本文選題：鼠疫耶爾森氏菌 + F1-V��； 參考：《第三軍醫(yī)大學(xué)》2005年碩士論文

【摘要】：鼠疫桿菌(Yersinia pestis)是腺鼠疫、敗血癥鼠疫和肺鼠疫的自然疫源性病原,曾經(jīng)以“瘟疫”的形式給人類的健康造成了極大的危害,歷史上共造成約2 億人死亡,現(xiàn)在世界各地每年仍有鼠疫病例報道。由于生物恐怖襲擊事件的發(fā)生,已引起世界各國對鼠疫的高度警惕�？植酪u擊常采用氣溶膠感染,可直接引起原發(fā)性肺鼠疫流行,因而更需要研究安全有效的鼠疫疫苗。傳統(tǒng)的鼠疫疫苗包括滅活全菌體死疫苗、減毒活疫苗。雖然滅活全菌體死疫苗在預(yù)防腺型鼠疫有一定效果,但對肺型鼠疫無效;減毒活疫苗又具有潛在的毒性。 近年來,DNA 疫苗由于能夠誘導(dǎo)出較好的體液免疫和細(xì)胞免疫而日益受到重視,被譽為疫苗的第三次革命。tPA 信號肽可以促進(jìn)異種蛋白的分泌表達(dá),增強表達(dá)抗原的免疫原性。本文對鼠疫耶爾森氏菌新型DNA 疫苗進(jìn)行了初步研究,選取鼠疫菌保護性抗原F1、V 蛋白編碼基因和人tPA 信號肽為研究對象,PCR 擴增鼠疫菌F1 和V 編碼基因,分別與克隆載體pGEM-T 連接后測序,將測序正確的片段與真核表達(dá)質(zhì)粒pVAX1連接,分別構(gòu)建重組真核表達(dá)質(zhì)粒pVAX1/F1、pVAX1/V 和pVAX1/F1-V,擴增tPA 信號肽,分別插入到F1、V 和F1-V 目的基因的上游,構(gòu)建分泌型重組真核表達(dá)質(zhì)粒tPA-pVAX1/F1、tPA-pVAX1/V 和tPA-pVAX1/F1-V。重組質(zhì)粒轉(zhuǎn)染COS-7 細(xì)胞,細(xì)胞免疫化學(xué)方法和Western blot 方法鑒定結(jié)果顯示重組質(zhì)�？杀磉_(dá)F1、V 和F1-V 蛋白。在此基礎(chǔ)上,重組質(zhì)粒分別加細(xì)胞因子佐劑GM-CSF 免疫BALB/c 小鼠,每組10只,并設(shè)PBS 組為陰性對照,100μg/只,雙側(cè)股四頭肌肌肉注射,隔周免疫一次,加強免疫3 次,檢測細(xì)胞免疫和體液免疫指標(biāo),ELISA 方法檢測小鼠血清中總IgG 水平,免疫組顯著高于PBS 對照組(P0.01),在初次免疫后tPA-pVAX1/V 和tPA-pVAX1/F1-V組可以誘導(dǎo)出較高的抗體水平,而其它組要至少二次免疫后才能達(dá)到相應(yīng)水平�？贵w亞型分類檢測結(jié)果顯示tPA-pVAX1/V 和tPA-pVAX1/F1-V 組的IgG2a 水平顯著提高,說明分泌型tPA-V 和tPA-F1-V DNA疫苗免疫在機體內(nèi)成功地增強了Th1型細(xì)胞免疫為主的免疫應(yīng)答。ELISPOT 方法檢測了特異性活化的可以分泌IFN-γ的CD8+ T 淋巴細(xì)胞數(shù),免疫組小鼠形成斑點的細(xì)胞數(shù)均顯著多于PBS 對照組(P0.01),以tPA-pVAX1/V 和
[Abstract]:Yersinia pestis (Yersinia pestis) is the natural pathogen of plague, septicemia and pneumonic plague, which has caused great harm to human health in the form of "plague", and has caused a total of 200 million deaths in history. Plague cases are still reported every year around the world. Due to the occurrence of bioterrorist attacks, countries around the world have been highly alert to plague. Aerosol infection is often used in terrorist attacks, which can directly cause the epidemic of primary pneumonic plague, so it is necessary to study a safe and effective plague vaccine. Traditional Yersinia pestis vaccine includes inactivated whole cell dead vaccine and attenuated live vaccine. Although the inactivated whole cell death vaccine has certain effect in preventing glandular plague, it has no effect on pulmonary plague, and attenuated live vaccine has potential toxicity. In recent years, DNA vaccine has been paid more and more attention because of its ability to induce better humoral and cellular immunity. It is known as the third revolution of vaccine. TPA signal peptide can promote the secretion and expression of xenogeneic proteins and enhance the immunogenicity of expressed antigens. A novel Yersinia pestis DNA vaccine was studied in this paper. The gene encoding F1V protein of Yersinia pestis protection antigen and human tPA signal peptide were selected to amplify the F1 and V coding genes of Yersinia pestis. The recombinant eukaryotic expression plasmids pVAX1 / F1pVAX1 / V and pVAX1 / F1-V were constructed, and the tPA signal peptides were amplified and inserted into the upstream of F1V and F1-V target genes, respectively. The secretory recombinant eukaryotic expression plasmids tPA-pVAX1 / F1 + tPA-pVAX1 / V and tPA-pVAX1 / F1-V were constructed. The recombinant plasmid was transfected into COS-7 cells. The results of cell immunocytochemistry and Western blot analysis showed that the recombinant plasmid could express F1V and F1-V proteins. On this basis, 10 BALB/c mice were immunized with recombinant plasmids with cytokine adjuvant GM-CSF, 10 mice in each group were immunized with PBS group as negative control (100 渭 g / mouse), bilateral quadriceps femoris were injected intramuscularly, once every other week, and 3 times were strengthened. The levels of total IgG in serum of mice were detected by Elisa. The levels of total IgG in the immunized group were significantly higher than those in the PBS control group (P 0.01). The higher antibody level could be induced in the tPA-pVAX1/V and tPA-pVAX1/F1-V groups after the first immunization. Other groups had to be immunized at least twice before they reached the level. The results of classification of antibody subtypes showed that the level of IgG2a in tPA-pVAX1/V and tPA-pVAX1/F1-V groups was significantly increased. The results showed that the secretory tPA-V and tPA-F1-V DNA vaccine immunization successfully enhanced the immune response of Th1 type cellular immunity. Elispot method detected the number of specifically activated CD8 T lymphocytes which could secrete IFN- 緯. The number of cells forming spots in immunized mice was significantly higher than that in PBS control group (P 0.01).
【學(xué)位授予單位】：第三軍醫(yī)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：1815860