貉IgG克隆及Fc活性多肽功能分析

发布时间：2018-11-05 09:33

【摘要】：为获得貉IgG抗体序列,分析其活性多肽功能,从而为貉源疾病治疗研究奠定免疫学基础。本研究通过RTPCR成功克隆貉IgG保守区基因,借助3′RACE以及overlap-PCR技术成功获得其IgG全长。运用在线软件分析其Fc区功能位点,人工合成IgG-Fc区多肽NTVSITCLVK(N10K),PSVYVLPPSQ(P10Q)及阴性对照多肽MSTAVSKCAT(NC)。多肽在不同浓度下与小鼠外周血淋巴细胞共培养18h检测上清液中细胞因子含量(TNF-α,IL-1β,IL-6),发现在质量浓度为10mg/L时,多肽N10K可以刺激淋巴细胞分泌更多的TNF-α(271.95ng/L),IL-1β(27.42ng/L),而多肽P10Q可以促进淋巴细胞产生较多的IL-6(35.51ng/L)。本研究成功克隆貉IgG重链核酸序列,初步确定貉IgG-Fc区多肽P10Q及N10K对外周血淋巴细胞的体外免疫诱导活性,同时为今后貉源疾病预防及治疗奠定基础,为研究DNA疫苗、Fc生物佐剂、多肽疫苗及单克隆抗体制备提供了试验依据。
[Abstract]:In order to obtain the sequence of raccoon dog IgG antibody and analyze the function of its active polypeptide, the immunological foundation was established for the treatment of raccoon dog disease. In this study, the conserved gene of raccoon dog IgG was cloned by RTPCR, and the whole length of IgG was obtained by 3'RACE and overlap-PCR. The functional sites of Fc region were analyzed by on-line software. NTVSITCLVK (N10K), PSVYVLPPSQ (P10Q) and MSTAVSKCAT (NC). (negative control polypeptide) were synthesized by artificial synthesis of IgG-Fc region polypeptide NTVSITCLVK (N10K), PSVYVLPPSQ (P10Q) and negative control polypeptide MSTAVSKCAT (NC). The levels of cytokines (TNF- 伪, IL-1 尾, IL-6) in the supernatant were determined by co-culture of polypeptides with peripheral blood lymphocytes of mice at different concentrations for 18 h. It was found that when the mass concentration was 10mg/L, the levels of cytokines in the supernatant were determined. Polypeptide N10K can stimulate lymphocytes to secrete more TNF- 伪 (271.95ng/L) and IL-1 尾 (27.42ng/L), while polypeptide P10Q can stimulate lymphocytes to produce more IL-6 (35.51ng/L). In this study, IgG heavy chain nucleic acid sequences were cloned successfully, and the in vitro immunogenicity of IgG-Fc polypeptides P10Q and N10K on peripheral blood lymphocytes was preliminarily determined, which laid a foundation for the prevention and treatment of raccoon dog origin diseases in the future and for the study of DNA vaccine. The preparation of Fc biological adjuvant, polypeptide vaccine and monoclonal antibody provides experimental basis.
【作者单位】：泰安市动物卫生监督所;山东农业大学动物科技学院;肥城市畜牧兽医局;烟台市牟平区姜格庄街道畜牧兽医站;山东省滨州畜牧兽医研究院;
【基金】：山东省毛皮动物创新团队滨州综合试验站资助项目(SAT-180-011-15)
【分类号】：S858.92

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