以乙型肝炎病毒核心蛋白为载体的新型疫苗研究
发布时间：2018-08-16 09:51
【摘要】： 本研究旨在利用HBc自身所具有的一系列优点,包括具有高度的免疫原性,是一种T细胞依赖性和T细胞非依赖性抗原,没有遗传限制;能形成颗粒样结构,允许外源插入,可以将上述性质传递给与之融合的外源序列,并且将这些外源序列以重复且高密度的方式展示在自身表面;可以允许对自身进行替换、添加、缺失等修饰;能在E.coli中进行大量的表达,并能快速制备等,借助以激发体液免疫为主的炭疽表位和以激发细胞免疫为主的结核抗原为模型,构建以HBc为疫苗载体的新型炭疽表位疫苗和新型结核亚单位疫苗,探讨HBc作为疫苗载体辅助增强体液和细胞免疫反应的机制,构建体液免疫和细胞免疫通用的新型疫苗及新发突发传染病病原体快速反应的载体,以更好地适应现代疫苗,尤其是军用生物防御疫苗的需求。 本研究中首先利用重叠延伸PCR的方法,通过4轮PCR拼接,1轮PCR扩增,得到了编码乙型肝炎病毒核心抗原(HBcAg)氨基端144个氨基酸的核酸序列HBc-N144。基于所得到的测序正确的质粒pET21a-HBc-N144,在HBc-N144疫苗载体主要的免疫显性区域(MIR区)的78-79位氨基酸之间插入了本室前期鉴定得到的炭疽保护性抗原PA结构域2中的2β2-2β3loop序列,这段序列被认为与介导炭疽致死因子(LF)和水肿因子(EF)进入胞质,PA与受体结合,稳定PA七聚体的稳定性等生物学作用相关,而且被认为包含一个炭疽保护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技术验证了抗体与这种嵌合蛋白的结合,通过透射电子显微镜技术检测了这种嵌合蛋白装配成颗粒的情况。负染和透射电子显微镜的结果表明,携带有炭疽表位的HBc/炭疽表位嵌合蛋白可以形成电子显微镜下可见的直径约为30nm的颗粒样结构,这与HBc分子天然状态下形成的颗粒结构在形态学上没有明显差异。此后,在动物模型上验证了这种HBc/炭疽表位疫苗HBc-N144-PA-loop2的免疫学性质。免疫小鼠模型上的血清学检测结果表明HBc-N144-PA-loop2能激发针对炭疽保护性抗原(PA)的特异性抗体。为了进一步验证所获得的HBc/炭疽表位疫苗的免疫学性质,在相对小鼠更为理想的评价模型,豚鼠模型上检测了特异性抗体的消长情况,发现HBc/炭疽表位疫苗HBc-N144-PA-loop2不仅可以激发抗PA抗体(实质上为PA表位特异性抗体),也能激发针对炭疽毒素的中和性抗体,并且在40LD50剂量的炭疽芽胞攻毒实验中,部分保护免疫的豚鼠对抗炭疽芽胞的侵袭。而之前本室用含有此中和性表位“SFFD”的特异性噬菌体克隆作为抗原免疫小鼠,仅观察到了抗PA抗体的产生,未观察到明显的毒素中和活性。此外,在豚鼠试验中也观察到了HBc一定的佐剂效应,HBc-N144-PA-loop2不加佐剂组和使用氢氧化铝佐剂组可以激发几乎一样高的抗PA抗体(即表位特异性抗体)和中和性抗体,并且能保护57%的免疫豚鼠对抗炭疽芽胞的侵袭。相对于重组PA(rPA)激发的免疫效果而言,HBc-N144-PA-loop2组在抗PA抗体、中和性抗体、保护率等方面均略低,考虑到PA是83kDa的大分子,包含多个表位,而HBc-N144-PA-loop2仅含有其中的一个表位,用HBc作为炭疽表位疫苗的载体是可行的,但HBc-N144-PA-loop2激发免疫反应的能力还需优化。 为提高HBc-N144-PA-loop2作为炭疽表位疫苗的效力,本研究以序列正确的pET21a-HBc-N144-PA-loop2为基础,通过反向PCR技术对HBc载体进行了MIR区79-81位氨基酸的缺失突变和添加天然HBc羧基端145-149位氨基酸的延长突变,得到了HBc分子MIR区的缺失突变体H144DL2和羧基端延长突变体H149L2,并去掉了HBc-N144-PA-loop2包含的多余氨基酸和组氨酸标签,得到H144L2;通过检测改造后的HBc/炭疽表位疫苗激发体液免疫的各项指标的变化,评价了HBc的改造效果。研究发现经突变的嵌合蛋白仍可以形成电镜下可见的颗粒状结构;HBc分子MIR区的缺失突变(H144DL2)可以使抗PA抗体有所增加,抗HBc抗体较HBc-N144组显著下降,并显著低于H144L2组激发的抗HBc抗体;此外HBc分子MIR区的缺失突变体H144DL2还显著激发了仅次于rPA组的中和性抗体,并且组内所有动物的免疫血清都能被检测到中和性抗体,各动物体间中和性抗体的滴度值分布也相对集中,并且能100%保护免疫的小鼠对抗炭疽致死毒素的攻击。HBc羧基端的延长突变体H149L2未能增加抗PA抗体,抗体效价低于H144L2的水平;抗HBc抗体的滴度稍有增加;相对HBc-N144组和空白对照组可显著激发中和性抗体,组内动物免疫血清中也均被检测到了中和性抗体,但抗体滴度低于H144DL2组;小鼠的死亡时间延长。佐剂效应的研究结果表明,H144L2不加佐剂组相对使用铝佐剂和弗氏佐剂组激发了最高的抗PA抗体,能保护50%的小鼠,小鼠的死亡时间由攻毒后6h延长到了10h,这提示,H144DL2不加佐剂可能是一个较好的HBc/炭疽表位疫苗剂型。此外,本部分还优化了重组蛋白的表达纯化工艺,排除了内毒素的干扰。 本研究还在HBc中插入结核抗原ESAT-6,初步探讨了HBc辅助增强特异性细胞免疫反应的能力。以本室构建的携带有ESAT-6的质粒PAS22-ESAT-6和pET21a-H144L2为模板,借助HBc分子核酸序列中的MscⅠ酶切位点,将全长288bp的ESAT-6基因序列通过平端连接引入到HBc的78-79位氨基酸之间,得到以HBc为疫苗载体的新型结核亚单位疫苗HE6。通过WB检测了重组蛋白HE6与抗体的结合,通过负染和透射电镜技术验证了颗粒的形成。由于机体抗结核杆菌的感染主要以T细胞介导的细胞免疫为主,所以能否有效地激发机体的T细胞免疫,尤其是CD4+Th1型细胞的免疫反应是评价结核疫苗效力的关键。本研究在小鼠模型上评价了HE6的免疫学性质。通过对小鼠的特异性免疫反应进行检测,发现所构建的HBc/结核亚单位疫苗HE6使用和不使用佐剂组均可激发高效价的ESAT-6特异的IgG2a和IgG1,而且抗体的滴度水平均显著高于单独使用rESAT-6加铝佐剂免疫组。rESAT-6加铝佐剂免疫组激发的IgG2a效价最低,仅约为2.81×102,这提示单独使用rESAT-6加铝佐剂免疫组不能很好地激发Th1型反应。虽然HE6使用和不使用铝佐剂组激发的IgG2a的抗体滴度均超过103,但IgG2a/IgG1比值无显著性差异。HE6使用和不使用铝佐剂组的ELISPOT结果显著高于空白组和ESAT-6单独加铝佐剂免疫组,表明HBc的使用显著增加了分泌IFN-γ的T细胞数目,提示HBc的使用能有助于增强Th1型反应。流式的结果表明,HE6不加佐剂组中rESAT-6特异的IFN-γ+CD4+和IFN-γ+CD8+T细胞的频率相对rESAT-6单独加铝佐剂免疫组和空白组有显著增加,HE6使用铝佐剂组中ESAT-6特异的IFN-γ+CD4+和IFN-γ+CD8+T细胞的频率相对空白组有显著增加,HE6不加佐剂组和使用铝佐剂组中ESAT-6特异的IFN-γ+CD4+和IFN-γ+CD8+T细胞的频率没有显著性差异。此结果与ELISPOT结果一致,这提示,HBc作为疫苗载体能有效地辅助增强结核抗原ESAT-6特异的细胞免疫,虽然疫苗结构还有待优化,但HE6是一种有前景的候选结核亚单位疫苗。 综上所述,本研究筛选到了一个较优的HBc疫苗载体结构(H144D),优化了相关的制备方法;通过插入炭疽表位,在小鼠和豚鼠模型上证明了HBc作为疫苗载体具有辅助增强特异性体液免疫的能力;通过插入结核抗原,在小鼠模型上证明了HBc作为疫苗载体具有辅助增强特异性细胞免疫的能力;HBc作为一种辅助增强体液和细胞免疫通用的疫苗载体,有良好的应用前景。
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
本文编号：2185627
[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.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392
【共引文献】
相关硕士学位论文 前1条
1 高美琴;炭疽杆菌eag基因的功能研究[D];华中农业大学;2009年
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