甲型副伤寒沙门氏菌膜影的免疫保护性研究
发布时间：2018-09-01 14:55
【摘要】：甲型副伤寒是一种严重的侵袭性胞内感染疾病,主要通过粪-口途径传播。近年来,我国甲型副伤寒的发生率呈逐年上升的趋势,随着临床耐药株及多重耐药株的出现,使临床治疗更加困难。目前还没有真正安全有效的甲型副伤寒疫苗。传统的三联灭活疫苗虽然有效,但提供的有效免疫反应持续时间短且具有副作用；目前研发的甲型副伤寒疫苗多为减毒活疫苗或亚单位疫苗,减毒活疫苗虽然免疫原性高但具有回复突变的危险,亚单位疫苗通常免疫原性较低,需要一种合适的佐剂共同使用,而且生产成本高。细菌膜影(Bacterial Ghost, BG)由噬菌体PhiX174E蛋白裂解革兰氏阴性细菌而产生,与活菌具有相同的功能性抗原表位,不但具有佐剂活性,而且能同时诱导全身、粘膜、细胞免疫反应。本课题尝试构建了甲型副伤寒沙门氏菌(Salmonella paratyphi A, S. paratyphi A)BG,并对其进行相应生物学特性研究,通过Balb/c小鼠模型检测S.paratyphi A BG的免疫原性与免疫保护性。 本文通过PCR扩增,意外获得包括噬菌体PhiX174裂解蛋白E基因5’端75bp及7个功能未知氨基酸序列的片段(Em),构建pBV220-Em温控型表达载体,电转S. paratyphi A CMCC50973,通过热诱导获得S. paratyphi A BG。探索诱导后生长曲线及裂解率,透射电镜(Transmission Electron Microscope, TEM)观察BG形态。为测定S. paratyphi A BG的免疫原性,将S. paratyphi A灭活疫苗作为阳性对照及PBS作为阴性对照,在0、2、4周通过口服或皮下注射进行免疫,每次免疫前一天对小鼠尾静脉取血并分离血清,全菌ELISA检测小鼠不同时间血清抗原特异性IgG抗体滴度。第三次免疫后第10天,每组随机挑取5只小鼠处死、取脾、分离脾淋巴细胞,ELISPOT检测IL-4、IFN-y分泌细胞情况。为测定S. paratyphi A BG的免疫保护性,首先测定最低绝对致死剂量(100%minimal lethal dose, MLD)以确定攻毒剂量,于第三次免疫后第10天,将每组剩余的5只小鼠用2倍MLD S. paratyphi A进行腹腔注射攻毒,攻毒后一周,处死存活小鼠,无菌条件下取脾、肝、肺等器官并研磨,涂布无抗性平板,评价单位质量器官上S. paratyphi A活菌量,并用特异性S. paratyphi A噬菌体鉴定分离到的细菌,最后全菌ELISA检测攻毒后小鼠血清抗原特异性IgG抗体滴度。 研究结果显示,当pBV220-Em/S.paratyphi A初始OD600值低于0.7时,诱导后细菌OD600值在3小时内基本保持不变；当初始OD600值高于0.7时,诱导后细菌OD600值会持续升高直到稳定期。TEM观察BG,发现热诱导后pBV220-Em/S.paratyphi A中部或两极有胞质内溶物外排的迹象。三免后小鼠血清抗原特异性IgG几何平均抗体滴度结果显示：口服免疫BG组为7.76×104,口服免疫灭活疫苗组为2.56×104；皮下注射BG组为1.08×106,皮下注射灭活疫苗组为1.24×106。小鼠脾淋巴细胞ELISPOT检测结果表明,口服、皮下注射BG或灭活疫苗组,脾脏IL-4分泌细胞数均高于IFN-y分泌细胞数。S. paratyphi A关于Balb/c小鼠的MLD为5×105个,所以免疫组小鼠攻毒剂量确定为1×106个。免疫保护性检测结果显示,口服BG能提供20%的保护率,而口服灭活疫苗没有保护效果,皮下注射的BG或灭活疫苗均能提供100%的保护效果。攻毒后的小鼠各脏器中,S. paratyphi A菌量分布情况为：肝脏脾脏肺脏。噬菌体鉴定结果表明,攻毒后小鼠各脏器所分布菌极大多数是S. paratyphi A。较第三次免疫,攻毒后小鼠血清特异性IgG抗体滴度约提高2-8倍。 研究结果表明,本研究成功构建的S. paratyphi A BG,免疫原性强,免疫形式主要为体液免疫,皮下注射后能提供很强的保护效果。虽然BG能提供较强的免疫保护力,然而攻毒后小鼠各脏器还是存在一定量S. paratyphi A,这可能与BG诱导的免疫形式及S. paratyphi A的侵袭机制有关,但这需要做进一步的研究。总之,本文构建的S. paratyphi A BG,具有较好的免疫原性及较强免疫保护性,为S. paratyphi A BG疫苗研制奠定基础。
[Abstract]:Paratyphoid A is a serious invasive intracellular infectious disease, mainly transmitted through fecal-oral route. In recent years, the incidence of paratyphoid A in China is increasing year by year. With the emergence of clinical drug-resistant strains and multi-drug-resistant strains, clinical treatment is more difficult. Although the triple inactivated vaccine is effective, the effective immune response provided by the vaccine is short and has side effects; most of the paratyphoid A vaccines developed at present are live attenuated or subunit vaccines. Although the attenuated live vaccine has high immunogenicity, it has the risk of mutagenesis, and the subunit vaccines usually have low immunogenicity and need a kind of vaccine. Bacterial Ghost (BG) is produced by the cleavage of Gram-negative bacteria by phage PhiX174E protein. It has the same functional antigen epitope as living bacteria. It not only has adjuvant activity, but also can induce systemic, mucosal and cellular immune responses. Salmonella paratyphi A (S. paratyphi A) BG and its corresponding biological characteristics were studied. The immunogenicity and immune protection of S. paratyphi A BG were detected by Balb/c mouse model.
In this paper, 75 BP fragments of 5'-terminal of PhiX174 cleavage protein E gene and 7 unknown amino acid sequences (Ems) were accidentally obtained by PCR amplification. Temperature-controlled expression vector pBV220-Em was constructed and S. paratyphi A CMCC50973 was electrotransformed. S. paratyphi A BG was obtained by thermal induction. To determine the immunogenicity of S. paratyphi A BG, S. paratyphi A inactivated vaccine was used as positive control and PBS as negative control. The mice were immunized by oral or subcutaneous injection at 0, 2, 4 weeks. The blood was taken from the tail vein one day before each immunization and the serum was separated. The whole bacteria ELISA was used to detect the mice. On the 10th day after the third immunization, 5 mice in each group were randomly selected and killed. Spleen and spleen lymphocytes were isolated. IL-4 and IFN-y secreting cells were detected by ELISPOT. To determine the immune protection of S. paratyphi A BG, the lowest absolute lethal dose (100% minimal lethal dose, MLD) was determined. Five remaining mice in each group were injected intraperitoneally with twice MLD S. paratyphi A on the 10th day after the third immunization. The surviving mice were sacrificed one week after the treatment. The spleen, liver, lung and other organs were harvested under aseptic conditions and ground. The S. paratyphi A viable quantity per unit mass organ was evaluated with the specific S. P. The isolated bacteria were identified by aratyphi A phage and the titer of antigen specific IgG antibody in serum of mice was detected by whole-bacterial ELISA.
The results showed that when the initial OD600 value of pBV220-Em/S.paratyphi A was lower than 0.7, the bacterial OD600 value remained basically unchanged for three hours after induction; when the initial OD600 value was higher than 0.7, the bacterial OD600 value would continue to increase until the stable period. TEM observation of BG showed that there were intracytoplasmic solutes in the middle or both poles of pBV220-Em/S.paratyphi A after thermal induction. The results of geometric mean antibody titer of serum antigen-specific IgG in mice after three immunizations showed that: oral immunization BG group was 7.76 *104, oral inactivated immunization vaccine group was 2.56 *104, subcutaneous injection BG group was 1.08 *106, subcutaneous injection inactivated vaccine group was 1.24 *106. The number of IL-4 secreting cells in spleen was higher than that in IFN-y secreting cells in BG group or inactivated vaccine group. The distribution of S. paratyphi A in the organs of mice after exposure to the virus was as follows: liver, spleen and lung. The results of phage identification showed that most of the bacteria distributed in the organs of mice after exposure to the virus were S. paratyphi A. Compared with the third immunization, the serum specific IgG antibody of mice after exposure to the virus was dripped. The increase is about 2-8 times.
The results showed that the S. paratyphi A BG constructed successfully in this study had strong immunogenicity, and the immune form was mainly humoral immunity, which could provide strong protective effect after subcutaneous injection. In short, the S. paratyphi A BG constructed in this paper has good immunogenicity and strong immune protection, which lays a foundation for the development of S. paratyphi A BG vaccine.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
本文编号：2217539
[Abstract]:Paratyphoid A is a serious invasive intracellular infectious disease, mainly transmitted through fecal-oral route. In recent years, the incidence of paratyphoid A in China is increasing year by year. With the emergence of clinical drug-resistant strains and multi-drug-resistant strains, clinical treatment is more difficult. Although the triple inactivated vaccine is effective, the effective immune response provided by the vaccine is short and has side effects; most of the paratyphoid A vaccines developed at present are live attenuated or subunit vaccines. Although the attenuated live vaccine has high immunogenicity, it has the risk of mutagenesis, and the subunit vaccines usually have low immunogenicity and need a kind of vaccine. Bacterial Ghost (BG) is produced by the cleavage of Gram-negative bacteria by phage PhiX174E protein. It has the same functional antigen epitope as living bacteria. It not only has adjuvant activity, but also can induce systemic, mucosal and cellular immune responses. Salmonella paratyphi A (S. paratyphi A) BG and its corresponding biological characteristics were studied. The immunogenicity and immune protection of S. paratyphi A BG were detected by Balb/c mouse model.
In this paper, 75 BP fragments of 5'-terminal of PhiX174 cleavage protein E gene and 7 unknown amino acid sequences (Ems) were accidentally obtained by PCR amplification. Temperature-controlled expression vector pBV220-Em was constructed and S. paratyphi A CMCC50973 was electrotransformed. S. paratyphi A BG was obtained by thermal induction. To determine the immunogenicity of S. paratyphi A BG, S. paratyphi A inactivated vaccine was used as positive control and PBS as negative control. The mice were immunized by oral or subcutaneous injection at 0, 2, 4 weeks. The blood was taken from the tail vein one day before each immunization and the serum was separated. The whole bacteria ELISA was used to detect the mice. On the 10th day after the third immunization, 5 mice in each group were randomly selected and killed. Spleen and spleen lymphocytes were isolated. IL-4 and IFN-y secreting cells were detected by ELISPOT. To determine the immune protection of S. paratyphi A BG, the lowest absolute lethal dose (100% minimal lethal dose, MLD) was determined. Five remaining mice in each group were injected intraperitoneally with twice MLD S. paratyphi A on the 10th day after the third immunization. The surviving mice were sacrificed one week after the treatment. The spleen, liver, lung and other organs were harvested under aseptic conditions and ground. The S. paratyphi A viable quantity per unit mass organ was evaluated with the specific S. P. The isolated bacteria were identified by aratyphi A phage and the titer of antigen specific IgG antibody in serum of mice was detected by whole-bacterial ELISA.
The results showed that when the initial OD600 value of pBV220-Em/S.paratyphi A was lower than 0.7, the bacterial OD600 value remained basically unchanged for three hours after induction; when the initial OD600 value was higher than 0.7, the bacterial OD600 value would continue to increase until the stable period. TEM observation of BG showed that there were intracytoplasmic solutes in the middle or both poles of pBV220-Em/S.paratyphi A after thermal induction. The results of geometric mean antibody titer of serum antigen-specific IgG in mice after three immunizations showed that: oral immunization BG group was 7.76 *104, oral inactivated immunization vaccine group was 2.56 *104, subcutaneous injection BG group was 1.08 *106, subcutaneous injection inactivated vaccine group was 1.24 *106. The number of IL-4 secreting cells in spleen was higher than that in IFN-y secreting cells in BG group or inactivated vaccine group. The distribution of S. paratyphi A in the organs of mice after exposure to the virus was as follows: liver, spleen and lung. The results of phage identification showed that most of the bacteria distributed in the organs of mice after exposure to the virus were S. paratyphi A. Compared with the third immunization, the serum specific IgG antibody of mice after exposure to the virus was dripped. The increase is about 2-8 times.
The results showed that the S. paratyphi A BG constructed successfully in this study had strong immunogenicity, and the immune form was mainly humoral immunity, which could provide strong protective effect after subcutaneous injection. In short, the S. paratyphi A BG constructed in this paper has good immunogenicity and strong immune protection, which lays a foundation for the development of S. paratyphi A BG vaccine.
【学位授予单位】：昆明理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392
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