本文選題：SARS　切入點(diǎn)：S1基因　出處：《東北農(nóng)業(yè)大學(xué)》2011年碩士論文　論文類型：學(xué)位論文

【摘要】：本研究首先利用原核系統(tǒng)表達(dá)SARS-CoV S1蛋白和霍亂毒素B亞單位CTB蛋白制備了相應(yīng)的多克隆抗體。然后,成功構(gòu)建了真核表達(dá)質(zhì)粒pVAX-S1、pVAX-CTB;并利用SOE-PCR技術(shù)將S1基因和CTB基因進(jìn)行連接,得到融合基因真核表達(dá)質(zhì)粒pVAX-S1-CTB。利用脂質(zhì)體2000將各種真核表達(dá)質(zhì)粒轉(zhuǎn)染BHK-21細(xì)胞。間接免疫熒光的結(jié)果表明,各種真核表達(dá)質(zhì)粒均能在哺乳動物細(xì)胞中表達(dá),為后續(xù)動物免疫實(shí)驗(yàn)提供了理論基礎(chǔ)。 利用6周齡昆明鼠進(jìn)行DNA疫苗免疫效果研究。在動物免疫實(shí)驗(yàn)中設(shè)計(jì)6個處理組,即PBS組(免疫滅菌PBS)、pVAX載體對照組(免疫真核載體pVAX1)、病毒對照S1組(免疫真核表達(dá)質(zhì)粒pVAX-S1)、佐劑對照CTB組(免疫真核表達(dá)質(zhì)粒pVAX-CTB)、聯(lián)合免疫S1~+CTB組(聯(lián)合免疫pVAX-S1和pVAX-CTB)和融合免疫S1-CTB組(免疫融合基因真核表達(dá)質(zhì)粒pVAX-S1-CTB)。檢測小鼠脾臟和外周血特異性T淋巴細(xì)胞增值效果、CD4~+和CD8~+T淋巴細(xì)胞數(shù)量變化、特異性CTL活性、IL-4和IFN-γ產(chǎn)量以及血清中特異性IgG抗體效價(jià)。 瞬時轉(zhuǎn)染實(shí)驗(yàn)結(jié)果表明,重組真核表達(dá)質(zhì)粒pVAX-S1、pVAX-CTB和pVAX-S1-CTB均可在哺乳動物細(xì)胞中表達(dá)。免疫后42d,兩疫苗組T淋巴細(xì)胞增殖效果明顯高于各對照組,差異極顯著(P0.01)。聯(lián)合組脾臟和外周血CD4~+T細(xì)胞均在免疫后42d達(dá)到最大值;聯(lián)合組脾臟和外周血CD8~+T細(xì)胞均在免疫后42d達(dá)到最大值。特異性細(xì)胞毒性作用結(jié)果表明,免疫后42d聯(lián)合組及融合組細(xì)胞毒性效果明顯,融合組效果尤佳。小鼠血清抗S1蛋白IgG含量檢測結(jié)果表明,兩疫苗組IgG含量均呈一定的增長趨勢;免疫后42d,融合組IgG含量明顯高于其他各組,差異極顯著(p0.01)。外周血IFN-γ和IL-4含量檢測結(jié)果表明,免疫后42d聯(lián)合組及融合組以上兩種細(xì)胞因子含量明顯高于其他各對照組,且融合組刺激產(chǎn)生更多的細(xì)胞因子,二者差異顯著(p0.05)。 綜上所述,通過本試驗(yàn)研究表明,CTB基因在配合SARS-CoV S1抗原基因免疫過程中,不但能夠促進(jìn)機(jī)體的細(xì)胞免疫應(yīng)答能力,而且能夠提高機(jī)體體液免疫應(yīng)答的能力。本實(shí)驗(yàn)結(jié)果為進(jìn)一步研究CTB的生物活性提供了實(shí)驗(yàn)依據(jù),同時為探討開發(fā)研制新型免疫增強(qiáng)型抗SARS病毒核酸疫苗提供了理論基礎(chǔ)。
[Abstract]:In this study, SARS-CoV S1 protein and cholera toxin B subunit CTB protein were first expressed in prokaryotic system. Then the eukaryotic expression plasmid pVAX-S1 pVAX-CTB was successfully constructed, and the S1 gene and CTB gene were ligated by SOE-PCR technique. The eukaryotic expression plasmid pVAX-S1-CTB was obtained. Various eukaryotic expression plasmids were transfected into BHK-21 cells by liposome 2000. The results of indirect immunofluorescence showed that all eukaryotic expression plasmids could be expressed in mammalian cells. It provides a theoretical basis for the follow-up animal immune experiment. The immune effect of DNA vaccine in 6-week-old Kunming mice was studied. Those in PBS group (immunized PBSX pVAX vector control group), virus control S1 group (immune eukaryotic expression plasmid pVAX-S1), adjuvant control CTB group (immunized eukaryotic expression plasmid pVAX-CTBN), combined immunized S1 ~ CTB group (combined immunized pVAX-S1 and pVAX-CTB), In fusion immunized S1-CTB group (eukaryotic expression plasmid pVAX-S1-CTB), the changes of CD4 ~ and CD8 ~ T lymphocytes in spleen and peripheral blood of mice were detected. The specific CTL activity and the production of IL-4 and IFN- 緯, as well as the titer of specific IgG antibody in serum. The results of transient transfection showed that the recombinant eukaryotic expression plasmids pVAX-S1pVAX-CTB and pVAX-S1-CTB could be expressed in mammalian cells. After 42 days of immunization, the proliferation effect of T lymphocytes in the two vaccine groups was significantly higher than that in the control group. The difference was very significant (P 0.01). The CD4T cells in spleen and peripheral blood reached the maximum at 42 days after immunization in the combined group, and the CD8 ~ T cells in the spleen and peripheral blood of the combined group reached the maximum value at 42 days after immunization. The results of specific cytotoxicity showed that CD4T cells in the spleen and peripheral blood of the combined group reached the maximum value at 42 days after immunization. The cytotoxic effect of the combined group and the fusion group was obvious 42 days after immunization, especially in the fusion group. The detection of serum anti-S1 protein IgG content in mice showed that the IgG content of both vaccine groups showed a certain increasing trend. At 42 days after immunization, the IgG content in fusion group was significantly higher than that in other groups, and the difference was very significant (p 0.01). The results of detection of IFN- 緯 and IL-4 in peripheral blood showed that the levels of above two cytokines in combination group and fusion group were significantly higher than those in other control groups at 42 days after immunization. In the fusion group, more cytokines were produced by stimulation, and the difference between the two groups was significant (p 0.05). To sum up, this study shows that CTB gene can not only promote the cellular immune response ability in the process of immunization with SARS-CoV S1 antigen gene, The results provide a theoretical basis for the further study of the biological activity of CTB and the development of a novel immune-enhanced nucleic acid vaccine against SARS virus.
【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1

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