弓形虫单克隆抗体的制备及生物学活性的初步鉴定

发布时间：2018-02-24 06:44

本文关键词： 弓形虫 P30 克隆表达单克隆抗体杂交瘤细胞　出处：《广西大学》2012年硕士论文　论文类型：学位论文

【摘要】：刚地弓形虫(T.gondii)是一种全球性的人畜共患寄生虫,该种寄生虫病的流行与传播不仅严重地影响着畜牧业的可持续发展,同时也对人类的生命与健康造成严重的威胁及危害。本实验目的在于利用分子生物学中的蛋白表达技术及单克隆抗体的制备等分子及免疫学相关技术,选用弓形虫速殖子抗原及膜表面主要致病性抗原P30作为免疫原,制备出弓形虫单克隆抗体为接下来建立弓形虫病快速、简便诊断方法和进一步的研究打下基础。通过对弓形虫P30基因的PCR扩增、克隆、测序,成功的构建了原核表达重组质粒pET32a-P30,并成功的诱导表达了弓形虫P30重组蛋白抗原,经纯化制备出了可溶性弓形虫P30重组蛋白抗原。同时采用小鼠腹腔收集弓形虫速殖子方法,用胰蛋白酶法纯化制备了弓形虫速殖子抗原,为后续试验提供了免疫抗原。用弓形虫P30重组蛋白抗原和弓形虫速殖子抗原分别免疫小鼠、经细胞融合,利用建立的间接ELISA方法对融合胞融克隆进行检测筛选,成功的用表达的P30蛋白制备了2株单克隆抗体杂交瘤细胞分别命名为5D5、2A1；用速殖子抗原制备了2株单克隆抗体杂交瘤细胞,分别命名为5B6、2D2。对这4株杂交瘤细胞分泌的单克隆抗体的生物学特性进行了鉴定：经间接ELISA测定5B6、2D2、5D5、2A1四株杂交瘤细胞单克隆抗体上清的效价分别为：1：800、1：1600、1：3200、1：6400；腹水效价分别为：1：104、1：104、1：105、1：105；经单克隆抗体亚型鉴定,4株单克隆抗体5B6、2D2、5D5、2A1亚型分别为：Ig G1,Ig M,Ig G1和Ig M；染色体计数结果显示：4株杂交瘤细胞染色体计数为90-110条,均大于亲本细胞；经间接ELISA检测(?)SPSS统计软件进行方差分析,四株杂交瘤细胞第一代、第十代细胞培养上清OD450值差异不显著,均能稳定分泌单克隆抗体。广西地区是人畜弓形虫病流行的地区,本实验成功的制备了抗弓形虫速殖子抗原和弓形虫P30重组蛋白抗原的单克隆抗体,并对其生物学特性进行了鉴定,为进一步研究弓形虫病的快速灵敏的诊断检测方法以及弓形虫病的免疫防治奠定了基础。
[Abstract]:T. gondii (Toxoplasma gondii) is a global zoonotic parasite, the prevalence and spread of this parasitic disease not only seriously affect the sustainable development of animal husbandry. At the same time, it also poses a serious threat to human life and health. The purpose of this experiment is to utilize molecular and immunological techniques such as protein expression in molecular biology and preparation of monoclonal antibodies. Using Toxoplasma gondii tachyzoite antigen and membrane surface major pathogenic antigen P30 as immunogen, monoclonal antibodies against Toxoplasma gondii were prepared, which laid the foundation for the establishment of a rapid, simple diagnostic method and further research on Toxoplasma gondii. The prokaryotic expression plasmid pET32a-P30 was successfully constructed by PCR amplification, cloning and sequencing of the P30 gene of Toxoplasma gondii, and the recombinant protein antigen of Toxoplasma gondii P30 was successfully induced. The soluble recombinant protein antigen of Toxoplasma gondii P30 was prepared by purification, and the Toxoplasma gondii Tachyzoite antigen was purified by the method of collecting Toxoplasma gondii tachyzoites by abdominal cavity of mice and purified by trypsin method, which provided the immune antigen for further test. Toxoplasma gondii P30 recombinant protein antigen and Toxoplasma gondii Tachyzoites antigen were used to immunize mice respectively. The fusion clones were screened by indirect ELISA method. Two monoclonal antibody hybridoma cell lines were successfully prepared from expressed P30 protein and named 5D5O2A1, respectively, and two monoclonal antibody hybridoma cells were prepared by using tachyzoite antigen as 5B6O2D2. The biological characteristics of monoclonal antibodies secreted by these four hybridoma cells were identified. The titers of monoclonal antibodies supernatants of 5B6D2D2D5H2A1 were: 1: 1 800, 1: 1 600: 1 200: 1: 6400; ascites titers were 1: 1 10 4: 1 10 4: 1 10 5: 1: 105; The subtypes of monoclonal antibody 5B6O2D2D5D5O2A1 were: 1 / Ig G1 / Ig / Ig / G / G, and chromosome counts showed that the chromosome counts of the 4 strains were 90-110, respectively, and the results of chromosome counting showed that the chromosome counts of the four strains of hybridoma were 90-110, and the results of chromosome counting showed that the chromosome number of the four strains of hybridoma was 90-110. All of them were larger than parent cells, and were detected by indirect ELISA. The results of ANOVA showed that the OD450 values of the supernatants of the first generation and 10th passage of the four hybridoma cells were not significantly different, and all of them could secrete monoclonal antibodies stably. Toxoplasma gondii is endemic in Guangxi. Monoclonal antibodies against Toxoplasma Tachyzoites antigen and Toxoplasma P30 recombinant protein antigen were successfully prepared and their biological characteristics were identified. It lays a foundation for further study on the rapid and sensitive diagnosis and detection of Toxoplasma gondii and the immunoprophylaxis of Toxoplasma gondii.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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