本文關(guān)鍵詞： 弓形蟲 P30 克隆 表達(dá) 單克隆抗體 雜交瘤細(xì)胞　出處：《廣西大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：剛地弓形蟲(T.gondii)是一種全球性的人畜共患寄生蟲,該種寄生蟲病的流行與傳播不僅嚴(yán)重地影響著畜牧業(yè)的可持續(xù)發(fā)展,同時也對人類的生命與健康造成嚴(yán)重的威脅及危害。本實(shí)驗(yàn)?zāi)康脑谟诶梅肿由飳W(xué)中的蛋白表達(dá)技術(shù)及單克隆抗體的制備等分子及免疫學(xué)相關(guān)技術(shù),選用弓形蟲速殖子抗原及膜表面主要致病性抗原P30作為免疫原,制備出弓形蟲單克隆抗體為接下來建立弓形蟲病快速、簡便診斷方法和進(jìn)一步的研究打下基礎(chǔ)。 通過對弓形蟲P30基因的PCR擴(kuò)增、克隆、測序,成功的構(gòu)建了原核表達(dá)重組質(zhì)粒pET32a-P30,并成功的誘導(dǎo)表達(dá)了弓形蟲P30重組蛋白抗原,經(jīng)純化制備出了可溶性弓形蟲P30重組蛋白抗原。同時采用小鼠腹腔收集弓形蟲速殖子方法,用胰蛋白酶法純化制備了弓形蟲速殖子抗原,為后續(xù)試驗(yàn)提供了免疫抗原。 用弓形蟲P30重組蛋白抗原和弓形蟲速殖子抗原分別免疫小鼠、經(jīng)細(xì)胞融合,利用建立的間接ELISA方法對融合胞融克隆進(jìn)行檢測篩選,成功的用表達(dá)的P30蛋白制備了2株單克隆抗體雜交瘤細(xì)胞分別命名為5D5、2A1；用速殖子抗原制備了2株單克隆抗體雜交瘤細(xì)胞,分別命名為5B6、2D2。 對這4株雜交瘤細(xì)胞分泌的單克隆抗體的生物學(xué)特性進(jìn)行了鑒定：經(jīng)間接ELISA測定5B6、2D2、5D5、2A1四株雜交瘤細(xì)胞單克隆抗體上清的效價分別為：1：800、1：1600、1：3200、1：6400；腹水效價分別為：1：104、1：104、1：105、1：105；經(jīng)單克隆抗體亞型鑒定,4株單克隆抗體5B6、2D2、5D5、2A1亞型分別為：Ig G1,Ig M,Ig G1和Ig M；染色體計數(shù)結(jié)果顯示：4株雜交瘤細(xì)胞染色體計數(shù)為90-110條,均大于親本細(xì)胞；經(jīng)間接ELISA檢測(?)SPSS統(tǒng)計軟件進(jìn)行方差分析,四株雜交瘤細(xì)胞第一代、第十代細(xì)胞培養(yǎng)上清OD450值差異不顯著,均能穩(wěn)定分泌單克隆抗體。 廣西地區(qū)是人畜弓形蟲病流行的地區(qū),本實(shí)驗(yàn)成功的制備了抗弓形蟲速殖子抗原和弓形蟲P30重組蛋白抗原的單克隆抗體,并對其生物學(xué)特性進(jìn)行了鑒定,為進(jìn)一步研究弓形蟲病的快速靈敏的診斷檢測方法以及弓形蟲病的免疫防治奠定了基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R392

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