副粘病毒Tianjin株缺損性干擾顆粒用作新型生物佐劑的初步研究
發(fā)布時間：2019-01-11 18:57
【摘要】：目的新型疫苗如基因工程多肽或亞單位疫苗和合成肽疫苗等的弱免疫原性使得佐劑在疫苗研制中的應(yīng)用越來越廣泛,并隨著疫苗推廣應(yīng)用到其他領(lǐng)域如抗感染,抗腫瘤的輔助治療。因此尋求理想的佐劑對于疫苗的研制發(fā)展有著關(guān)鍵性的意義。常見的佐劑有不溶性鋁鹽類膠體、油水乳劑、微生物及其代謝產(chǎn)物、核酸及其類似物、細(xì)胞因子、免疫刺激復(fù)合物、蜂膠、脂質(zhì)體等。缺損性干擾.(defective interfering, DI)顆粒是一種含有部分基因組的病毒顆粒,它是病毒在復(fù)制過程中發(fā)生錯誤而出現(xiàn)的,其基因組只是亞基因組片段。其自身屬性決定其亞基因組中攜帶了眾多正常病毒顆粒所沒有的信息。研究發(fā)現(xiàn)仙臺病毒DI顆粒具有強(qiáng)大的刺激樹突狀細(xì)胞(dendritic cell, DC)成熟的能力。副粘病毒Tianjin株(Paramyxovirus, Tianjin Strain)是1999年從天津醫(yī)科大學(xué)動物實驗中心爆發(fā)急性上呼吸道感染的普通棉耳狨猴肺臟中分離出來的毒株,經(jīng)鑒定屬于副粘病毒科、副粘病毒亞科、呼吸道病毒屬,與仙臺病毒(Sendai virus,SeV)親緣關(guān)系最近,不屬于仙臺病毒現(xiàn)有的3個進(jìn)化分支而自成獨(dú)立的一支。因此本文對副粘病毒Tianjin株DI顆粒作為新型生物佐劑的可行性進(jìn)行了初步研究。 方法將副粘病毒Tianjin株用雞胚培養(yǎng)法連續(xù)傳代三次,鋪設(shè)5%～45%蔗糖密度梯度超速離心分離提純副粘病毒Tianjin株DI顆粒,通過電鏡觀察,體外接種敏感細(xì)胞系大鼠腦膠質(zhì)瘤細(xì)胞C6株觀察細(xì)胞病變,接種雞胚收集尿囊液檢測血凝素效價等對DI顆粒進(jìn)行了鑒定,將脊髓灰質(zhì)炎病毒(PV)疫苗株的滅活抗原與DI顆粒佐劑等量混合后制備疫苗。選取4周齡ICR小鼠,經(jīng)皮下多點注射對小鼠進(jìn)行免疫,觀察小鼠的大體情況和免疫應(yīng)答水平。并設(shè)PV滅活抗原加氫氧化鋁凝膠佐劑組為陽性對照,PV滅活抗原無佐劑組以及生理鹽水組為陰性對照。觀察小鼠的存活情況；于免疫后兩周摘眼球取血分離血清,檢測免疫血清中PV特異性抗體；無菌取脾臟分離脾淋巴細(xì)胞進(jìn)行體外培養(yǎng),分別用MTT法進(jìn)行T、B淋巴細(xì)胞增殖試驗；檢測脾淋巴細(xì)胞接受PV滅活抗原再次刺激后IFN-γ、IL-4的產(chǎn)生情況,對DI顆粒能否增強(qiáng)PV免疫小鼠的免疫應(yīng)答以及免疫應(yīng)答的類型進(jìn)行評估。 結(jié)果實驗結(jié)果確定純化分離出的是與完整病毒有所差別的,無復(fù)制能力的缺損性顆粒成分。動物免疫指標(biāo)結(jié)果顯示副粘病毒Tianjin株DI顆粒佐劑組血清特異性PV抗體水平高于陰性對照組,但低于陽性對照組,差異有統(tǒng)計學(xué)意義；T淋巴細(xì)胞刺激指數(shù)(SI),脾淋巴細(xì)胞分泌IFN-γ的量高于其他組,差異有統(tǒng)計學(xué)意義,而B淋巴細(xì)胞SI和脾淋巴細(xì)胞分泌IL-4的量與其他組相比差異沒有統(tǒng)計學(xué)意義。 結(jié)論實驗證實副粘病毒Tianjin株DI顆粒具有一定的增強(qiáng)機(jī)體免疫應(yīng)答能力的作用,且以誘導(dǎo)Thl型細(xì)胞免疫應(yīng)答為主。提示副粘病毒Tianjin株DI顆粒具有佐劑活性,具有成為生物佐劑的潛在可能,為下一步的研究提供了一定的實驗和理論依據(jù)。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
本文編號：2407358
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392
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