發(fā)布時(shí)間：2018-01-12 18:19

  本文關(guān)鍵詞：乙肝病毒RNA復(fù)制子疫苗、DNA疫苗的構(gòu)建及其對小鼠免疫效果的比較　出處：《吉林大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 乙肝病毒 RNA 復(fù)制子疫苗 DNA 疫苗 體液免疫 細(xì)胞免疫

【摘要】：本研究的目的是構(gòu)建乙肝病毒的RNA 復(fù)制子疫苗,通過與普通疫苗和DNA 疫苗進(jìn)行比較,探索RNA 復(fù)制子疫苗最佳的形式和免疫途徑。 首先,充分利用生物信息學(xué)工具及多條重疊引物PCR 拼接的策略,設(shè)計(jì)并合成了兩個(gè)抗原基因:S2-S 和MAG。將兩種抗原基因的PCR產(chǎn)物分別與pGEM-T 載體連接,并篩選到序列正確的克隆質(zhì)粒pGEM-S2S 和pGEM-MAG。 本研究利用S2-S、MAG 作為兩個(gè)抗原基因,以IL-2 為分子佐劑,利用pcDNA3.1+構(gòu)建DNA疫苗,利用RNA復(fù)制子載體pSFV構(gòu)建RNA復(fù)制子疫苗,共構(gòu)建8個(gè)疫苗載體:pSFV-S2S,pSFV-MAG,pcDNA-S2S,pcDNA-MAG,pSFV-S2S-II-IL2,pSFV-MAG-II-IL2,pcDNA-S2S-II-IL2,pcDNA-MAG-II-IL2。其中II 是本研究中從載體pIRES1neo 上擴(kuò)增IVS和IRES 的片段(本研究中通稱為II)。 將上述八個(gè)疫苗載體分別轉(zhuǎn)染BHK-21 細(xì)胞,ELISA 檢測其表達(dá)情況,結(jié)果表明以RNA 復(fù)制子疫苗載體的表達(dá)效率高于DNA 疫苗。另外,RNA 復(fù)制子疫苗載體轉(zhuǎn)染BHK-21 細(xì)胞后,細(xì)胞呈現(xiàn)凋亡特征。將所構(gòu)建的疫苗載體以不同的劑量分三次免疫BALB/c 小鼠,ELISA檢測體液免疫效果。建立穩(wěn)定表達(dá)S2S 和MAG 的P815 細(xì)胞作為CTL檢測的靶細(xì)胞,通過CTL 方法檢測細(xì)胞免疫效果。結(jié)果表明,RNA 復(fù)制子疫苗體液免疫和細(xì)胞免疫效果均優(yōu)于DNA 疫苗,但二者的體液免疫效果不及基因工程亞單位疫苗。
[Abstract]:The purpose of this study is to construct hepatitis B virus RNA replicon vaccine. Through comparing with the common vaccine and DNA vaccine, we explore the best form and immune way of RNA replicon vaccine.
First, make full use of bioinformatics tools and multiple overlapping primers PCR splicing strategy, designed and synthesized two antigen genes: S2-S and MAG. PCR antigen gene products of the two are respectively connected with the pGEM-T vector, and screened the correct sequence of plasmid pGEM-S2S and pGEM-MAG.
This study uses S2-S and MAG as the two antigen gene, using IL-2 as a molecular adjuvant DNA vaccine constructed by pcDNA3.1+, using the RNA replicon vector pSFV to construct the RNA replicon vaccine, we constructed 8 vaccine vectors: pSFV-S2S, pSFV-MAG, pcDNA-S2S, pcDNA-MAG, pSFV-S2S-II-IL2, pSFV-MAG-II-IL2, pcDNA-S2S-II-IL2, pcDNA-MAG-II-IL2. and II are in this study from the vector pIRES1neo amplified IVS and IRES fragments (known as II in this study).
The eight vaccine vector were transfected into BHK-21 cells, detect the expression of ELISA, the results showed that the expression efficiency of replicon vaccine vector with RNA was higher than that of DNA vaccine. In addition, RNA replicon vaccine vector were transfected into BHK-21 cells, the cells exhibited the characteristics of apoptosis. The vaccine carrier with different doses of three immune BALB/c mice, ELISA detection of humoral immune effect. The establishment of stable expression of S2S and MAG of P815 cells as the target cells of CTL detection, by detecting the effect of cellular immunity in CTL method. The results show that the RNA replicon vaccine humoral and cellular immune effects were better than the DNA vaccine, but less than two of the humoral immunity effect of gene engineering subunit the vaccine.

【學(xué)位授予單位】：吉林大學(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：1415424