本文選題：水貂　切入點(diǎn)：細(xì)胞因子　出處：《吉林農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：水貂(Neovison vison)是一種珍貴的小型毛皮動(dòng)物,屬于哺乳綱食肉目,鼬科鼬屬。近年來伴隨著毛皮動(dòng)物養(yǎng)殖業(yè)的迅速發(fā)展,水貂的健康與否嚴(yán)重影響著社會(huì)經(jīng)濟(jì)效益。我國水貂感染細(xì)小病毒、犬瘟熱和阿留申病毒病的趨勢(shì)逐年上升,疫苗免疫效果不盡人意。究其原因,除了病毒變異外,動(dòng)物機(jī)體的免疫狀態(tài)也會(huì)引起免疫失敗。確切了解動(dòng)物機(jī)體的免疫狀態(tài)能夠?yàn)閮?yōu)化免疫程序和免疫時(shí)間提供了重要依據(jù),所以建立疫苗免疫效果評(píng)價(jià)技術(shù)體系對(duì)于特種動(dòng)物疫病防控具有深刻的現(xiàn)實(shí)意義。本試驗(yàn)主要研究?jī)?nèi)容如下:1.水貂IL-4、IFN-γ和TNF-α基因的克隆以及序列分析為了獲得水貂IL-4、IFN-γ和TNF-α全基因序列,對(duì)無菌分離得到水貂外周血淋巴細(xì)胞(PBMC)經(jīng)過植物血凝素(PHA)誘導(dǎo)后,將細(xì)胞體外培養(yǎng)24 h,離心收集,提取淋巴細(xì)胞總RNA。根據(jù)GenBank中登錄的不同種屬動(dòng)物的IL-4、IFN-γ和TNF-α全基因序列,設(shè)計(jì)并合成特異性引物,RT-PCR擴(kuò)增獲得水貂IL-4、IFN-γ和TNF-α全長(zhǎng)基因序列,全長(zhǎng)依次為399 bp、501 bp、702 bp,并進(jìn)行序列分析與比對(duì)。本試驗(yàn)為水貂IL-4、IFN-γ和TNF-α基因的進(jìn)一步研究奠定了基礎(chǔ)。2.水貂IL-4、IFN-γ和TNF-αmRNA熒光定量RT-PCR檢測(cè)方法的建立為檢測(cè)CDV強(qiáng)毒株和CDV弱毒株感染水貂PBMC后對(duì)幾種相關(guān)細(xì)胞因子mRNA轉(zhuǎn)錄的影響,本試驗(yàn)根據(jù)已經(jīng)擴(kuò)增得到的IL-4、IFN-γ和TNF-α全基因序列和Genebank上登錄的管家基因3-磷酸甘油脫氫酶(GAPDH)序列,制備質(zhì)粒標(biāo)準(zhǔn)品。建立檢測(cè)IL-4、IFN-γ和TNF-αmRNA的熒光定量RT-PCR檢測(cè)方法,構(gòu)建標(biāo)準(zhǔn)曲線。以PHA、CDV強(qiáng)毒株(CDV-Hebei)和CDV弱毒株(CDV3)感染后的外周血淋巴細(xì)胞體外培養(yǎng),在不同時(shí)間點(diǎn)收集細(xì)胞,作為臨床樣品進(jìn)行檢測(cè)和分析。試驗(yàn)結(jié)果表明:PHA可誘導(dǎo)水貂外周血淋巴細(xì)胞高效表達(dá)IL-4和IFN-γ;CDV病毒可抑制淋巴細(xì)胞分泌IL-4、促進(jìn)IFN-γ的分泌。本研究同時(shí)為水貂相關(guān)細(xì)胞因子mRNA的定量分析提供了有效的方法。3.水貂IFN-γ單克隆抗體的制備以及間接夾心ELISA檢測(cè)方法的建立將水貂IFN-γ基因序列的成熟蛋白基因構(gòu)建到原核表達(dá)載體pColdⅡ中,經(jīng)鑒定pCold-MiIFN-γ為可溶性表達(dá),用MiIFN-γ成熟蛋白免疫BALB/c小鼠,經(jīng)過4次免疫,取小鼠的脾臟細(xì)胞和SP2/0骨髓瘤細(xì)胞進(jìn)行融合,以該重組可溶蛋白pCold-MiIFN-γ作為檢測(cè)抗原進(jìn)行間接ELISA檢測(cè),共篩選出可穩(wěn)定分泌抗體的單細(xì)胞克隆株24株。經(jīng)敏感性檢測(cè),篩選出5株親和力較好的雜交瘤細(xì)胞株,分別命名為31A、31B、31G、E44和G46株,利用Western-blot檢測(cè)均能夠形成特異性反應(yīng)。構(gòu)建pcDNA3.1-MiIFN-γ轉(zhuǎn)染于Vero細(xì)胞,應(yīng)用篩選出的5株單克隆抗體作為一抗,進(jìn)行間接免疫熒光,結(jié)果證實(shí)其中2株為細(xì)胞分泌的水貂IFN-γ的特異性單克隆抗體。經(jīng)單克隆抗體亞型鑒定,這兩株分別為IgG2a和IgG2b,輕鏈均為κ鏈。于BALB/c小鼠腹腔注射雜交瘤細(xì)胞,制備單克隆抗體,親和層析法純化該抗體。將犬瘟熱病毒強(qiáng)毒和弱毒感染的淋巴細(xì)胞上清包被96孔板,應(yīng)用純化的抗體作為一抗,HRP標(biāo)記兔抗鼠IgG為二抗,選取0-96 h中的不同時(shí)間點(diǎn)進(jìn)行夾心ELISA檢測(cè),結(jié)果顯示,在感染初期,強(qiáng)毒CDV-Hebei和疫苗毒CDV3感染后IFN-γ表達(dá)水平均在48 h上升到最高峰,72 h表達(dá)明顯被抑制,疫苗株感染后IFN-γ變化趨勢(shì)較強(qiáng)毒小。
[Abstract]:Mink (Neovison vison) is a small precious fur animal, belongs to mammalia Carnivora, Mustelidae Mustela. In recent years, with the rapid development of fur animal breeding industry, mink health and seriously affected the social and economic benefits. China's mink parvovirus infection, virus disease of canine distemper and Aleutian trend year by year rise, not vaccine immune effect. The reason, in addition to variation of the virus, the immune status of the animal's body will cause the immune failure. The exact understanding of animal immune state can provide an important basis for the optimization of the immunization schedule and time, so the establishment of evaluation system of immune effect has profound practical significance for the construction of animal epidemic disease the prevention and control of the test. The main contents are as follows: 1. mink IL-4, IFN- gamma and alpha TNF- gene cloning and sequence analysis of IL-4 IFN- in order to obtain the mink, gamma and TNF- The whole sequence of alpha, the sterile isolated from mink peripheral blood lymphocytes (PBMC) by phytohemagglutinin (PHA) after induction, the cells cultured in vitro for 24 h, centrifuged, the total cell RNA. was extracted according to different animal species IL-4 GenBank login, the whole sequence of IFN- and TNF-, the synthesis of specific the primers were designed and amplified, RT-PCR mink IL-4, full-length sequence of IFN- gamma and TNF- alpha length were 399 BP, 501 BP, 702 BP, and the sequence was analyzed and compared. The test for mink IL-4, IFN- gamma and TNF- alpha gene further research.2. mink IL-4 assay IFN- y and TNF- a mRNA fluorescent quantitative RT-PCR for detection of virulent strain CDV and CDV weak effects on several related cytokines of mRNA transcription and virus infection of mink PBMC after the test according to the amplified IL-4, IFN- gamma and TNF- alpha gene sequence and Genebank In the housekeeping gene 3- phosphate dehydrogenase (GAPDH) sequence, the preparation of standard plasmid. The establishment of IL-4 detection, fluorescence quantitative RT-PCR method for detection of IFN- y and TNF- a mRNA, to construct a standard curve. In PHA, the virulent CDV (CDV-Hebei) and CDV strain (CDV3) of peripheral blood lymphocyte culture in vitro infection after the cells were collected at different time points, as clinical samples were detected and analyzed. The experimental results show that PHA can induce mink peripheral blood lymphocytes and high expression of IL-4 and IFN- gamma; CDV virus can inhibit the secretion of IL-4, promote IFN- secretion. Antibodies provide effective method for.3. mink IFN- gamma monoclonal preparation and indirect sandwich ELISA method for detection of mature protein gene of mink IFN- gene sequence was constructed into the prokaryotic expression vector of pCold in this study for quantitative analysis of mink related cytokines mRNA, by means of PCold-MiIFN- gamma for soluble expression of MiIFN- gamma mature protein immunized BALB/c mice after 4 times of immunization, the spleen cells of mice were fused with SP2/0 myeloma cells, the recombinant soluble pCold-MiIFN- protein gamma as detection antigen were detected with ELISA, 24 strains were screened in single cells stably secreting antibody clones. The detection sensitivity, good affinity screening of 5 strains of hybridoma cell lines, named 31A, 31B, 31G, E44 and G46 were detected by Western-blot are able to form a specific reaction. Construction of pcDNA3.1-MiIFN- gamma transfection in Vero cells, 5 strains of monoclonal antibody screened as primary antibody by indirect immune fluorescence, specific monoclonal antibody results confirmed the mink IFN- gamma 2 strains of cells. The monoclonal antibody subtype identification, these two strains were IgG2a and IgG2b, were light chain kappa chain in BALB/c. Hybridoma cells in mice by intraperitoneal injection, preparation of monoclonal antibody, the antibody affinity chromatography. The canine distemper virus virulent and attenuated infected lymphocyte supernatant coated 96 well plates using purified antibody as the first antibody, HRP labeled Rabbit anti mouse anti IgG was two, 0-96 selected time points of H sandwich ELISA detection results showed that in the early stage of infection, IFN- expression level was in the 48 h increased to the highest peak of the virulent CDV-Hebei and vaccine CDV3 72 after infection, the expression of h was significantly inhibited, the vaccine strain IFN- after infection trend of strong toxicity. Gamma

【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S858.92

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