified mouse anti-ALV-J polyantibody was detected by direct Elisa. The antibody was labeled as enzyme labeled antibody after storage. The double antibody sandwich Elisa method was established by using the mouse McAb preserved in our laboratory as the capture antibody coating Elisa plate and the mouse polyantibody as the enzyme labeled second antibody. The working conditions of double antibody sandwich Elisa were optimized to detect ALV-J in avian serum after subculture of DF1. The results showed that the concentration of the sealant was 5%, the coating amount of the McAb was 1 渭 g / mL, the dilution of the second antibody was 1: 1600, the time of the second antibody was 1 h and the color reaction time of the substrate was 15 min. The results of specificity test, repeatability test, sensitivity test and critical value determination show that this method has good characteristics with Newcastle disease virus, Marek virus, infectious bursal virus, Infectious bronchitis virus has no cross reaction, and a scientific positive rate can be obtained by detecting a large number of clinical samples, which can provide the basis for clinical detection of ALV-J.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S855.3

【引證文獻(xiàn)】

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1 潘偉;高玉龍;秦立廷;祁小樂(lè);高宏雷;孫芬芬;王永強(qiáng);鄧曉蕓;王笑梅;;蛋雞J亞群禽白血病病毒HLJ09MDJ-1株的分離鑒定[A];中國(guó)畜牧獸醫(yī)學(xué)會(huì)獸醫(yī)公共衛(wèi)生學(xué)分會(huì)第二次學(xué)術(shù)研討會(huì)論文集[C];2010年

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本文編號(hào)：2129599