發(fā)布時(shí)間：2018-04-30 17:00

  本文選題：結(jié)核分枝桿菌 + 融合蛋白��； 參考：《復(fù)旦大學(xué)》2011年碩士論文

【摘要】：結(jié)核病是一種古老的疾病,早在十七世紀(jì)就已經(jīng)存在。目前,由于全世界感染人口基數(shù)大,耐藥結(jié)核病例大大增加和HIV/TB共感染情況嚴(yán)重,結(jié)核病正對(duì)人類構(gòu)成巨大的威脅。為了預(yù)防結(jié)核病,全球已經(jīng)半個(gè)多世紀(jì)前廣泛接種了卡介苗BCG,但是BCG的免疫保護(hù)效果并不理想。隨著生物技術(shù)的發(fā)展,很多新型疫苗正在陸續(xù)開發(fā),例如重組卡介苗,減毒疫苗,蛋白疫苗和DNA疫苗等。他們各自都有不同的優(yōu)勢(shì),但是否可以超越BCG的免疫保護(hù)效果還有待臨床證明。 在人群中給予結(jié)核桿菌疫苗免疫是防治結(jié)核感染、控制局部爆發(fā)流行最有效的措施。結(jié)核疫苗主要有兩大類：活的結(jié)核桿菌疫苗和亞單位疫苗。結(jié)核亞單位疫苗雖然免疫原性沒有活的結(jié)核桿菌疫苗強(qiáng),但其免疫效力不受預(yù)先接觸環(huán)境分枝桿菌和結(jié)核分枝桿菌的影響,而且背景清楚,安全性好,制備簡(jiǎn)單,可以用于成人強(qiáng)化免疫,是控制結(jié)核病爆發(fā)的理想疫苗。亞單位疫苗有三種形式：蛋白質(zhì)疫苗、DNA疫苗和以病毒為載體的疫苗[1,2],其中蛋白質(zhì)疫苗相對(duì)最為安全,易于被人們接受。目前,已經(jīng)有超過20種的不同抗原用于誘導(dǎo)抗結(jié)核的免疫應(yīng)答[3,4]. 分泌性抗原Ag85a屬于Ag85蛋白家族,被認(rèn)為具有較好的免疫原性。結(jié)核桿菌是一種巨噬細(xì)胞內(nèi)寄生菌。結(jié)核病的發(fā)生與免疫系統(tǒng)密切相關(guān)。故人體對(duì)結(jié)核桿菌感染的免疫,以細(xì)胞免疫為主。在抗結(jié)核感染中,CD4+和CD8+T細(xì)胞起主要作用。結(jié)核桿菌活化的T細(xì)胞所釋放出的淋巴因子,包括白介素-2(IL-2),γ-干擾素(IFN-γ),它們直接或間接殺傷結(jié)核桿菌。 本研究采用來源于結(jié)核桿菌的重要抗原Ag85a和結(jié)核免疫相關(guān)的細(xì)胞因子IFN-y與IL-2,以結(jié)核分枝桿菌標(biāo)準(zhǔn)株H37R v基因組DNA為模板,擴(kuò)增Ag85a基因,分別與鼠源IFN-y和IL-2的基因融合與表達(dá)質(zhì)粒pET28a構(gòu)建成重組質(zhì)粒pet28a-Ag85a-IFN-γ和pet28a-Ag85a-IL-2。在大腸桿菌BL21(DE3)中表達(dá)去除信號(hào)肽的融合蛋白,純化得到Ag85a—IFN-γ和Ag85a—IL-2的融合蛋白。用該蛋白皮下免疫C57BL/6小鼠,并進(jìn)行免疫效果評(píng)價(jià),以期篩選高效的加強(qiáng)免疫的蛋白亞單位疫苗。通過ELISA方法檢測(cè)血清中IgG,IgG1和IgG2c水平,我們發(fā)現(xiàn)融合蛋白Ag85a-IFN-γ和Ag85a-IL-2實(shí)驗(yàn)組的免疫球蛋白水平均高于單獨(dú)抗原,并且融合蛋白組IgG2c/IgG1的比值大于單獨(dú)抗原,并且Ag85a—IL-2的免疫保護(hù)效果優(yōu)于Ag85a-IFN-γ;流式細(xì)胞術(shù)檢測(cè)胞內(nèi)特異性CD4+和CD8T+細(xì)胞,Ag85a-IFN-γ和Ag85a-IL-2實(shí)驗(yàn)組CD8+/CD4+比例高于單獨(dú)抗原。 本實(shí)驗(yàn)成功構(gòu)建并表達(dá)了Ag85a-IFN-γ和Ag85a-IL-2的融合蛋白,融合蛋白的免疫效果優(yōu)于單獨(dú)Ag85a抗原,并且Ag85a—IL-2的免疫效果優(yōu)于Ag85a—IFN-y。
[Abstract]:Tuberculosis is an ancient disease that existed as early as the seventeenth century. At present, because of the large number of infected people in the world, the increase of drug-resistant tuberculosis cases and the serious co-infection of HIV/TB, tuberculosis is posing a great threat to human beings. In order to prevent tuberculosis, BCG was widely inoculated around the world more than half a century ago, but the immune protection effect of BCG is not satisfactory. With the development of biotechnology, many new vaccines are being developed, such as recombinant BCG vaccine, attenuated vaccine, protein vaccine and DNA vaccine. They each have different advantages, but whether they can outperform the immune protection of BCG has yet to be clinically proven. Immunization with Mycobacterium tuberculosis vaccine in the population is the most effective measure to prevent and treat tuberculosis infection and to control the local outbreak. There are two main types of TB vaccine: live Mycobacterium tuberculosis vaccine and subunit vaccine. Although the immunogenicity of tuberculosis subunit vaccine is not as strong as that of live mycobacterium tuberculosis vaccine, its immune efficacy is not affected by the preexposure to environmental mycobacterium and mycobacterium tuberculosis, and the background is clear, the safety is good, and the preparation is simple. It is an ideal vaccine to control the outbreak of tuberculosis. There are three forms of subunit vaccine: protein vaccine DNA vaccine and virus-vector vaccine [1], in which protein vaccine is the safest and is easy to be accepted. At present, more than 20 different antigens have been used to induce anti-tuberculosis immune response. Secretory antigen Ag85a belongs to Ag85 protein family and is considered to have good immunogenicity. Mycobacterium tuberculosis is a parasite in macrophages. Tuberculosis is closely related to the immune system. Therefore, the human body to Mycobacterium tuberculosis infection immunity, mainly cellular immunity. CD4 and CD8 T cells play a major role in anti-tuberculosis infection. The lymphoid factors released by activated T cells of Mycobacterium tuberculosis, including interleukin-2, interferon 緯 -IFN- 緯, kill mycobacterium tuberculosis directly or indirectly. In this study, the important antigen Ag85a derived from Mycobacterium tuberculosis and the cytokines IFN-y and IL-2 related to tuberculosis immunity were used to amplify the Ag85a gene using the genomic DNA of Mycobacterium tuberculosis standard strain H37R v as template. The recombinant plasmids pet28a-Ag85a-IFN- 緯 and pet28a-Ag85a-IL-2were constructed by fusion and expression plasmid pET28a with mouse IFN-y and IL-2, respectively. The fusion protein of Ag85a-IFN- 緯 and Ag85a-IL-2 was purified by expressing the fusion protein which removed the signal peptide in E. coli BL21DDE3. The fusion protein of Ag85a-IFN- 緯 and Ag85a-IL-2 was purified. C57BL/6 mice were immunized subcutaneously with this protein, and the immunological effect was evaluated in order to screen highly effective protein subunit vaccine. The levels of IgG1 and IgG2c in serum were detected by ELISA method. We found that the immunoglobulin levels of the fusion protein Ag85a-IFN- 緯 and Ag85a-IL-2 group were higher than those of the single antigen, and the ratio of IgG2c/IgG1 in the fusion protein group was higher than that of the single antigen. The protective effect of Ag85a-IL-2 was better than that of Ag85a-IFN- 緯, and the ratio of CD8 / CD4 in CD4 and CD8T cells detected by flow cytometry was higher than that in Ag85a-IL-2 group. The fusion protein of Ag85a-IFN- 緯 and Ag85a-IL-2 was successfully constructed and expressed in this experiment. The immune effect of the fusion protein was better than that of single Ag85a antigen, and the immune effect of Ag85a-IL-2 was better than that of Ag85a-IFN-y.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392

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