本文選題：結(jié)核分枝桿菌 + 亞單位疫苗��； 參考：《山東大學(xué)》2010年博士論文

【摘要】： 結(jié)核病(Tuberculosis, TB)是由結(jié)核分枝桿菌(Mycobacterium tuberculosis,Mtb)引起的一種古老而漫長的感染性疾病,由其導(dǎo)致的死亡率居全球第二位,給各國尤其是發(fā)展中國家?guī)砹顺林氐呢?fù)擔(dān)。據(jù)WHO統(tǒng)計,2007年全球新增結(jié)核病人937萬,因結(jié)核病死亡人數(shù)達(dá)132萬(不包括HIV陽性患者)。新增病例中亞洲和非洲分別占55%和31%,其中中國以130萬新增病例位居第二位,結(jié)核病防控形勢非常嚴(yán)峻。 目前,卡介苗是唯一一種獲得批準(zhǔn)的廣泛使用的結(jié)核病預(yù)防疫苗,對于兒童卡介苗具有良好的保護(hù)效果,但對于成人其保護(hù)效果不容樂觀,對于抑制潛伏期結(jié)核的復(fù)發(fā)更是毫無作用。因此研制新的結(jié)核病疫苗迫在眉睫,未來的結(jié)核病疫苗不僅要對剛出生嬰兒以及未感染結(jié)核菌的人群提供良好的預(yù)防作用,對于已經(jīng)感染結(jié)核菌但尚未發(fā)病的已感染人群也要具有增強(qiáng)細(xì)胞免疫、加速清除結(jié)核菌的保護(hù)作用。 本研究用分子生物學(xué)技術(shù)構(gòu)建了兩種克隆表達(dá)載體pET30a-Ag85b和pET30a-HspX,表達(dá)并純化了結(jié)核分支桿菌H37Rv的保護(hù)性抗原Ag85b和休眠期高表達(dá)蛋白HspX,并與另外兩種分泌性蛋白CFP-10和ESAT-6(已制備成融合蛋白CFP-10：ESAT-6)混合后,與鋁佐劑和CpG DNA佐劑聯(lián)合應(yīng)用,制備成新型結(jié)核病亞單位疫苗。此種亞單位疫苗的制備為國內(nèi)外首次報道。對此疫苗進(jìn)行有效性評價,觀察此疫苗的免疫原性和保護(hù)力；同時用OVA和Al(OH)3作為免疫原免疫豚鼠初步建立豚鼠Ⅰ型過敏反應(yīng)模型和相關(guān)的定量評價指標(biāo)，為今后疫苗的Ⅰ型過敏評價奠定基礎(chǔ)。 一、新型結(jié)核病亞單位疫苗的制備 運(yùn)用分子生物學(xué)技術(shù)成功構(gòu)建了兩種克隆表達(dá)載體pET30a-Ag85b和pET30a-HspX,并將兩種載體分別轉(zhuǎn)化入表達(dá)宿主菌大腸桿菌BL-21,通過IPTG誘導(dǎo)成功表達(dá)了兩種蛋白。經(jīng)驗證,Ag85b為包涵體表達(dá)而HspX為上清表達(dá)；經(jīng)過一系列前期處理,Ag85b上樣樣品經(jīng)過Source30 Q陰離子交換柱層析進(jìn)行一步純化,HspX上樣樣品經(jīng)過QHP陰離子交換柱層析,疏水層析,QHP陰離子交換柱層析進(jìn)行三步純化,分別得到了較純的的蛋白樣品。經(jīng)過SDS-PAGE、蛋白質(zhì)測序和WB等一系列實驗,初步驗證了蛋白分子量、純度和等電點等基本理化性質(zhì)以及蛋白的生物學(xué)活性，，為后期的有效性評價包括免疫原性評價和保護(hù)力評價奠定了基礎(chǔ)。 兩種蛋白樣品與事先制備好的另一種融合蛋白CFP-10：ESAT-6按照一定比例進(jìn)行混合，之后按照順序與鋁佐劑和CpG佐劑進(jìn)行混合制備成新型結(jié)核病亞單位疫苗。 二、新型結(jié)核病亞單位疫苗的免疫原性評價 將上述疫苗免疫小鼠對其免疫原性進(jìn)行初步評價。對BALB／c小鼠進(jìn)行頸背部皮下免疫,每周一次共免疫三次,末次免疫后一周,小鼠摘眼球放血處死,無菌取脾分離小鼠脾淋巴細(xì)胞；檢測小鼠血清中針對三種蛋白的特異性IgG水平；體外用三種抗原分別刺激小鼠脾淋巴細(xì)胞后,通過淋巴細(xì)胞增殖實驗和ELISPOT實驗檢測小鼠脾淋巴細(xì)胞的增殖能力和脾淋巴細(xì)胞中能分泌抗原特異性IFN-γ的細(xì)胞數(shù)量；分離小鼠腹腔巨噬細(xì)胞,體外用三種抗原刺激后,檢測分泌IL-12的水平。結(jié)果表明,混合抗原與兩種佐劑同時應(yīng)用的組(Ag+Al+CpG)小鼠血清中特異性IgG水平、小鼠脾淋巴細(xì)胞的增殖能力、小鼠脾淋巴細(xì)胞能夠分泌針對三種蛋白的特異性IFN-γ細(xì)胞數(shù)量以及巨噬細(xì)胞分泌IL-12水平均顯著高于陰性對照組,與單純混合抗原組相比也有顯著升高。這表明新型疫苗在小鼠體內(nèi)能同時引起較高的細(xì)胞免疫和體液免疫,同時也表明重組蛋白必須要與佐劑聯(lián)合使用才能滿足亞單位疫苗的要求,這也是為什么近幾年進(jìn)入臨床評價的亞單位疫苗無一例外的采用了強(qiáng)大的佐劑系統(tǒng)。 三、新型結(jié)核病亞單位疫苗的保護(hù)性評價 用結(jié)核桿菌H37Rv通過腹股溝皮下注射的方式攻擊豚鼠,造成結(jié)核菌感染后模型，然后用新型亞單位疫苗通過后腿肌肉注射方式進(jìn)行免疫,每兩周免疫一次,共免疫三次,末次免疫后兩周,豚鼠腹腔注射戊巴比妥鈉進(jìn)行安樂死,取肝臟、脾臟和肺臟進(jìn)行病理檢查,計算病變評分；取脾臟研磨后接種改良羅氏培養(yǎng)基四周后計數(shù)菌落數(shù)計算脾菌負(fù)荷量。脾菌負(fù)荷量和臟器病變評分顯示其保護(hù)效果有限,雖然好于生理鹽水對照組和單純抗原組,但尚未有統(tǒng)計學(xué)差異。導(dǎo)致保護(hù)效果有限的原因可能與疫苗制備工藝尚不成熟,各組分配比以及佐劑混合順序不同有關(guān),此外,結(jié)核菌感染后模型的不完善也可能是原因之一,腹股溝皮下注射H37Rv建立豚鼠感染后模型的方法有待進(jìn)一步優(yōu)化。本研究中的效力學(xué)實驗并未設(shè)置BCG陽性對照組原因在于BCG對于結(jié)核菌感染后人群并無保護(hù)效果,而本實驗效力學(xué)評價使用的豚鼠模型為感染后模型。 四、疫苗Ⅰ型過敏反應(yīng)陽性模型的建立 疫苗的安全性評價是疫苗臨床前評價的重要內(nèi)容,為了給疫苗免疫毒性中Ⅰ型過敏反應(yīng)的評價提供一個陽性模型,同時建立Ⅰ型過敏反應(yīng)定量評價指標(biāo),本研究以O(shè)VA和Al(OH)3作為免疫原免疫豚鼠,初次免疫采取背部皮下注射的方式,然后通過后腿肌肉注射的方式進(jìn)行三次加強(qiáng)免疫；末次免疫后一周,對模型組豚鼠用OVA通過后腿靜脈注射的方式進(jìn)行激發(fā),觀察并記錄豚鼠的Ⅰ型過敏反應(yīng)癥狀；對激發(fā)后的豚鼠采取支氣管肺泡灌洗液,離心后收集上清檢測Ⅰ型過敏反應(yīng)介導(dǎo)介質(zhì)組胺和白三烯水平,對未激發(fā)的模型組豚鼠直接采取腹腔灌洗液分離腹腔肥大細(xì)胞,體外經(jīng)OVA激發(fā)后檢測上清中組胺和白三烯水平。 結(jié)果表明,模型組豚鼠在經(jīng)OVA激發(fā)后出現(xiàn)明顯的Ⅰ型過敏反應(yīng)癥狀如撓耳、撓臉、擺頭、豎毛、咳嗽、噴嚏、呼吸困難、步態(tài)不穩(wěn)、痙攣等,對支氣管肺泡灌洗液中組胺和白三烯水平進(jìn)行檢測發(fā)現(xiàn)兩者水平均有顯著升高；未激發(fā)的豚鼠的腹腔肥大細(xì)胞體外經(jīng)OVA刺激后分泌組胺和白三烯水平也顯著升高。這些結(jié)果表明我們成功的建立了豚鼠I型過敏反應(yīng)模型,并且建立了幾種定量評價I型過敏反應(yīng)的指標(biāo),今后對于疫苗的I型過敏反應(yīng)評價,我們除了觀察I型過敏反應(yīng)癥狀等定性指標(biāo)外,還可以對支氣管肺泡灌洗液中組胺和白三烯水平以及腹腔肥大細(xì)胞所分泌組胺和白三烯水平進(jìn)行定量檢測。 五、結(jié)論 本研究首次將結(jié)核菌保護(hù)性抗原Ag85b、潛伏期蛋白HspX和另外兩種早期分泌蛋白CFP10：ESAT6混合,并與兩種佐劑CpG DNA和Al(OH)3聯(lián)用,制備成一種新型結(jié)核病亞單位疫苗。 對這種新型疫苗進(jìn)行免疫原性和結(jié)核菌感染后的保護(hù)力評價,發(fā)現(xiàn)具有較強(qiáng)的免疫原性但對于結(jié)核菌感染后豚鼠保護(hù)力有限,我們計劃經(jīng)過后續(xù)對蛋白表達(dá)形式的優(yōu)化和疫苗配比的改進(jìn)以期獲得更好的保護(hù)效果,從而為結(jié)核菌已感染人群提供一種理想的治療性疫苗。同時本疫苗含有結(jié)核菌早期分泌蛋白成分也使其可能成為針對未感染人群的一種預(yù)防性疫苗或者BCG的加強(qiáng)疫苗。 初步建立了一種豚鼠Ⅰ型過敏反應(yīng)模型,建立了BALF上清中和腹腔肥大細(xì)胞體外刺激培養(yǎng)液中組胺和白三烯的檢測方法,建立的Ⅰ型過敏反應(yīng)模型和組胺、白三烯等定量檢測指標(biāo)可為疫苗的Ⅰ型過敏反應(yīng)評價提供新的方法。
[Abstract]:Tuberculosis (TB) is an ancient and long, long, infectious disease caused by Mycobacterium tuberculosis (Mtb). The death rate is the second largest in the world, bringing a heavy burden to all countries, especially the developing countries. According to WHO statistics, the total number of new TB patients worldwide in 2007 was killed by tuberculosis. The number of dead people was 1 million 320 thousand (excluding HIV positive). Among the new cases, Asia and Africa accounted for 55% and 31% respectively, of which 1 million 300 thousand of the new cases in China ranked second, and the situation of tuberculosis prevention and control was very severe.
At present, Bacillus Calmette Guerin is the only widely used vaccine for tuberculosis prevention, which has good protective effect on BCG in children, but it is not optimistic for adults and has no effect on inhibiting the recurrence of latent tuberculosis. Therefore, the development of a new tuberculosis vaccine is imminent and the future tuberculosis epidemic is imminent. The vaccine should not only provide a good preventive effect on the newborn babies and those who have not infected the Mycobacterium tuberculosis, but also have the enhancement of cellular immunity to the infected people who have been infected but have not yet been infected, and accelerate the protection of the tuberculosis.
In this study, two cloned expression vectors, pET30a-Ag85b and pET30a-HspX, were constructed with molecular biology techniques, expressing and purifying the protective antigen Ag85b of Mycobacterium tuberculosis H37Rv and the hibernation high expression protein HspX, and mixed with two other secretory proteins, CFP-10 and ESAT-6 (prepared fusion protein CFP-10:ESAT-6). In combination with CpG DNA adjuvant, a new subunit vaccine for tuberculosis was prepared. The preparation of this subunit vaccine was first reported at home and abroad. The effectiveness of the vaccine was evaluated, the immunogenicity and protective ability of the vaccine were observed. At the same time, OVA and Al (OH) 3 were used as immunogenic immunogenic guinea pigs to establish guinea pig type I anaphylaxis model. And related quantitative evaluation indicators will lay the foundation for future type I allergy evaluation of vaccines.
Preparation of a new subunit vaccine for tuberculosis
Two kinds of cloning expression vectors pET30a-Ag85b and pET30a-HspX were successfully constructed by molecular biology technology, and two vectors were transformed into Escherichia coli BL-21 respectively, and two proteins were successfully expressed through IPTG. It was proved that Ag85b was expressed as inclusion body and HspX was expressed in the supernatant; after a series of preprocessing, Ag85b Samples were purified by Source30 Q anion exchange column chromatography. Samples of HspX samples were purified by QHP anion exchange column chromatography, hydrophobicity chromatography and QHP anion exchange column chromatography. A series of pure protein samples were obtained respectively. After a series of experiments, such as SDS-PAGE, protein sequencing and WB, the protein molecules were preliminarily verified. The basic physicochemical properties of the quantity, purity and isoelectric point, as well as the biological activity of the protein, lay the foundation for the evaluation of the later effectiveness, including the evaluation of immunogenicity and the evaluation of protective ability.
The two protein samples were mixed in a certain proportion with a pre prepared fusion protein CFP-10:ESAT-6, then mixed with aluminum and CpG adjuvant in order to prepare a new subunit vaccine for tuberculosis.
Two. Evaluation of the immunogenicity of a new subunit vaccine against tuberculosis.
The immunogenicity of the mice immunized with the above vaccine was preliminarily evaluated. The BALB / c mice were immunized with the back of the neck. The mice were immunized three times a week. One week after the last immunization, the mice were killed and the spleen lymphocytes were isolated from the spleen, and the specific IgG level of the three proteins in the mice serum was detected. After three antigens were used to stimulate the spleen lymphocytes of mice, the proliferation of lymphocyte and the number of antigen specific IFN- gamma cells in splenic lymphocytes were detected by lymphocyte proliferation test and ELISPOT test, and the level of IL-12 was detected after three kinds of antigen stimulation in vitro of peritoneal macrophages in mice. The results showed that the specific IgG level in the serum of the mixed antigen and the two adjuvants (Ag+Al+CpG), the proliferation of mouse spleen lymphocyte, the number of specific IFN- gamma cells secreted by the spleen lymphocyte and the secretion of IL-12 in the macrophage were significantly higher than that of the negative control group. This shows that the new vaccine can cause higher cellular immunity and humoral immunity in mice, and that the recombinant protein must be combined with the adjuvant in order to meet the requirements of subunit vaccine, which is why the subunit vaccine entered into clinical evaluation in recent years is no exception. A powerful adjuvant system is used.
Three, the protective evaluation of a new subunit vaccine against tuberculosis.
The guinea pig was attacked by subcutaneous injection of Mycobacterium tuberculosis H37Rv, resulting in the model of Mycobacterium tuberculosis after infection, and then immunized with a new subunit vaccine through the hind leg muscle injection, immunized once every two weeks, three times, and two weeks after the last immunization, and pentobarbital was injected into the abdominal cavity of guinea pigs for euthanasia, liver and spleen. The pathological examination was carried out with the lungs to calculate the lesion score, and the spleen load was calculated after the inoculation of the spleen after lapping the improved Roche culture medium. The spleen load and organ lesion score showed that the protective effect was limited, although it was better than the normal saline control group and the former group of Dan Chunkang, but there was no statistical difference. The cause of the limited fruit may be not mature with the vaccine preparation process, the distribution ratio of each group and the mixing order of the adjuvant are different. In addition, the imperfect model of the Mycobacterium tuberculosis may be one of the reasons. The method of subcutaneous injection of H37Rv in the groin to establish the model of the guinea pig after infection needs to be further optimized. The reason for the setting of BCG positive control group was that BCG had no protective effect on the population after tuberculosis infection, and the guinea pig model used in the effectiveness evaluation of this experiment was the post infection model.
Four, the establishment of an allergic reaction model for vaccine type I
The safety evaluation of the vaccine is an important part of the pre clinical evaluation of the vaccine. In order to provide a positive model for the evaluation of type I anaphylaxis in vaccine immuno toxicity, and to establish a quantitative evaluation index of type I anaphylaxis, this study takes OVA and Al (OH) 3 as the immunogenicity of the immunogen mouse, the first immunization by subcutaneous injection of the back. After the last week, the guinea pigs in the model group were stimulated by the injection of OVA through the hind leg vein to observe and record the symptoms of type I anaphylaxis in the guinea pig. The bronchoalveolar lavage fluid of the guinea pigs after the stimulation was taken and the supernatant was collected to detect type I allergy after the centrifuge was centrifuged after the last immunization. The reaction mediates the level of histamine and leukotriene, and the abdominal mast cells are isolated from the non excited model group of guinea pigs directly by intraperitoneal lavage solution, and the levels of histamine and leukotrienes in the supernatant are detected by OVA in vitro.
The results showed that the guinea pigs in the model group had obvious symptoms of type I anaphylaxis, such as ear, flex, head, hair, coughing, sneezing, dyspnea, gait instability, spasm and so on. The levels of histamine and leukotrienes in bronchoalveolar lavage fluid were significantly increased, and the abdominal cavity of unstimulated Guinea pigs was found. The secretory histamine and leukotriene levels of the mast cells also increased significantly after OVA stimulation in vitro. These results suggest that we have successfully established the guinea pig I anaphylaxis model, and established several indicators for quantitative evaluation of the I anaphylaxis. In the future, we evaluate the I anaphylaxis of the vaccine, in addition to observing the symptoms of the I allergic reaction. In addition, the levels of histamine and leukotrienes in bronchoalveolar lavage fluid and the levels of histamine and leukotrienes secreted by mast cells in the abdominal cavity can also be quantified.
Five. Conclusion
For the first time, a new type of subunit vaccine for tuberculosis was prepared by mixing the protective antigen Ag85b, latent protein HspX and two other early secretory protein CFP10:ESAT6 and combined with two kinds of adjuvant, CpG DNA and Al (OH) 3.
To evaluate the immunogenicity of the new vaccine and the protective effect of Mycobacterium tuberculosis infection, it is found that the vaccine has a strong immunogenicity but limited protection force for the guinea pig after tuberculosis infection. We plan to improve the protein expression and improve the ratio of the vaccine in order to obtain better protective effect, so that the Mycobacterium tuberculosis has been felt. The infected population provides an ideal therapeutic vaccine. The vaccine contains early secretory protein components from Mycobacterium tuberculosis and may also make it a preventive vaccine against uninfected people or a strengthened vaccine for BCG.
A guinea pig type I anaphylaxis model was established. The detection methods of histamine and leukotriene in the culture medium of BALF supernatant and peritoneal mast cells in vitro were established. The model of type I anaphylaxis and histamine, leukotrienes and other quantitative indicators could provide a new method for the evaluation of type I anaphylaxis.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

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