猪带绦虫六钩蚴TSOL18抗原表位及组织定位的研究

发布时间：2018-02-27 10:31

本文关键词： 猪带绦虫六钩蚴 TSOL18 单克隆抗体噬菌体展示技术抗原表位抗原定位　出处：《甘肃农业大学》2010年博士论文　论文类型：学位论文

【摘要】：囊尾蚴病是由猪带绦虫的幼虫——囊尾蚴寄生于人或猪而引起的人畜共患寄生虫病,该病不仅给畜牧业造成一定的经济损失,而且还威胁着人类健康,是经济落后国家和地区重要的公共卫生问题。TSOL18是猪带绦虫六钩蚴阶段特异性表达的抗原,主要在六钩蚴入侵中间宿主的早期阶段分泌,其免疫血清在体外能有效杀死六钩蚴,体外表达的重组TSOL18能完全保护猪不被猪带绦虫虫卵感染。随着免疫学的发展,人们认识到有效的保护是抗原表位的参与。因此,阐明猪带绦虫TSOL18蛋白的抗原表位和组织分布对于该病的新型诊断试剂以及分子疫苗设计方面意义重大。本研究挑取实验室保存的重组菌株Pichia pastoris pPIC9K-TSOL18进行5L发酵罐发酵培养,甲醇诱导72 h后将表达上清液进行SDS-PAGE,用薄层扫描分析,目的蛋白约占上清总蛋白含量的80%以上,目的蛋白表达量达2.00g/L。重组蛋白TSOL18经Sephadex G-100分子筛凝胶层析纯化后,其纯度达90%,满足制备单抗的抗原要求。将纯化的TSOL18作为免疫原,制备了12株可分泌TSOL18单克隆抗体的杂交瘤细胞株,经分型试纸条鉴定,12株单抗分属于IgG1、IgG2a、IgG2b、IgM抗体亚型,轻链均为K型；ELISA叠加试验表明,12株单抗分别识别2个不同抗原表位,2株不同抗原表位单克隆抗体的腹水效价分别达1×102和1×106；杂交瘤细胞株的染色体组型、单抗的稳定性等的分析表明,所获得的12株单抗均能稳定分泌,满足常规实验的要求。采用次氯酸钠溶液、胆汁和胰酶获得激活的六钩蚴,虫卵的脱壳率为95%,六钩蚴的活力为72%,然后用TSOL18免疫的猪血清和TSOL18单抗对激活的六钩蚴分别进行体外杀伤实验,台盼蓝染色判定六钩蚴活力。结果证实在有补体的情况下,多抗和单抗6天时对六钩蚴的杀伤率分别为73%和52%。为了分析单抗的抗原结合位点,本研究利用TSOL18单克隆抗体筛选噬菌体随机12肽库,通过四轮“吸附-洗脱-扩增”的亲和筛选,对噬菌体12肽库进行了富集。随机挑取的59个噬斑提取DNA后测序,根据测序结果推导出的TSOL18单抗识别的表位氨基酸序列分别为ETTKLQRFQAML (L1)和DHTLF (L2),两个序列出现频率分别是83%和15%。ELISA方法检测噬菌体克隆与单抗MAb或BSA的结合反应表明,两个表位均与单抗特异性结合。在验证噬菌体蛋白抑制实验中,加入不同滴度的纯化噬菌体Ll或L2克隆,以阻断TSOL18 MAb与TSOL18抗原的结合,结果显示,随着噬菌体滴度的降低,对TSOL18 MAb与TSOL18抗原结合的抑制率也逐渐降低,呈剂量效应关系。将筛选出的特异性噬菌体克隆大量扩增,纯化蛋白免疫小鼠,检测血清与TSOL18蛋白的反应性。ELISA结果显示,筛选的两个表位均能与TSOL18抗原发生反应。同时用两个表位作抗原检测表位与TSOL18免疫的猪血清的反应性。ELISA结果显示,两个表位均与被检血清反应。用筛选到的表位检测猪囊尾蚴病血清,ELISA敏感性分别为85%(L1)和79%(L2)。利用噬菌体展示技术筛选TSOL18抗原表位为猪囊尾蚴病免疫和诊断技术的进一步研究奠定了基础。采用胶体金标记技术对猪带绦虫六钩蚴宿主保护性抗原TSOL18进行定位,发现金颗粒沉积于小钩、穿刺腺细胞的细胞质和分泌颗粒周围,而对照血清的六钩蚴胶体金染色未见特异性金颗粒沉积。
[Abstract]:Cysticercosis is parasitic larvae by cysticercus of Taenia solium in pigs or human caused zoonotic parasitic disease of the disease, not only cause economic losses to the livestock industry, but also a threat to human health, economy is behind.TSOL18 public health problems in countries and regions is important with pig antigen specificity the expression of the six stage larvae tapeworm, mainly secreted in the early stage of six ancylostome invasion of the intermediate host, the immune serum can effectively kill six larvae in vitro, the recombination TSOL18 can completely protect pigs by eggs of Taenia solium infection. With the development of immunology, people realize that effective protection is the antigen the epitope involved. Therefore, to clarify the Taenia solium epitope of the TSOL18 protein and tissue distribution of new diagnostic reagents for the disease and molecular vaccine design is significant.
This study was preserved in the laboratory of pastoris pPIC9K-TSOL18 recombinant strain Pichia 5L fermentor fermentation and methanol induction after 72 h expression supernatant SDS-PAGE analysis by TLC, the target protein accounted for more than 80% of the total protein content of the supernatant, the protein expression of 2.00g/L. recombinant protein TSOL18 by Sephadex G-100 gel chromatography later, the purity of 90%, meet the monoclonal antibody antigen.
The purified TSOL18 as immunogen, preparation of the 12 strains could secrete TSOL18 monoclonal antibody hybridoma cell line by typing test strip identification, 12 McAbs belong to IgG1, IgG2a, IgG2b, IgM antibody subtype, light chain were K type; ELISA stack test showed that the 12 mAbs were identified. 2 different epitopes, 2 strains of different epitopes of monoclonal antibody titers of ascites were 1 * 102 and 1 * 106; karyotype analysis of hybridoma cell lines, monoclonal antibody stability showed that the 12 mAbs could stably secrete, meet the general requirements of experiments.
Using sodium hypochlorite solution, bile and trypsin activation of six larvae, the egg shell rate was 95%, six ancylostome activity is 72%, then the pig serum immunized with TSOL18 and TSOL18 monoclonal antibody on activation of six larvae were carried out in vitro experiment, trypan blue staining was used to determine six ancylostome results confirmed the vitality. With the absence of complement, polyclonal antibody and monoclonal antibody 6 days killing rates of six larvae were 73% and 52%.
In order to analyze the antigen binding sites, this study used TSOL18 monoclonal antibody phage random peptide library of 12 through four rounds of "adsorption elution amplification" of affinity screening, 12 phage display peptide library was enriched. The 59 plaques were randomly selected from DNA sequenced according to the sequencing results of TSOL18 monoclonal antibody derived recognition the epitope of amino acid sequences were ETTKLQRFQAML (L1) and DHTLF (L2), two sequences are indicated with 83% frequency response and 15%.ELISA method to detect the phage clones with mAb MAb or BSA, two epitopes with single anti specifically. In the verification of phage protein inhibition experiments, purified phage Ll or L2 clone with different titer, results indicated the combined blockade of TSOL18 MAb and TSOL18 antigen, with the decrease of phage titer, inhibition of binding to TSOL18 MAb and TSOL18 antigen were also decreased, There is a dose effect relationship.
The specific phage clones were screened to amplify and immunize mice with the purified protein, and serum TSOL18 reactive protein.ELISA results showed that the screening of the two epitopes can react with TSOL18 antigen. At the same time with the two epitopes for reactive.ELISA results of porcine serum antigen detection and TSOL18 immune epitope show that the two epitopes were detected with serum response. The epitope detection of swine cysticercosis with serum screening, the sensitivity of ELISA was 85% (L1) and 79% (L2). Screening of TSOL18 antigen laid the foundation for a further study of immune and diagnostic techniques for cysticercosis by phage.
Using colloidal gold labeling technique of Taenia solium oncosphere six host protective antigen TSOL18 localization, particle deposition in cash around the puncture hooks, glandular cells of cytoplasm and secretory granules and six ancylostome colloidal gold staining serum specific gold particle deposition.

【学位授予单位】：甘肃农业大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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