發(fā)布時(shí)間：2018-06-10 18:31

  本文選題：流感病毒 + DNA疫苗　； 參考：《吉林大學(xué)》2007年博士論文

【摘要】： 本研究以pVAXI載體為骨架,構(gòu)建出含有微小病毒內(nèi)部核糖體進(jìn)入位點(diǎn)(IRES)基因的DNA疫苗雙表達(dá)載體pVI。為驗(yàn)證該載體的表達(dá)效果,又將增強(qiáng)型綠色熒光蛋白(EGFP)基因和新霉素磷酸轉(zhuǎn)移酶(neor)基因作為報(bào)告基因連接到pVI載體的上下游多克隆位點(diǎn)處,構(gòu)建出真核表達(dá)載體pEIN。將其轉(zhuǎn)染COS-7細(xì)胞,得到具有G418抗性的細(xì)胞克隆,同時(shí)鏡下觀(guān)察到綠色熒光,表明該載體可成功表達(dá)兩個(gè)外源基因。 在此基礎(chǔ)上,將甲型流感病毒New Caledonia/20/99(H1N1)株接種于雞胚的尿囊液中,Trizol法提取流感病毒總RNA,以其為模板,RT-PCR法分離出甲型流感病毒M2基因,同時(shí)將GM-CSF基因做為佐劑基因連接到pVI載體上,構(gòu)建出流感病毒DNA疫苗雙表達(dá)載體pMIG,測(cè)序后,將pMIG質(zhì)粒轉(zhuǎn)染COS7細(xì)胞,Western blot結(jié)果表明表達(dá)了甲型流感病毒M2蛋白。 堿裂解法大提質(zhì)粒pMIG,用Sepharose-CL 4B柱層析純化質(zhì)粒,紫外分光光度計(jì)測(cè)A260和A280比值,測(cè)定純度和濃度后,瓊脂糖電泳檢測(cè)DNA的完整性。 將Balb/C小鼠隨機(jī)分成5組,每組10只,分組如下:流感疫苗對(duì)照組、空載體pVAXI對(duì)照組、pVAX/GM-CSF對(duì)照組、pVAX/M2實(shí)驗(yàn)組、pMIG實(shí)驗(yàn)組。 將質(zhì)粒注射于小鼠后腿脛前肌,注射劑量100ug/100ul,流感裂解疫苗對(duì)照組4ug抗原/只的劑量。分0、2、4周免疫接種3次,三次免疫劑量與注射部位均相同,初免后第1周開(kāi)始每周定期對(duì)小鼠眼眶采血,檢測(cè)其免疫效果。 經(jīng)方差分析,流感裂解疫苗對(duì)照組免后第二周即開(kāi)始產(chǎn)生特異性血凝素抗體,到第四周達(dá)到峰值。在免后14周抗體還沒(méi)有消失。而其余各組均未檢測(cè)到抗體。 流式細(xì)胞術(shù)雙標(biāo)法檢測(cè)小鼠T-cell表面標(biāo)記CD4+、CD8+水平及CD4+/CD8+水平,T淋巴細(xì)胞表面標(biāo)記檢測(cè)表明,流感DNA疫苗組與流感裂解疫苗對(duì)照組相比,CD4+/CD8++比例,以及細(xì)胞因子(IL-4、IFNγ)水平上均有顯著的增強(qiáng)作用。 攻毒實(shí)驗(yàn)表明,流感DNA疫苗比流感裂解疫苗保護(hù)效力高出近一倍。
[Abstract]:In this study, pVAXI vector was used as the skeleton to construct a DNA vaccine double expression vector pVII containing the gene of internal ribosomal entry site (IRES) of parvovirus. To verify the expression effect of the vector, the enhanced green fluorescent protein (EGFP) gene and neomycin phosphotransferase (neo) gene were linked to the upstream and downstream polyclonal sites of pVI vector, and the eukaryotic expression vector pEINwas constructed. It was transfected into COS-7 cells to obtain G418 resistant cell clones. Green fluorescence was observed under microscope, which indicated that the vector could express two foreign genes successfully. Influenza A virus strain New Caledonia / 20 / 99H1 N1 was inoculated into chicken embryo allantoic fluid to extract total RNA of influenza virus by Trizol method. The M2 gene of influenza A virus was isolated by RT-PCR, and the GM-CSF gene was linked to PVI vector as adjuvant gene. The double expression vector pMIG of influenza virus DNA vaccine was constructed. After sequencing, the plasmid pMIG was transfected into COS7 cells by Western blot. The results showed that the plasmid pMIG was extracted by alkaline lysis method and purified by Sepharose-CL4B column chromatography. The ratio of A260 to A280 was measured by ultraviolet spectrophotometer, and the purity and concentration were measured. The DNA integrity was detected by agarose electrophoresis. Balb / C mice were randomly divided into 5 groups, 10 in each group, as follows: influenza vaccine control group. Empty vector pVAXI control group pVAX / GM-CSF control group pVAX / M2 experimental group was injected into the anterior tibial muscle of the hind leg of mice at the dose of 100ug-100ul. the dose of 4ug antigen / mouse in the influenza lytic vaccine control group. The immune dose was the same as that of the injection site. The blood samples were collected from the orbit of mice at the first week after the first immunization, and the immunological effects were tested by ANOVA. Specific hemagglutinin antibodies began to be produced in the influenza lytic vaccine control group at the second week after immunization and reached a peak at the fourth week. The antibody did not disappear at 14 weeks after immunization. However, no antibody was detected in other groups. The detection of T-cell surface labeled CD4 / CD8 and CD4 / CD8 level by flow cytometry showed that the ratio of CD 4 / CD 8 in the influenza DNA vaccine group was higher than that in the influenza lytic vaccine group, and the ratio of CD 4 / CD 8 in the influenza DNA vaccine group was higher than that in the control group. The level of cytokine IL-4 and IFN- 緯 was significantly enhanced. The results showed that the protective efficacy of influenza DNA vaccine was nearly twice as high as that of influenza cleavage vaccine.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2007
【分類(lèi)號(hào)】：R392.1

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本文編號(hào)：2004190