【摘要】： 本研究旨在利用HBc自身所具有的一系列優(yōu)點(diǎn),包括具有高度的免疫原性,是一種T細(xì)胞依賴性和T細(xì)胞非依賴性抗原,沒有遺傳限制;能形成顆粒樣結(jié)構(gòu),允許外源插入,可以將上述性質(zhì)傳遞給與之融合的外源序列,并且將這些外源序列以重復(fù)且高密度的方式展示在自身表面;可以允許對自身進(jìn)行替換、添加、缺失等修飾;能在E.coli中進(jìn)行大量的表達(dá),并能快速制備等,借助以激發(fā)體液免疫為主的炭疽表位和以激發(fā)細(xì)胞免疫為主的結(jié)核抗原為模型,構(gòu)建以HBc為疫苗載體的新型炭疽表位疫苗和新型結(jié)核亞單位疫苗,探討HBc作為疫苗載體輔助增強(qiáng)體液和細(xì)胞免疫反應(yīng)的機(jī)制,構(gòu)建體液免疫和細(xì)胞免疫通用的新型疫苗及新發(fā)突發(fā)傳染病病原體快速反應(yīng)的載體,以更好地適應(yīng)現(xiàn)代疫苗,尤其是軍用生物防御疫苗的需求。 本研究中首先利用重疊延伸PCR的方法,通過4輪PCR拼接,1輪PCR擴(kuò)增,得到了編碼乙型肝炎病毒核心抗原(HBcAg)氨基端144個(gè)氨基酸的核酸序列HBc-N144。基于所得到的測序正確的質(zhì)粒pET21a-HBc-N144,在HBc-N144疫苗載體主要的免疫顯性區(qū)域(MIR區(qū))的78-79位氨基酸之間插入了本室前期鑒定得到的炭疽保護(hù)性抗原PA結(jié)構(gòu)域2中的2β2-2β3loop序列,這段序列被認(rèn)為與介導(dǎo)炭疽致死因子(LF)和水腫因子(EF)進(jìn)入胞質(zhì),PA與受體結(jié)合,穩(wěn)定PA七聚體的穩(wěn)定性等生物學(xué)作用相關(guān),而且被認(rèn)為包含一個(gè)炭疽保護(hù)性抗原主要的中和性表位“SFFD”,得到了嵌合的HBc/炭疽表位疫苗HBc-N144-PA-loop2。研究中借助Western blotting技術(shù)驗(yàn)證了抗體與這種嵌合蛋白的結(jié)合,通過透射電子顯微鏡技術(shù)檢測了這種嵌合蛋白裝配成顆粒的情況。負(fù)染和透射電子顯微鏡的結(jié)果表明,攜帶有炭疽表位的HBc/炭疽表位嵌合蛋白可以形成電子顯微鏡下可見的直徑約為30nm的顆粒樣結(jié)構(gòu),這與HBc分子天然狀態(tài)下形成的顆粒結(jié)構(gòu)在形態(tài)學(xué)上沒有明顯差異。此后,在動物模型上驗(yàn)證了這種HBc/炭疽表位疫苗HBc-N144-PA-loop2的免疫學(xué)性質(zhì)。免疫小鼠模型上的血清學(xué)檢測結(jié)果表明HBc-N144-PA-loop2能激發(fā)針對炭疽保護(hù)性抗原(PA)的特異性抗體。為了進(jìn)一步驗(yàn)證所獲得的HBc/炭疽表位疫苗的免疫學(xué)性質(zhì),在相對小鼠更為理想的評價(jià)模型,豚鼠模型上檢測了特異性抗體的消長情況,發(fā)現(xiàn)HBc/炭疽表位疫苗HBc-N144-PA-loop2不僅可以激發(fā)抗PA抗體(實(shí)質(zhì)上為PA表位特異性抗體),也能激發(fā)針對炭疽毒素的中和性抗體,并且在40LD50劑量的炭疽芽胞攻毒實(shí)驗(yàn)中,部分保護(hù)免疫的豚鼠對抗炭疽芽胞的侵襲。而之前本室用含有此中和性表位“SFFD”的特異性噬菌體克隆作為抗原免疫小鼠,僅觀察到了抗PA抗體的產(chǎn)生,未觀察到明顯的毒素中和活性。此外,在豚鼠試驗(yàn)中也觀察到了HBc一定的佐劑效應(yīng),HBc-N144-PA-loop2不加佐劑組和使用氫氧化鋁佐劑組可以激發(fā)幾乎一樣高的抗PA抗體(即表位特異性抗體)和中和性抗體,并且能保護(hù)57%的免疫豚鼠對抗炭疽芽胞的侵襲。相對于重組PA(rPA)激發(fā)的免疫效果而言,HBc-N144-PA-loop2組在抗PA抗體、中和性抗體、保護(hù)率等方面均略低,考慮到PA是83kDa的大分子,包含多個(gè)表位,而HBc-N144-PA-loop2僅含有其中的一個(gè)表位,用HBc作為炭疽表位疫苗的載體是可行的,但HBc-N144-PA-loop2激發(fā)免疫反應(yīng)的能力還需優(yōu)化。 為提高HBc-N144-PA-loop2作為炭疽表位疫苗的效力,本研究以序列正確的pET21a-HBc-N144-PA-loop2為基礎(chǔ),通過反向PCR技術(shù)對HBc載體進(jìn)行了MIR區(qū)79-81位氨基酸的缺失突變和添加天然HBc羧基端145-149位氨基酸的延長突變,得到了HBc分子MIR區(qū)的缺失突變體H144DL2和羧基端延長突變體H149L2,并去掉了HBc-N144-PA-loop2包含的多余氨基酸和組氨酸標(biāo)簽,得到H144L2;通過檢測改造后的HBc/炭疽表位疫苗激發(fā)體液免疫的各項(xiàng)指標(biāo)的變化,評價(jià)了HBc的改造效果。研究發(fā)現(xiàn)經(jīng)突變的嵌合蛋白仍可以形成電鏡下可見的顆粒狀結(jié)構(gòu);HBc分子MIR區(qū)的缺失突變(H144DL2)可以使抗PA抗體有所增加,抗HBc抗體較HBc-N144組顯著下降,并顯著低于H144L2組激發(fā)的抗HBc抗體;此外HBc分子MIR區(qū)的缺失突變體H144DL2還顯著激發(fā)了僅次于rPA組的中和性抗體,并且組內(nèi)所有動物的免疫血清都能被檢測到中和性抗體,各動物體間中和性抗體的滴度值分布也相對集中,并且能100%保護(hù)免疫的小鼠對抗炭疽致死毒素的攻擊。HBc羧基端的延長突變體H149L2未能增加抗PA抗體,抗體效價(jià)低于H144L2的水平;抗HBc抗體的滴度稍有增加;相對HBc-N144組和空白對照組可顯著激發(fā)中和性抗體,組內(nèi)動物免疫血清中也均被檢測到了中和性抗體,但抗體滴度低于H144DL2組;小鼠的死亡時(shí)間延長。佐劑效應(yīng)的研究結(jié)果表明,H144L2不加佐劑組相對使用鋁佐劑和弗氏佐劑組激發(fā)了最高的抗PA抗體,能保護(hù)50%的小鼠,小鼠的死亡時(shí)間由攻毒后6h延長到了10h,這提示,H144DL2不加佐劑可能是一個(gè)較好的HBc/炭疽表位疫苗劑型。此外,本部分還優(yōu)化了重組蛋白的表達(dá)純化工藝,排除了內(nèi)毒素的干擾。 本研究還在HBc中插入結(jié)核抗原ESAT-6,初步探討了HBc輔助增強(qiáng)特異性細(xì)胞免疫反應(yīng)的能力。以本室構(gòu)建的攜帶有ESAT-6的質(zhì)粒PAS22-ESAT-6和pET21a-H144L2為模板,借助HBc分子核酸序列中的MscⅠ酶切位點(diǎn),將全長288bp的ESAT-6基因序列通過平端連接引入到HBc的78-79位氨基酸之間,得到以HBc為疫苗載體的新型結(jié)核亞單位疫苗HE6。通過WB檢測了重組蛋白HE6與抗體的結(jié)合,通過負(fù)染和透射電鏡技術(shù)驗(yàn)證了顆粒的形成。由于機(jī)體抗結(jié)核桿菌的感染主要以T細(xì)胞介導(dǎo)的細(xì)胞免疫為主,所以能否有效地激發(fā)機(jī)體的T細(xì)胞免疫,尤其是CD4+Th1型細(xì)胞的免疫反應(yīng)是評價(jià)結(jié)核疫苗效力的關(guān)鍵。本研究在小鼠模型上評價(jià)了HE6的免疫學(xué)性質(zhì)。通過對小鼠的特異性免疫反應(yīng)進(jìn)行檢測,發(fā)現(xiàn)所構(gòu)建的HBc/結(jié)核亞單位疫苗HE6使用和不使用佐劑組均可激發(fā)高效價(jià)的ESAT-6特異的IgG2a和IgG1,而且抗體的滴度水平均顯著高于單獨(dú)使用rESAT-6加鋁佐劑免疫組。rESAT-6加鋁佐劑免疫組激發(fā)的IgG2a效價(jià)最低,僅約為2.81×102,這提示單獨(dú)使用rESAT-6加鋁佐劑免疫組不能很好地激發(fā)Th1型反應(yīng)。雖然HE6使用和不使用鋁佐劑組激發(fā)的IgG2a的抗體滴度均超過103,但I(xiàn)gG2a/IgG1比值無顯著性差異。HE6使用和不使用鋁佐劑組的ELISPOT結(jié)果顯著高于空白組和ESAT-6單獨(dú)加鋁佐劑免疫組,表明HBc的使用顯著增加了分泌IFN-γ的T細(xì)胞數(shù)目,提示HBc的使用能有助于增強(qiáng)Th1型反應(yīng)。流式的結(jié)果表明,HE6不加佐劑組中rESAT-6特異的IFN-γ+CD4+和IFN-γ+CD8+T細(xì)胞的頻率相對rESAT-6單獨(dú)加鋁佐劑免疫組和空白組有顯著增加,HE6使用鋁佐劑組中ESAT-6特異的IFN-γ+CD4+和IFN-γ+CD8+T細(xì)胞的頻率相對空白組有顯著增加,HE6不加佐劑組和使用鋁佐劑組中ESAT-6特異的IFN-γ+CD4+和IFN-γ+CD8+T細(xì)胞的頻率沒有顯著性差異。此結(jié)果與ELISPOT結(jié)果一致,這提示,HBc作為疫苗載體能有效地輔助增強(qiáng)結(jié)核抗原ESAT-6特異的細(xì)胞免疫,雖然疫苗結(jié)構(gòu)還有待優(yōu)化,但HE6是一種有前景的候選結(jié)核亞單位疫苗。 綜上所述,本研究篩選到了一個(gè)較優(yōu)的HBc疫苗載體結(jié)構(gòu)(H144D),優(yōu)化了相關(guān)的制備方法;通過插入炭疽表位,在小鼠和豚鼠模型上證明了HBc作為疫苗載體具有輔助增強(qiáng)特異性體液免疫的能力;通過插入結(jié)核抗原,在小鼠模型上證明了HBc作為疫苗載體具有輔助增強(qiáng)特異性細(xì)胞免疫的能力;HBc作為一種輔助增強(qiáng)體液和細(xì)胞免疫通用的疫苗載體,有良好的應(yīng)用前景。
[Abstract]:The purpose of this study is to take advantage of a series of advantages of HBc itself, including high immunogenicity, T cell-dependent and T cell-independent antigens without genetic restriction, granular structure, allowed for foreign insertion, which can transmit these properties to the fusion sequence, and these foreign sequences to Repeated and high-density display on its own surface; allowed to replace itself, add, delete and other modifications; can carry out a large number of expression in E.coli, and rapid preparation, etc., with the help of humoral immunity-based anthrax epitopes and stimulated cell immunity-based tuberculosis antigen as a model to construct a vaccine vector HBc. A novel anthrax epitope vaccine and a new type of tuberculosis subunit vaccine were developed to explore the mechanism of HBc as a vaccine carrier to assist in enhancing humoral and cellular immune responses, and to construct a novel vaccine for humoral and cellular immunity and a vector for rapid response to pathogens of emerging infectious diseases, so as to better adapt to modern vaccines, especially military biological defense. Demand for vaccines.
In this study, the nucleic acid sequence encoding 144 amino acids of hepatitis B virus core antigen (HBcAg) was obtained by four rounds of PCR splicing and one round of PCR amplification. The correct sequencing plasmid pET21a-HBc-N144 was found in the major immunodominant region (MIR region) of HBc-N144 vaccine vector. The 2-2-2-3 loop sequence of PA domain 2, a previously identified anthrax protective antigen, was inserted between 78-79 amino acids. This sequence is believed to be involved in mediating the entry of anthrax lethal factor (LF) and edema factor (EF) into cytoplasm, binding of PA to receptors, stabilization of the stability of PA heptamer, and is considered to include one. The chimeric HBc/anthrax epitope vaccine HBc-N144-PA-loop 2 was obtained from the neutralizing epitope SFFD of three anthrax protective antigens. The binding of the antibody to the chimeric protein was verified by Western blotting. The assembly of the chimeric protein into particles was examined by transmission electron microscopy. The results of electron microscopy showed that the HBc/anthrax epitope chimeric protein with anthrax epitope could form a granular structure with a diameter of about 30 nm under electron microscopy, which was not significantly different from the granular structure formed in the natural state of HBc molecule. Immunological properties of the epitope vaccine HBc-N144-PA-loop 2. Serological tests on the immunized mice showed that HBc-N144-PA-loop 2 could stimulate specific antibodies against the protective antigen of anthrax (PA). In order to further verify the immunological properties of the obtained HBc/anthrax epitope vaccine, the model of guinea pig was more ideal for evaluation in mice. HBc/anthrax epitope vaccine HBc-N144-PA-loop 2 can not only stimulate anti-PA antibody (essentially PA epitope specific antibody), but also stimulate neutralizing antibody against anthrax toxin. In the anthrax spore attack experiment at dose of 40LD50, some protective immune guinea pigs against anthrax were found. The specific phage clones containing the neutralizing epitope "SFFD" were used as antigen to immunize mice. The production of anti-PA antibody and the neutralizing activity of toxin were not observed. In addition, the adjuvant effect of HBc was observed in guinea pig test. HBc-N144-PA-loop 2 did not add adjuvant group or make it. Almost the same level of anti-PA antibodies (i.e. epitope-specific antibodies) and neutralizing antibodies were stimulated in the adjuvant group, and 57% of the immunized guinea pigs were protected against the invasion of anthrax spores. Considering that PA is a macromolecule of 83 kDa and contains multiple epitopes, and HBc-N144-PA-loop 2 contains only one of them, it is feasible to use HBc as a vector for anthrax epitope vaccine, but the ability of HBc-N144-PA-loop 2 to stimulate immune response needs to be optimized.
In order to improve the efficacy of HBc-N144-PA-loop 2 as an anthrax epitope vaccine, the deletion mutation of 79-81 amino acids in the MIR region and the prolonged mutation of 145-149 amino acids in the carboxyl terminal of HBc molecule were carried out by reverse PCR based on the correct sequence of pET21a-HBc-N144-PA-loop 2. Variant H144DL2 and H149L2 with prolonged carboxyl terminal were obtained by removing the labels of redundant amino acids and histidines from HBc-N144-PA-loop 2. The effect of HBc modification was evaluated by detecting the changes of humoral immunity induced by modified HBc/anthrax epitope vaccine. The anti-PA antibody was increased by deletion mutation in the MIR region of HBc molecule (H144DL2), and the anti-HBc antibody was significantly decreased compared with that of HBc-N144 group, and was significantly lower than that of anti-HBc antibody stimulated by H144L2 group. In addition, the deletion mutant H144DL2 in the MIR region of HBc molecule also significantly stimulated neutral antibody second only to that of rPA group. The neutralizing antibodies were detected in all the immune sera, and the titer distribution of neutralizing antibodies was relatively concentrated among the animals, and the mice immunized with 100% protection were attacked by anthrax lethal toxin. The prolonged HBc carboxyl terminal mutant H149L2 failed to increase the anti-PA antibody, and the antibody titer was lower than that of H144L2. Compared with HBc-N144 group and blank control group, the titer of antibody increased slightly, and the neutral antibody was also detected in the animal immune serum, but the antibody titer was lower than that of H144DL2 group. The death time of mice was prolonged. The results of adjuvant effect study showed that H144L2 group used aluminum adjuvant and no adjuvant relatively. Freund's adjuvant group stimulated the highest anti-PA antibody and protected 50% of the mice. The death time of the mice was prolonged from 6 hours to 10 hours. This suggested that H144DL2 without adjuvant might be a better formulation of HBc/anthrax epitope vaccine. In addition, the expression and purification process of recombinant protein was optimized to eliminate the interference of endotoxin.
ESAT-6, a tuberculosis antigen, was inserted into HBc to explore the ability of HBc to assist in enhancing specific cellular immune responses. The 288 BP ESAT-6 gene sequence was linked through a flat end ligation using plasmids PAS22-ESAT-6 and pET21a-H144L2 constructed in our laboratory as templates. A new type of tuberculosis subunit vaccine HE6 with HBc as vaccine carrier was obtained by introducing 78-79 amino acids into HBc. The binding of recombinant protein HE6 to antibody was detected by WB, and the formation of granules was verified by negative staining and transmission electron microscopy. Whether the immune response of T cells, especially CD4+Th1 cells, can be effectively stimulated is the key to evaluate the efficacy of the TB vaccine. The immunological properties of HE6 were evaluated in a mouse model. Both groups could stimulate ESAT-6-specific IgG2a and IgG1 with high titer, and the antibody titer was significantly higher than that of the group immunized with rESAT-6 and aluminum adjuvant alone. The IgG2a titer of the group immunized with rESAT-6 and aluminum adjuvant was the lowest, only about 2.81 *102, which suggested that the group immunized with rESAT-6 and aluminum adjuvant alone could not stimulate Th1 type reaction well. However, the antibody titers of IgG2a stimulated by HE6 and non-aluminum adjuvant groups exceeded 103, but there was no significant difference in the ratio of IgG2a to IgG1. The results of ELISPOT in HE6 group and non-aluminum adjuvant group were significantly higher than those in blank group and ESAT-6 group, indicating that HBc significantly increased the number of IFN-gamma secreting T cells. Flow cytometry showed that the frequencies of rESAT-6-specific IFN-gamma+CD4+ and IFN-gamma+CD8+T cells in HE6-free group were significantly higher than those in rESAT-6-alone immunized group and blank group. The frequencies of ESAT-6-specific IFN-gamma+CD4+ and IFN-gamma+CD8+T cells in HE6-free group were relatively blank. There was no significant difference in the frequency of ESAT-6-specific IFN-gamma+CD4+ and IFN-gamma+CD8+T cells between the HE6-free group and the aluminium-adjuvant group. The results were consistent with the ELISPOT results, suggesting that HBc as a vaccine vector can effectively assist in enhancing the specific cellular immunity of tuberculosis antigen ESAT-6, although the vaccine structure needs to be optimized. However, HE6 is a promising candidate subunit vaccine for tuberculosis.
To sum up, a better HBc vaccine vector structure (H144D) was screened and the related preparation methods were optimized; the ability of HBc as a vaccine vector to assist in enhancing specific humoral immunity was demonstrated in mice and guinea pigs by inserting anthrax epitopes; and the ability of HBc as a vaccine vector to assist in enhancing specific humoral immunity was demonstrated in mice by inserting tuberculosis antigen. As a vaccine carrier, HBc has the ability of assisting to enhance specific cellular immunity. As a universal vaccine carrier for assisting to enhance humoral and cellular immunity, HBc has a good application prospect.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2010
【分類號】：R392

【共引文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 高美琴;炭疽桿菌eag基因的功能研究[D];華中農(nóng)業(yè)大學(xué);2009年

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本文編號：2185627