水貂IL-4、IFN-γ和TNF-α mRNA荧光定量RT-PCR和IFN-γ间接夹心ELISA检测方法的建立及应用

发布时间：2018-03-08 11:15

本文选题：水貂　切入点：细胞因子　出处：《吉林农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：水貂(Neovison vison)是一种珍贵的小型毛皮动物,属于哺乳纲食肉目,鼬科鼬属。近年来伴随着毛皮动物养殖业的迅速发展,水貂的健康与否严重影响着社会经济效益。我国水貂感染细小病毒、犬瘟热和阿留申病毒病的趋势逐年上升,疫苗免疫效果不尽人意。究其原因,除了病毒变异外,动物机体的免疫状态也会引起免疫失败。确切了解动物机体的免疫状态能够为优化免疫程序和免疫时间提供了重要依据,所以建立疫苗免疫效果评价技术体系对于特种动物疫病防控具有深刻的现实意义。本试验主要研究内容如下:1.水貂IL-4、IFN-γ和TNF-α基因的克隆以及序列分析为了获得水貂IL-4、IFN-γ和TNF-α全基因序列,对无菌分离得到水貂外周血淋巴细胞(PBMC)经过植物血凝素(PHA)诱导后,将细胞体外培养24 h,离心收集,提取淋巴细胞总RNA。根据GenBank中登录的不同种属动物的IL-4、IFN-γ和TNF-α全基因序列,设计并合成特异性引物,RT-PCR扩增获得水貂IL-4、IFN-γ和TNF-α全长基因序列,全长依次为399 bp、501 bp、702 bp,并进行序列分析与比对。本试验为水貂IL-4、IFN-γ和TNF-α基因的进一步研究奠定了基础。2.水貂IL-4、IFN-γ和TNF-αmRNA荧光定量RT-PCR检测方法的建立为检测CDV强毒株和CDV弱毒株感染水貂PBMC后对几种相关细胞因子mRNA转录的影响,本试验根据已经扩增得到的IL-4、IFN-γ和TNF-α全基因序列和Genebank上登录的管家基因3-磷酸甘油脱氢酶(GAPDH)序列,制备质粒标准品。建立检测IL-4、IFN-γ和TNF-αmRNA的荧光定量RT-PCR检测方法,构建标准曲线。以PHA、CDV强毒株(CDV-Hebei)和CDV弱毒株(CDV3)感染后的外周血淋巴细胞体外培养,在不同时间点收集细胞,作为临床样品进行检测和分析。试验结果表明:PHA可诱导水貂外周血淋巴细胞高效表达IL-4和IFN-γ;CDV病毒可抑制淋巴细胞分泌IL-4、促进IFN-γ的分泌。本研究同时为水貂相关细胞因子mRNA的定量分析提供了有效的方法。3.水貂IFN-γ单克隆抗体的制备以及间接夹心ELISA检测方法的建立将水貂IFN-γ基因序列的成熟蛋白基因构建到原核表达载体pColdⅡ中,经鉴定pCold-MiIFN-γ为可溶性表达,用MiIFN-γ成熟蛋白免疫BALB/c小鼠,经过4次免疫,取小鼠的脾脏细胞和SP2/0骨髓瘤细胞进行融合,以该重组可溶蛋白pCold-MiIFN-γ作为检测抗原进行间接ELISA检测,共筛选出可稳定分泌抗体的单细胞克隆株24株。经敏感性检测,筛选出5株亲和力较好的杂交瘤细胞株,分别命名为31A、31B、31G、E44和G46株,利用Western-blot检测均能够形成特异性反应。构建pcDNA3.1-MiIFN-γ转染于Vero细胞,应用筛选出的5株单克隆抗体作为一抗,进行间接免疫荧光,结果证实其中2株为细胞分泌的水貂IFN-γ的特异性单克隆抗体。经单克隆抗体亚型鉴定,这两株分别为IgG2a和IgG2b,轻链均为κ链。于BALB/c小鼠腹腔注射杂交瘤细胞,制备单克隆抗体,亲和层析法纯化该抗体。将犬瘟热病毒强毒和弱毒感染的淋巴细胞上清包被96孔板,应用纯化的抗体作为一抗,HRP标记兔抗鼠IgG为二抗,选取0-96 h中的不同时间点进行夹心ELISA检测,结果显示,在感染初期,强毒CDV-Hebei和疫苗毒CDV3感染后IFN-γ表达水平均在48 h上升到最高峰,72 h表达明显被抑制,疫苗株感染后IFN-γ变化趋势较强毒小。
[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

【学位授予单位】：吉林农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.92

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