甲型副傷寒沙門氏菌膜影的免疫保護性研究
發(fā)布時間：2018-09-01 14:55
【摘要】：甲型副傷寒是一種嚴重的侵襲性胞內(nèi)感染疾病,主要通過糞-口途徑傳播。近年來,我國甲型副傷寒的發(fā)生率呈逐年上升的趨勢,隨著臨床耐藥株及多重耐藥株的出現(xiàn),使臨床治療更加困難。目前還沒有真正安全有效的甲型副傷寒疫苗。傳統(tǒng)的三聯(lián)滅活疫苗雖然有效,但提供的有效免疫反應持續(xù)時間短且具有副作用；目前研發(fā)的甲型副傷寒疫苗多為減毒活疫苗或亞單位疫苗,減毒活疫苗雖然免疫原性高但具有回復突變的危險,亞單位疫苗通常免疫原性較低,需要一種合適的佐劑共同使用,而且生產(chǎn)成本高。細菌膜影(Bacterial Ghost, BG)由噬菌體PhiX174E蛋白裂解革蘭氏陰性細菌而產(chǎn)生,與活菌具有相同的功能性抗原表位,不但具有佐劑活性,而且能同時誘導全身、粘膜、細胞免疫反應。本課題嘗試構(gòu)建了甲型副傷寒沙門氏菌(Salmonella paratyphi A, S. paratyphi A)BG,并對其進行相應生物學特性研究,通過Balb/c小鼠模型檢測S.paratyphi A BG的免疫原性與免疫保護性。 本文通過PCR擴增,意外獲得包括噬菌體PhiX174裂解蛋白E基因5’端75bp及7個功能未知氨基酸序列的片段(Em),構(gòu)建pBV220-Em溫控型表達載體,電轉(zhuǎn)S. paratyphi A CMCC50973,通過熱誘導獲得S. paratyphi A BG。探索誘導后生長曲線及裂解率,透射電鏡(Transmission Electron Microscope, TEM)觀察BG形態(tài)。為測定S. paratyphi A BG的免疫原性,將S. paratyphi A滅活疫苗作為陽性對照及PBS作為陰性對照,在0、2、4周通過口服或皮下注射進行免疫,每次免疫前一天對小鼠尾靜脈取血并分離血清,全菌ELISA檢測小鼠不同時間血清抗原特異性IgG抗體滴度。第三次免疫后第10天,每組隨機挑取5只小鼠處死、取脾、分離脾淋巴細胞,ELISPOT檢測IL-4、IFN-y分泌細胞情況。為測定S. paratyphi A BG的免疫保護性,首先測定最低絕對致死劑量(100%minimal lethal dose, MLD)以確定攻毒劑量,于第三次免疫后第10天,將每組剩余的5只小鼠用2倍MLD S. paratyphi A進行腹腔注射攻毒,攻毒后一周,處死存活小鼠,無菌條件下取脾、肝、肺等器官并研磨,涂布無抗性平板,評價單位質(zhì)量器官上S. paratyphi A活菌量,并用特異性S. paratyphi A噬菌體鑒定分離到的細菌,最后全菌ELISA檢測攻毒后小鼠血清抗原特異性IgG抗體滴度。 研究結(jié)果顯示,當pBV220-Em/S.paratyphi A初始OD600值低于0.7時,誘導后細菌OD600值在3小時內(nèi)基本保持不變；當初始OD600值高于0.7時,誘導后細菌OD600值會持續(xù)升高直到穩(wěn)定期。TEM觀察BG,發(fā)現(xiàn)熱誘導后pBV220-Em/S.paratyphi A中部或兩極有胞質(zhì)內(nèi)溶物外排的跡象。三免后小鼠血清抗原特異性IgG幾何平均抗體滴度結(jié)果顯示：口服免疫BG組為7.76×104,口服免疫滅活疫苗組為2.56×104；皮下注射BG組為1.08×106,皮下注射滅活疫苗組為1.24×106。小鼠脾淋巴細胞ELISPOT檢測結(jié)果表明,口服、皮下注射BG或滅活疫苗組,脾臟IL-4分泌細胞數(shù)均高于IFN-y分泌細胞數(shù)。S. paratyphi A關(guān)于Balb/c小鼠的MLD為5×105個,所以免疫組小鼠攻毒劑量確定為1×106個。免疫保護性檢測結(jié)果顯示,口服BG能提供20%的保護率,而口服滅活疫苗沒有保護效果,皮下注射的BG或滅活疫苗均能提供100%的保護效果。攻毒后的小鼠各臟器中,S. paratyphi A菌量分布情況為：肝臟脾臟肺臟。噬菌體鑒定結(jié)果表明,攻毒后小鼠各臟器所分布菌極大多數(shù)是S. paratyphi A。較第三次免疫,攻毒后小鼠血清特異性IgG抗體滴度約提高2-8倍。 研究結(jié)果表明,本研究成功構(gòu)建的S. paratyphi A BG,免疫原性強,免疫形式主要為體液免疫,皮下注射后能提供很強的保護效果。雖然BG能提供較強的免疫保護力,然而攻毒后小鼠各臟器還是存在一定量S. paratyphi A,這可能與BG誘導的免疫形式及S. paratyphi A的侵襲機制有關(guān),但這需要做進一步的研究�？傊�,本文構(gòu)建的S. paratyphi A BG,具有較好的免疫原性及較強免疫保護性,為S. paratyphi A BG疫苗研制奠定基礎(chǔ)。
[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.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392
本文編號：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.
【學位授予單位】：昆明理工大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R392
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