【作者】 张虎成；

【导师】 陈薇；

【作者基本信息】 中国人民解放军军事医学科学院 ， 微生物学， 2007， 博士

【摘要】 乳酸菌（Lactic Acid Bacteria，LAB）是一类能利用碳水化合物产生大量乳酸的革兰氏阳性细菌的通称，广泛存在于自然界中，被公认为是安全的食品级（generally regarded as safe，GRAS）微生物。黏膜系统是人体免疫系统的重要组成部分，利用细菌载体接种黏膜表面诱导黏膜和系统水平的有效免疫应答，是有效预防从黏膜入侵传染因子的方法之一。减毒活菌疫苗不适用于所有人群，并有恢复毒力的危险；而用乳酸菌作为活菌疫苗载体能有效的避免以上问题。利用乳酸菌作为活菌疫苗载体能顺利通过胃到达肠黏膜，刺激机体产生免疫反应，有望成为新型疫苗。在乳酸菌中，可以作为活菌疫苗载体有乳杆菌（Lactobacillus）和乳球菌（Lactococcus）。用乳杆菌作为活菌载体表达外源抗原时，可能会产生免疫耐受。而乳球菌不是人体肠道的正常菌，不会产生免疫耐受。国内外有用乳酸菌进行重组蛋白表达和活菌疫苗研究的报道，但缺乏系统研究。国内相关研究较少。用增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）作为报告基因，比较了两种启动子的启动效果。酸诱导启动子P170受到自身代谢产生的H⁺和生长量的诱导，即在pH低于6.5和在稳定期时诱导表达，不需要额外加入诱导物，宿主菌的菌体生长期和表达蛋白的生产期分开。P_nisA启动子是一个诱导效果很强的启动子，受到NisK和NisR的调控。nisin是一种安全的食品添加剂，其作为诱导物时的浓度在一定范围内与外源蛋白表达量呈正比例关系，容易控制。分别将egfp放在P170和P_nisA启动子后构建胞内和胞外表达载体，电转化入乳酸乳球菌（Lc．lactis）中，用SDS-PAGE检测及荧光显微镜观察发现无论是胞内表达还是胞外表达，P_nisA启动子的启动效果均强于P170启动效果。将pa置于P_nisA启动子后构建胞内和胞外表达载体，电转化入Lc．lactis1.2030中。用ELISA检测到PA的胞外表达情况，在培养12小时后检测到PA的表达，在16小时后趋于稳定；Western Blotting检测到胞内PA的表达，用SDS-PAGE检测PA随时间变化的关系，加入诱导物后4小时有表达，在16小时候趋于稳定。胞内表达效果较胞外表达效果好。同时构建了在PA的N端和C端含6个His-tag的表达载体。为了避免抗性基因在体内的转移和质粒的不稳定行，利用组氨醇磷酸化酶基因（hisB）作为同源交换序列，以pMD18-T为基础构建了在Lc．lactis中的自杀型整合载体pHEC。随后将P_nisA启动子和pa插入hisB正中间构建了含pa的自杀整合型载体pHEC-Pnis-PA，不含分泌信号肽基因，表达的PA不分泌到胞外。随后筛选得到了单交换突变体Lc．lactis1.2030S和双交换突变体Lc．lactis1.2030D，用SDS-PAGE检测有PA的表达。同样的用乳杆菌表达载体在植物乳杆菌（Lb．plantarum）中表达了N端含有6个his标签的PA。实验结果证实了应用hisB作为同源交换序列将外源基因整合到Lc．lactis染色体DNA的方法构建活菌载体疫苗是可行的。证明了构建的载体在Lc．lactis和Lb．plantarum中表达是可以接受的。以乳酸菌为活菌疫苗载体的策略为抗原在体内的运输提供一种新的途径。更多还原

【Abstract】 Lactic acid bacteria （LAB）, a group of G⁺ bacteria that can ferment carbohydrates to produce large quantities of lactic, are considered GRAS （Generally Regarded As Safe） organisms that could safely be used for the expression of foreign proteins. Mucosa system plays an important role in whole immune responses of our body. Most pathogens enter the body through mucosal surfaces, and the development of vaccine protective at such sites should be a very effective means. The attenuated pathogenic vectors may impair their immunogenicity and raises questions about the safety. Probiotics, such as Lactobacillus and Lactococcus, offer an original alternative as antigen delivery vehicles. Administered orally or nasally route, it would not necessitate the professional health care infrastructure required for injectable preparations. LAB, promising new general vaccine vector, survive the low pH in stomach and entry into intestines mucosa. Lc. lactis has been used successfully to induce secretary and protective systemic responses against pathogen orally or nasally in some reports. As commensal bacteria, some Lactobacillus strains maybe cause immune tolerance.The enhanced green fluorescent protein （EGFP） as a reporter was expressed in different vectors induced by two promoters in Lc. lactis, H⁺ induced promoter P170 and nisin-induced promoter P_nisA. P170 is strongly activated at pH below 6.5 in the transition to stationary phase, without addiing an exogenous inducer. The growth phase is separated from the protein production phase. P_nisA is strongly induced by nisin, widely used as food preservative because of its broad host spectrum. P_nisA is regulated by a phosphorylase NisK and a response regulator NisR. Studies with increasing the concentration of nisin showed a linear dose-curve of foreign protein expressed. The gene of egfp was placed behind the two inducible promoters P170 and P_nisA in intracellular or extracellular vectors. The results showed that the expression of EGFP can be induced by addition of the amounts of nisin （10 ng/ml） to the culture medium, not so efficient expression induced by P170 intracellularly or extracellularly. After the gene of protective antigen （PA） of B. anthracis pa was placed behind the promoter P_nisA, the expression of PA was obtained both intracellular and extracellular by the same methods. By enzyme-linked immunosorbent assay （ELISA） we found that PA was expressed extracellularly after 12 hours culture and no more expressed after 16 hours. Using Western Blotting （WB）, we found PA was expressed intracellulary. SDS-PAGE showed that PA was expressed after 4 hours induced by nisin added, and after 16 hours induced, PA was expressed no more than before.In order to avoid the erythromycin resistance gene {ery) and and lose of plasmid, a suicide crossover vector based on pMD18-T was constructed. Because the histidinol phosphate phosphatase is coded by hisB and one gene located elsewhere in the Lc. lactis, hisB is used as a homologous crossover sequence for recombination in the genome of Lc. lactis. As pMD18-T doesn’t contain an origin of replication in Lc. lactis, it and its derivates are suicide vectors in Lc. lactis. After hisB and ery were inserted in pMD18-T, named pHEC, followed digested by SnaB I, PnisA - PA was ligased to the pHEC, named pHEC- PnisA- PA, which is suicide vector without signal peptide for Lc. lactis. Single crossover mutant Lc. lactis1 .2030S and double crossover mutant Lc. lactis1. 2030D were obtained, and SDS-PAGE showed the expression of PA. The expression vectors of PA with 6 His-tag on the N or C point for Lc. lactis1. 2030 were botained, and PA with 6 His-tag on the N point was expressed by pSIP300-PA-His in Lb. plantarum.The result verified that construction of live bacteria vaccine using the vector pHEC and hisB as homologous crossover sequence was possible, and all vectors constructed in this experiment were acceptable for expression in Lc. lactis or Lb. plantarum. The strategy of developing live bacterial vaccine based on LAB provided a new way of the delivery of antigens in vivo.更多还原