沙门菌鞭毛蛋白增强T细胞表位肽免疫效果的研究

发布时间：2018-05-01 05:30

本文选题：鼠伤寒沙门菌 + 鞭毛蛋白　；参考：《第二军医大学》2017年硕士论文

【摘要】：【研究目的】细菌鞭毛蛋白作为Toll样受体(Toll-likereceptors,TLRs)的配体,可以作为载体佐剂与特异性抗原连接,诱导免疫反应,本课题首先介绍一种多肽-蛋白偶联的流程,并用多肽-蛋白偶联产物制备一种偶联疫苗,其次通过皮下注射免疫BALB/c小鼠,比较鞭毛蛋白与特异性抗原偶联组和鞭毛蛋白与特异性抗原混合组的免疫效果,证实重组鞭毛蛋白与特异性抗原偶联后可增强抗原特异性免疫应答。通过重组鞭毛蛋白增强T细胞表位肽免疫效果的研究,为自身免疫性疾病治疗性疫苗的研究做铺垫。【研究方法】在前人制备已导入质粒pET28a-FliC-6His的BL21E.coli菌种中提取重组鞭毛蛋白(recombinantSalmonellaflagellin,rFliC),经GEHisGraviTrap提纯蛋白,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodiumdodecylsulfate-polyacrylamidegelelectrophoresis,SDS-PAGE)和蛋白免疫印迹法(Westernblot,WB)分析蛋白的纯度和性质。用化学连接剂Sulfo-EMCS在作为载体蛋白的rFliC上加马来酰亚胺基团,用间接Ellman反应测定载体蛋白上所加的马来酰亚胺基团数。载体蛋白利用马来酰亚胺基团与人工合成的OVA323多肽上半胱氨酸反应形成稳定的硫醚键产生偶联,偶联反应结束后,经Ellman测定剩余巯基,计算出消耗的巯基数和载体蛋白上马来酰亚胺基团数相比,得出载体蛋白和多肽的偶联比。为了检测多肽和载体蛋白偶联体对T细胞的免疫效果,用PBS、OVA323+rFliC(混合)、OVA323-rFliC(偶联)、OVA323+CFA/IFA分别免疫BALA/c小鼠,之后取小鼠脾脏,获取淋巴细胞,体外刺激。用酶联免疫吸附试验(Enzymelinkedimmunosorbentassays,ELISA)检测刺激后细胞上清中IL4、IFN-γ的分泌情况;用酶联免疫斑点吸附试验(Enzymelinkedimmunospot,ELISPOT)法检测获取的淋巴细胞在培养刺激过程中特异性分泌IL-4、IFN-γ的细胞数;用流式方法检测刺激后细胞CD4+IL-4+/CD4+IFN-γ+的细胞所占的比例。实验结果统计分析利用SPSS22.0软件,组间分析使用单因素方差分析法,当P0.05时说明组间存在显著差异。随后使用邦弗伦尼法比较实验组间两两的差异,当P0.05时说明两组间存在显著差异,本实验所有统计学数据用均数±标准差表示。【研究结果】通过异丙基硫代-β-D-半乳糖苷(Isopropylthio-β-D-galactoside,IPTG)诱导导入质粒pET28a-FliC-6His的BL21E.coli菌种表达rFliC,成功的获取了高纯度的rFliC。经化学连接剂在每摩尔rFliC上添加约5.55摩尔的马来酰亚胺,用马来酰亚胺修饰的rFliC制备出了偶联比大约为5.84的OVA323-rFliC多肽-蛋白偶联产物。ELISA检测发现,小鼠脾脏细胞体外刺激后OVA323-rFliC免疫组IL-4、IFN-γ的表达量较PBS组和OVA323+rFliC组显著增高,且差异有统计学意义;ELISPOT检测发现,小鼠脾脏细胞体外刺激过程中OVA323-rFliC免疫组特异性分泌IL-4、IFN-γ的细胞数较PBS组和OVA323+rFliC组显著增高,且差异有统计学意义;流式细胞术分析发现,小鼠脾脏细胞体外刺激后OVA323-rFliC免疫组CD4+IL-4+、CD4+IFNγ+细胞比例(%)较PBS组和OVA323+rFliC组显著增高,且差异有统计学意义。【主要结论】本研究将T细胞表位肽OVA323与rFliC体外偶联,构建OVA323-rFliC偶联体,并用偶联体免疫小鼠,通过检测T细胞特异性细胞免疫,证实rFliC可以作为T细胞佐剂,增强T细胞表位肽免疫效果,为rFliC作为自身免疫性疾病治疗性疫苗的载体佐剂的研究打好基础。
[Abstract]:[Objective] as a ligand of Toll like receptor (Toll-likereceptors, TLRs), bacterial flagellin can be used as a carrier adjuvant to connect with specific antigens and induce immune response. First, a polypeptide protein coupling process was introduced, and a coupling vaccine was prepared with peptide protein coupling products, followed by subcutaneous injection. The immunization effect of the combined group of flagellin and flagellin and specific antigen was compared between the pestilence BALB/c mice and the group of flagellin and specific antigen. It was proved that the recombinant flagellin could enhance the antigen specific immune response after coupling with the specific antigen. The immune effect of T cell epitopes was enhanced by the recombinant flagellin, and it was an autoimmune disease. The study of therapeutic vaccines was used as a paving. [research method] the recombinant flagellin (recombinantSalmonellaflagellin, rFliC) was extracted from the BL21E.coli strains which had been introduced into the plasmid pET28a-FliC-6His, and purified by GEHisGraviTrap, using twelve alkyl sodium sulfate polyacrylamide gel electrophoresis (sodiumdodecylsulfate-polyacryl). Amidegelelectrophoresis, SDS-PAGE) and protein immunoblotting (Westernblot, WB) for the analysis of the purity and properties of the protein. The maleimide group was added to the rFliC as the carrier protein by the chemical connecting agent Sulfo-EMCS, and the number of the maleimide group added on the carrier protein was determined by the indirect Ellman reaction. The carrier protein was used for maleimide. The group is coupled with the stable thioether bond formed by the reaction of cysteine on the synthetic OVA323 polypeptide. After the coupling reaction, the residual sulfhydryl group is determined by Ellman. The coupling ratio of the carrier protein and the polypeptide is obtained by comparing the number of the consumption of the sulfhydryl group with the number of maleimide group on the carrier protein. The immune effect of body to T cells, BALA/c mice were immunized with PBS, OVA323+rFliC (mixed), OVA323-rFliC (coupling), and OVA323+CFA/IFA respectively, then the spleen of the mice was taken and the lymphocytes were obtained and stimulated in vitro. The secretion of IL4 and IFN- gamma in the supernatant of the cells was detected by enzyme linked immunosorbent assay (Enzymelinkedimmunosorbentassays, ELISA); the enzyme was used for the secretion of IFN- gamma. Enzymelinkedimmunospot (ELISPOT) assay was used to detect the specific secretion of IL-4, IFN- gamma cells during the culture stimulation process. The proportion of CD4+IL-4+/CD4+IFN- gamma cells in the cells after stimulation was detected by flow method. The statistical analysis of experimental results was used by SPSS22.0 software and used in group analysis. Single factor ANOVA showed significant differences between groups when P0.05. Then the difference between the 22 of the experimental groups was compared with the bond group. When P0.05 showed significant differences between the two groups, all statistical data in this experiment were expressed with mean standard deviation of mean number. [results] through isopropyl thiosulfate beta -D- galactoside (Isopropylth Io- beta -D-galactoside, IPTG) induced the BL21E.coli strain of plasmid pET28a-FliC-6His to express rFliC, and successfully obtained a highly purified rFliC. by adding about 5.55 mole of maleimide on each mole rFliC, and the OVA323-rFliC polypeptide of the coupling ratio of about 5.84 was prepared by the maleimide modified rFliC. .ELISA detection found that the expression of IL-4, IFN- gamma in the OVA323-rFliC group was significantly higher than that in the PBS group and the OVA323+rFliC group after the stimulation of the spleen cells in vitro, and the difference was statistically significant. The ELISPOT test found that the OVA323-rFliC immune group secreted IL-4 in the immune group of the spleen cells in vitro, and the number of IFN- gamma cells was PBS. The group and the OVA323+rFliC group were significantly higher, and the difference was statistically significant. The flow cytometry showed that the proportion of CD4+IL-4+, CD4+IFN gamma + cells (%) in the OVA323-rFliC immunization group was significantly higher than that of the PBS group and the OVA323+rFliC group after the stimulation of the spleen cells in vitro, and the difference was statistically significant. [main conclusions] this study will take the T cell epitope peptide OVA323. In vitro coupling with rFliC to construct OVA323-rFliC bicouplet and immunize mice with biplet, by detecting specific cell immunity of T cells, it is proved that rFliC can be used as a adjuvant of T cells to enhance the immune effect of the epitope of T cells, which is a good basis for the study of rFliC as a carrier adjuvant of the therapeutic vaccine of autoimmune diseases.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R392

【相似文献】