本文關(guān)鍵詞： 弓形蟲 P3O基因 ROP2基因 復(fù)合基因 核酸疫苗　出處：《山東大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：隨著分子生物學(xué)技術(shù)在寄生蟲學(xué)研究領(lǐng)域的應(yīng)用和發(fā)展,弓形蟲疫苗從最早的全蟲疫苗到新近迅速發(fā)展起來的核酸疫苗均取得了很大進(jìn)展。但是,弓形蟲滅活疫苗免疫保護(hù)力低;減毒活疫苗易恢復(fù)毒力,不能用于人體;亞單位疫苗生產(chǎn)過程復(fù)雜,且抗原蛋白不能形成正確的天然構(gòu)象因此免疫活性不足,使得弓形蟲核酸疫苗成為一個(gè)新的研究方向。同時(shí),越來越多的證據(jù)表明,弓形蟲階段特異性抗原分子只能激發(fā)機(jī)體的階段特異性保護(hù)免疫。因此,使用來自不同階段的多抗原基因的復(fù)合基因疫苗可能刺激機(jī)體產(chǎn)生較全面的免疫保護(hù)性。 P30(SAG1)作為弓形蟲速殖子期特異的主要表面抗原之一,具有高度的免疫原性和免疫保護(hù)性,是弓形蟲感染免疫診斷和疫苗開發(fā)的的主要候選抗原。ROP2(Rhoptry2)蛋白是弓形蟲棒狀體分泌的一種蛋白,主要協(xié)助蟲體入侵宿主細(xì)胞,在弓形蟲生活史的速殖子期、緩殖子期和子孢子期中均有表達(dá),具有高度的保守性和免疫原性,能充分刺激機(jī)體的免疫系統(tǒng),引發(fā)保護(hù)性免疫反應(yīng),其作為疫苗候選分子具有巨大的潛力。 本研究應(yīng)用基因工程方法構(gòu)建弓形蟲單基因真核表達(dá)質(zhì)粒pcDNA3.1-P30;并將之與pcDNA3.1-P30-ROP2復(fù)合基因真核表達(dá)質(zhì)粒分別瞬時(shí)轉(zhuǎn)染人宮頸癌細(xì)胞系(Hela)細(xì)胞,轉(zhuǎn)染48小時(shí)后用Trizol提取細(xì)胞總RNA,以O(shè)ligo(dT)18為引物逆轉(zhuǎn)錄生成cDNA第一條鏈;對管家基因β—actin進(jìn)行PCR驗(yàn)證逆轉(zhuǎn)錄;然后分別對P30、P30-ROP2基因片段進(jìn)行PCR,證實(shí)真核表達(dá)載體pcDNA3.1能夠攜帶弓形蟲抗原基因P30及P30-ROP2在哺乳動(dòng)物細(xì)胞中轉(zhuǎn)錄表達(dá)。此后,用堿裂解法大量制備質(zhì)粒pcDNA3.1-P30及pcDNA3.1-P30-ROP2,以肌注方式接種BalB/c小鼠(100μg/只),分別于2周、4周后加強(qiáng)免疫一次,并設(shè)立PBS組、pcDNA3.1空載體組為對照,觀察分析核酸疫苗所誘導(dǎo)的小鼠的免疫效應(yīng)以及抵
[Abstract]:With the application and development of molecular biology technology in parasitology research, Toxoplasma gondii vaccine has made great progress from the earliest whole worm vaccine to the newly developed nucleic acid vaccine. Toxoplasma gondii inactivated vaccine has low immune protection; Attenuated live vaccine is easy to restore virulence, can not be used in human body; The production process of subunit vaccine is complicated, and the antigen protein can not form the correct natural conformation. Therefore, the immune activity is insufficient, which makes Toxoplasma nucleic acid vaccine a new research direction. At the same time, more and more evidence shows that. Toxoplasma gondii stage specific antigen molecules can only stimulate the body stage specific protection of immunity. The use of multiple gene vaccines from different stages of polyantigen genes may stimulate a more comprehensive immune protection. As one of the major surface antigens of Toxoplasma gondii Tachyzoites, P30 Sag1 has high immunogenicity and immunogenicity. Toxoplasma gondii (Toxoplasma gondii) is the main candidate antigen for immunological diagnosis and vaccine development of Toxoplasma gondii. It is a protein secreted by Toxoplasma gondii Rhoptry2, which mainly assists in invading host cells of Toxoplasma gondii. Toxoplasma gondii has been expressed in the tachyzoites, bradyzoites and sporozoites. It is highly conservative and immunogenicity, which can stimulate the immune system and induce protective immune response. It has great potential as a vaccine candidate. In this study, the eukaryotic expression plasmid pcDNA3.1-P30 of Toxoplasma gondii was constructed by genetic engineering. Human cervical cancer cell line Hela was transfected with the eukaryotic expression plasmid of pcDNA3.1-P30-ROP2 gene. After 48 hours of transfection, the total RNAs were extracted by Trizol, and the first strand of cDNA was generated by reverse transcription with Oligo(dT)18 as the primer. The housekeeper gene 尾 -actin was reverse transcripted by PCR. The P30 P30-ROP2 gene fragment was then analyzed by PCR. It was confirmed that the eukaryotic expression vector pcDNA3.1 could carry Toxoplasma gondii antigen gene P30 and P30-ROP2 into mammalian cells for transcriptional expression. The plasmids pcDNA3.1-P30 and pcDNA3.1-P30-ROP2were prepared by alkaline lysis method and inoculated intramuscularly into BalB/c mice (100 渭 g / mouse). The immune response of mice induced by nucleic acid vaccine was observed and compared with that of PBS group with pcDNA3.1 empty vector as control group. The immune response of mice induced by nucleic acid vaccine was observed and analyzed.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392

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本文編號：1443993