發(fā)布時間：2018-03-08 11:15

  本文選題：水貂　切入點：細胞因子　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物,屬于哺乳綱食肉目,鼬科鼬屬。近年來伴隨著毛皮動物養(yǎng)殖業(yè)的迅速發(fā)展,水貂的健康與否嚴(yán)重影響著社會經(jīng)濟效益。我國水貂感染細小病毒、犬瘟熱和阿留申病毒病的趨勢逐年上升,疫苗免疫效果不盡人意。究其原因,除了病毒變異外,動物機體的免疫狀態(tài)也會引起免疫失敗。確切了解動物機體的免疫狀態(tài)能夠為優(yōu)化免疫程序和免疫時間提供了重要依據(jù),所以建立疫苗免疫效果評價技術(shù)體系對于特種動物疫病防控具有深刻的現(xiàn)實意義。本試驗主要研究內(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,對無菌分離得到水貂外周血淋巴細胞(PBMC)經(jīng)過植物血凝素(PHA)誘導(dǎo)后,將細胞體外培養(yǎng)24 h,離心收集,提取淋巴細胞總RNA。根據(jù)GenBank中登錄的不同種屬動物的IL-4、IFN-γ和TNF-α全基因序列,設(shè)計并合成特異性引物,RT-PCR擴增獲得水貂IL-4、IFN-γ和TNF-α全長基因序列,全長依次為399 bp、501 bp、702 bp,并進行序列分析與比對。本試驗為水貂IL-4、IFN-γ和TNF-α基因的進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檢測方法的建立為檢測CDV強毒株和CDV弱毒株感染水貂PBMC后對幾種相關(guān)細胞因子mRNA轉(zhuǎn)錄的影響,本試驗根據(jù)已經(jīng)擴增得到的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IL-4、IFN-γ和TNF-αmRNA的熒光定量RT-PCR檢測方法,構(gòu)建標(biāo)準(zhǔn)曲線。以PHA、CDV強毒株(CDV-Hebei)和CDV弱毒株(CDV3)感染后的外周血淋巴細胞體外培養(yǎng),在不同時間點收集細胞,作為臨床樣品進行檢測和分析。試驗結(jié)果表明:PHA可誘導(dǎo)水貂外周血淋巴細胞高效表達IL-4和IFN-γ;CDV病毒可抑制淋巴細胞分泌IL-4、促進IFN-γ的分泌。本研究同時為水貂相關(guān)細胞因子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)建到原核表達載體pColdⅡ中,經(jīng)鑒定pCold-MiIFN-γ為可溶性表達,用MiIFN-γ成熟蛋白免疫BALB/c小鼠,經(jīng)過4次免疫,取小鼠的脾臟細胞和SP2/0骨髓瘤細胞進行融合,以該重組可溶蛋白pCold-MiIFN-γ作為檢測抗原進行間接ELISA檢測,共篩選出可穩(wěn)定分泌抗體的單細胞克隆株24株。經(jīng)敏感性檢測,篩選出5株親和力較好的雜交瘤細胞株,分別命名為31A、31B、31G、E44和G46株,利用Western-blot檢測均能夠形成特異性反應(yīng)。構(gòu)建pcDNA3.1-MiIFN-γ轉(zhuǎn)染于Vero細胞,應(yīng)用篩選出的5株單克隆抗體作為一抗,進行間接免疫熒光,結(jié)果證實其中2株為細胞分泌的水貂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。將犬瘟熱病毒強毒和弱毒感染的淋巴細胞上清包被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中的不同時間點進行夾心ELISA檢測,結(jié)果顯示,在感染初期,強毒CDV-Hebei和疫苗毒CDV3感染后IFN-γ表達水平均在48 h上升到最高峰,72 h表達明顯被抑制,疫苗株感染后IFN-γ變化趨勢較強毒小。
[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)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S858.92

【參考文獻】

相關(guān)期刊論文 前9條

1 王洋;胡博;張海玲;魯榮光;呂爽;劉昊;孫彥剛;馬凡舒;趙建軍;張蕾;薛向紅;史寧;白雪;徐淑娟;范思寧;凌明玉;李欣彤;閆喜軍;;水貂IFN-α、IFN-β及IFN-γmRNA熒光定量RT-PCR檢測方法的建立及應(yīng)用[J];動物醫(yī)學(xué)進展;2015年11期

2 葉麗萍;楊文濤;楊桂連;王春鳳;;細胞因子在抗雞球蟲病中的作用機制及應(yīng)用的研究進展[J];中國畜牧獸醫(yī);2014年12期

3 潘福星;王冬冬;曹志偉;馮培祥;劉宏;齊娟;孫忠晟;王祖榮;尹燕博;;比格犬IFN-α和IFN-β mRNA SYBR GreenⅠ熒光定量RT-PCR檢測方法的建立[J];中國預(yù)防獸醫(yī)學(xué)報;2014年04期

4 李睿;;白細胞介素-4的研究進展[J];華北煤炭醫(yī)學(xué)院學(xué)報;2011年03期

5 韓猛立;蔡曰鵬;黃新;何延華;薄新文;鐘發(fā)剛;;牛Th1/Th2型細胞因子實時熒光定量RT-PCR檢測方法的建立及應(yīng)用[J];中國獸醫(yī)學(xué)報;2011年04期

6 韋天超;田志軍;周艷君;安同慶;肖燕;彭金美;姜一峰;童光志;;豬IFN-γ mRNA Taq Man熒光定量RT-PCR檢測方法的建立[J];中國獸醫(yī)科學(xué);2009年02期

7 張賀楠;賴漢漳;齊巖;孔留五;張小桃;曹偉勝;廖明;;雞IFN-α和IFN-β及IFN-γ基因?qū)崟r熒光定量RT-PCR檢測方法的建立[J];中國獸醫(yī)科學(xué);2009年02期

8 白宇;童鐵鋼;劉光亮;肖一紅;王群;徐樹蘭;張維軍;吳東來;;馬γ-干擾素單克隆抗體的制備及其特性分析[J];農(nóng)業(yè)生物技術(shù)學(xué)報;2008年01期

9 陳國華;景志忠;竇永喜;蒙學(xué)蓮;張巧穎;宋世斌;;豬白細胞介素-4重組蛋白的純化及其生物學(xué)特性分析[J];畜牧獸醫(yī)學(xué)報;2007年10期

，

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