副粘病毒Tianjin株缺损性干扰颗粒用作新型生物佐剂的初步研究

发布时间：2019-01-11 18:57

【摘要】：目的新型疫苗如基因工程多肽或亚单位疫苗和合成肽疫苗等的弱免疫原性使得佐剂在疫苗研制中的应用越来越广泛,并随着疫苗推广应用到其他领域如抗感染,抗肿瘤的辅助治疗。因此寻求理想的佐剂对于疫苗的研制发展有着关键性的意义。常见的佐剂有不溶性铝盐类胶体、油水乳剂、微生物及其代谢产物、核酸及其类似物、细胞因子、免疫刺激复合物、蜂胶、脂质体等。缺损性干扰.(defective interfering, DI)颗粒是一种含有部分基因组的病毒颗粒,它是病毒在复制过程中发生错误而出现的,其基因组只是亚基因组片段。其自身属性决定其亚基因组中携带了众多正常病毒颗粒所没有的信息。研究发现仙台病毒DI颗粒具有强大的刺激树突状细胞(dendritic cell, DC)成熟的能力。副粘病毒Tianjin株(Paramyxovirus, Tianjin Strain)是1999年从天津医科大学动物实验中心爆发急性上呼吸道感染的普通棉耳狨猴肺脏中分离出来的毒株,经鉴定属于副粘病毒科、副粘病毒亚科、呼吸道病毒属,与仙台病毒(Sendai virus,SeV)亲缘关系最近,不属于仙台病毒现有的3个进化分支而自成独立的一支。因此本文对副粘病毒Tianjin株DI颗粒作为新型生物佐剂的可行性进行了初步研究。方法将副粘病毒Tianjin株用鸡胚培养法连续传代三次,铺设5%～45%蔗糖密度梯度超速离心分离提纯副粘病毒Tianjin株DI颗粒,通过电镜观察,体外接种敏感细胞系大鼠脑胶质瘤细胞C6株观察细胞病变,接种鸡胚收集尿囊液检测血凝素效价等对DI颗粒进行了鉴定,将脊髓灰质炎病毒(PV)疫苗株的灭活抗原与DI颗粒佐剂等量混合后制备疫苗。选取4周龄ICR小鼠,经皮下多点注射对小鼠进行免疫,观察小鼠的大体情况和免疫应答水平。并设PV灭活抗原加氢氧化铝凝胶佐剂组为阳性对照,PV灭活抗原无佐剂组以及生理盐水组为阴性对照。观察小鼠的存活情况；于免疫后两周摘眼球取血分离血清,检测免疫血清中PV特异性抗体；无菌取脾脏分离脾淋巴细胞进行体外培养,分别用MTT法进行T、B淋巴细胞增殖试验；检测脾淋巴细胞接受PV灭活抗原再次刺激后IFN-γ、IL-4的产生情况,对DI颗粒能否增强PV免疫小鼠的免疫应答以及免疫应答的类型进行评估。结果实验结果确定纯化分离出的是与完整病毒有所差别的,无复制能力的缺损性颗粒成分。动物免疫指标结果显示副粘病毒Tianjin株DI颗粒佐剂组血清特异性PV抗体水平高于阴性对照组,但低于阳性对照组,差异有统计学意义；T淋巴细胞刺激指数(SI),脾淋巴细胞分泌IFN-γ的量高于其他组,差异有统计学意义,而B淋巴细胞SI和脾淋巴细胞分泌IL-4的量与其他组相比差异没有统计学意义。结论实验证实副粘病毒Tianjin株DI颗粒具有一定的增强机体免疫应答能力的作用,且以诱导Thl型细胞免疫应答为主。提示副粘病毒Tianjin株DI颗粒具有佐剂活性,具有成为生物佐剂的潜在可能,为下一步的研究提供了一定的实验和理论依据。
[Abstract]:The weak immunogenicity of a new vaccine, such as a genetic engineering polypeptide or a subunit vaccine and a synthetic peptide vaccine, makes the application of the adjuvant in vaccine development more and more extensive, and as the vaccine is applied to other fields such as anti-infection and anti-tumor auxiliary treatment. Therefore, it is of great significance to seek the ideal adjuvant for the development and development of the vaccine. Common adjuvants include insoluble aluminum salt colloids, oil-water emulsions, microorganisms and their metabolites, nucleic acids and the like, cytokines, immunostimulating complexes, propolis, liposomes, and the like. Defect interference. The (DI) particles are a part of the genome-containing virus particles, which are caused by an error in the replication process, the genome of which is just a sub-genomic fragment. Its own properties determine that its subgenome carries information that is not available for many of the normal virus particles. The study found that the Sendai virus DI particles have a powerful ability to stimulate the maturation of dendritic cells (DC). Paramyxovirus, Tianyin Strain (Paramyxovirus, Tianchin Strain), a strain isolated from the lung of the common cotton ear of the acute upper respiratory tract infection from the animal experiment center of Tianjin Medical University in 1999, is identified as the Paramyxoviridae, Paramyxoviridae, Respiratory virus, and Sendai virus. SeV) is related recently and is not an independent branch of the existing three evolutionary branches of the Sendai virus. Therefore, a preliminary study of the feasibility of the secondary amyxovirus Tianjin strain DI particle as a new type of bio-adjuvant is studied in this paper. Methods The paramyxovirus Tianjin strain was subcultured in chicken embryo culture method for three times, and 5% ~ 45% sucrose density gradient ultracentrifugation was used to separate and purify the DI particles of the paramyxovirus Tianjin strain. The method comprises the following steps of: carrying out identification on the DI particles by using a chicken embryo collecting allantoic liquid to detect the titer of the hemagglutinin and the like, and mixing the inactivated antigen of the poliovirus (PV) vaccine strain with the DI particle adjuvant to prepare the plague 4-week-old ICR mice were selected, and the mice were immunized with subcutaneous multi-point injection, and the general condition and immune response water of the mice were observed. and the PV inactivated antigen and the aluminum hydroxide gel adjuvant group were set to be positive control, and the PV inactivated antigen non-adjuvant group and the normal saline group were negative pairs. The survival of the mice was observed. The blood was isolated from the eyeball for two weeks after the immunization, and the PV-specific antibody in the immune serum was detected; the spleen was isolated from the spleen, and the spleen was isolated and cultured in vitro, and the proliferation of T and B lymphocytes was performed with the MTT method, respectively. The results showed that the immune response of the PV-immunized mice and the type of the immune response were evaluated by detecting the production of the IFN-1 and IL-4 after the splenocytes were re-stimulated by the PV-inactivated antigen. As a result, the results of the experiment confirm that the purification and separation are different from the complete virus, and there is no defective one with no replication capacity. The results of animal immunization showed that the level of serum-specific PV antibody in the adjuvant group of Tianjin strain in the paramyxovirus was higher than that of the negative control group, but lower than that of the positive control group, the difference was of statistical significance, and the T-lymphocyte stimulation index (SI) and the amount of IFN-2 secreted by the spleen lymphocytes were higher than that of the negative control group. In other groups, the difference was statistically significant, while the amount of IL-4 secreted by the B-lymphocytes SI and the spleen lymphocytes was not statistically significant compared to the other groups. Conclusion The result shows that the di-particles of the paramyxovirus Tianjin strain have a certain effect on enhancing the immune response of the organism, and the cell-free of Thl-type cell is induced. It is suggested that the adjuvant activity of the di-particles of the paramyxovirus Tianjin strain has the potential to become a biological adjuvant, and a certain experiment is provided for the next study.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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