弓形虫模拟肽的筛选及应用研究

发布时间：2018-05-30 11:09

本文选题：弓形虫 + 噬菌体肽库技术　；参考：《华东师范大学》2005年硕士论文

【摘要】：弓形虫是一种全球性分布的寄生原虫,可感染鸟类、哺乳动物和人。人感染弓形虫之后一般表现为隐性,但孕妇感染弓形虫常会流产或者胎儿发育不全。此外感染了弓形虫的人在免疫力低下时易诱发多种疾病。弓形虫病是导致艾滋病患者死亡的元凶之一。弓形虫还是畜牧业的重大危害之一。目前弓形虫的诊断和防治仍有待完善,新型弓形虫疫苗的研制是目前研究的重点。本课题采用噬菌体随机肽库筛选弓形虫抗原的模拟表位。我们用纯化的兔感染弓形虫血清作“诱饵”,从12肽和环7肽噬菌体随机肽库中分别筛选与该多抗有特异性结合的噬菌体,经过三轮正向筛选和正常血清反向吸收,将噬菌体单克隆化。ELISA结果显示,三轮筛选噬菌体得到了较有效的富集,并且亲和力逐轮提高。竞争ELISA显示第三轮筛选的噬菌体能抑制天然弓形虫抗原与兔感染弓形虫血清反应。噬菌体单克隆分别和多个阳性血清、正常兔血清反应,获得阳性克隆35个(25个12肽,10个环7肽)。和其他寄生虫血清无交叉反应。阳性单克隆DNA测序结果显示有25个克隆(19个12肽,6个环7肽)具有“WH:/WHH”或“W×H”的基序,13个克隆(12肽)具有SP/S×P的基序。通过http://www.ncbi.nlm.nih.gov与天然弓形虫抗原的基因序列比对,发现这些克隆与弓形虫重要的保护性抗原,如表膜抗原、致密颗粒抗原、棒状体蛋白、外分泌抗原等具有同源序列。根据ELISA和序列比对结果选择10个(6个12肽,4个环7肽)克隆进行Western Blottin、dot-ELISA检测和免疫小鼠,结果显示阳性克隆能特异地和兔感染弓形虫血清反应,在约66kD处有一特异的条带,能够诱导小鼠产生和天然弓形虫抗原反应的抗体,诱导小鼠产生保护性免疫应答,与对照组相比明显延长了攻击弓形虫后的小鼠的存活时间。通过本课题研究,我们筛选到了能与弓形虫抗体特异性结合的噬菌体克隆,而且这些克隆无论在ELISA、Western Blotting等方法的鉴定中还是在动物试验上,都显示了它们能够模拟天然弓形虫抗原,而序列分析又为这个结论提供了基因水平的证据。我们推断这些克隆有可能发展成新的具有良好特异性的弓形虫检测原,也为弓形虫新型疫苗的研究打下了基础,提示噬菌体模拟肽可用于新型疫苗的研制。
[Abstract]:Toxoplasma gondii is a globally distributed parasite that infects birds, mammals and humans. Human infection with Toxoplasma gondii usually shows recessive, but pregnant women infected with Toxoplasma gondii often abortion or fetal dysplasia. In addition, people infected with Toxoplasma gondii are susceptible to various diseases when their immunity is low. Toxoplasma gondii is one of the main causes of AIDS deaths. Toxoplasma gondii is also one of the major hazards of animal husbandry. At present, the diagnosis and control of Toxoplasma gondii still need to be improved, and the development of new Toxoplasma vaccine is the focus of current research. A phage random peptide library was used to screen the mimic epitopes of Toxoplasma gondii antigen. Purified rabbit sera infected with Toxoplasma gondii were used as "bait". Phage peptides with 12 peptide and 7 peptide were screened from phage random peptide library, respectively. The phages with specific binding to the polyclonal antibody were screened for three rounds of positive screening and reverse absorption of normal serum. The results of phage monoclonal. Elisa showed that the phage was enriched by three rounds of screening and the affinity of phage was improved round by round. Competitive ELISA showed that the phage screened in the third round could inhibit the reaction of Toxoplasma gondii antigens to the sera of rabbits infected with Toxoplasma gondii. A total of 35 positive clones (25 12 peptides and 10 cyclic 7 peptides) were obtained from phage monoclonal reaction with several positive sera and normal rabbit serum. There was no cross reaction with other parasitic serum. The results of DNA sequencing showed that 25 clones (19 12 peptides, 6 cyclic 7 peptides) had motifs of "WH:/WHH" or "W 脳 H", and 13 clones had motifs of SP/S 脳 P. By comparing the gene sequences of http://www.ncbi.nlm.nih.gov with natural Toxoplasma gondii antigens, it was found that these clones had homologous sequences with important protective antigens, such as surface membrane antigen, dense granular antigen, coryliform protein, exocrine antigen and so on. According to the results of ELISA and sequence alignment, 10 clones (6 12 peptides and 4 cyclic 7 peptides) were selected for Western Blottinindot-ELISA assay and immunized mice. The results showed that the positive clones could react specifically with the sera of rabbits infected with Toxoplasma gondii, and there was a specific band at about 66kD. Compared with the control group, Toxoplasma gondii antibody and protective immune response were induced, and the survival time of the mice after Toxoplasma gondii attack was significantly prolonged. In this study, we have screened phage clones that can specifically bind to Toxoplasma gondii antibodies, and these clones have been identified both in the identification of Elisa Western Blotting and in animal experiments. Both show that they mimic natural Toxoplasma antigens, and sequence analysis provides evidence of the genetic level. We infer that these clones may develop into new Toxoplasma gondii detectives with good specificity and lay the foundation for the study of new Toxoplasma gondii vaccine, suggesting that phage mimic peptides can be used in the development of new vaccines.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392;S852.72

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