本文選題：鼠傷寒沙門菌 + 鞭毛蛋白��； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：【研究目的】細菌鞭毛蛋白作為Toll樣受體(Toll-likereceptors,TLRs)的配體,可以作為載體佐劑與特異性抗原連接,誘導(dǎo)免疫反應(yīng),本課題首先介紹一種多肽-蛋白偶聯(lián)的流程,并用多肽-蛋白偶聯(lián)產(chǎn)物制備一種偶聯(lián)疫苗,其次通過皮下注射免疫BALB/c小鼠,比較鞭毛蛋白與特異性抗原偶聯(lián)組和鞭毛蛋白與特異性抗原混合組的免疫效果,證實重組鞭毛蛋白與特異性抗原偶聯(lián)后可增強抗原特異性免疫應(yīng)答。通過重組鞭毛蛋白增強T細胞表位肽免疫效果的研究,為自身免疫性疾病治療性疫苗的研究做鋪墊�！狙芯糠椒ā吭谇叭酥苽湟褜�(dǎo)入質(zhì)粒pET28a-FliC-6His的BL21E.coli菌種中提取重組鞭毛蛋白(recombinantSalmonellaflagellin,rFliC),經(jīng)GEHisGraviTrap提純蛋白,利用十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(sodiumdodecylsulfate-polyacrylamidegelelectrophoresis,SDS-PAGE)和蛋白免疫印跡法(Westernblot,WB)分析蛋白的純度和性質(zhì)。用化學(xué)連接劑Sulfo-EMCS在作為載體蛋白的rFliC上加馬來酰亞胺基團,用間接Ellman反應(yīng)測定載體蛋白上所加的馬來酰亞胺基團數(shù)。載體蛋白利用馬來酰亞胺基團與人工合成的OVA323多肽上半胱氨酸反應(yīng)形成穩(wěn)定的硫醚鍵產(chǎn)生偶聯(lián),偶聯(lián)反應(yīng)結(jié)束后,經(jīng)Ellman測定剩余巰基,計算出消耗的巰基數(shù)和載體蛋白上馬來酰亞胺基團數(shù)相比,得出載體蛋白和多肽的偶聯(lián)比。為了檢測多肽和載體蛋白偶聯(lián)體對T細胞的免疫效果,用PBS、OVA323+rFliC(混合)、OVA323-rFliC(偶聯(lián))、OVA323+CFA/IFA分別免疫BALA/c小鼠,之后取小鼠脾臟,獲取淋巴細胞,體外刺激。用酶聯(lián)免疫吸附試驗(Enzymelinkedimmunosorbentassays,ELISA)檢測刺激后細胞上清中IL4、IFN-γ的分泌情況;用酶聯(lián)免疫斑點吸附試驗(Enzymelinkedimmunospot,ELISPOT)法檢測獲取的淋巴細胞在培養(yǎng)刺激過程中特異性分泌IL-4、IFN-γ的細胞數(shù);用流式方法檢測刺激后細胞CD4+IL-4+/CD4+IFN-γ+的細胞所占的比例。實驗結(jié)果統(tǒng)計分析利用SPSS22.0軟件,組間分析使用單因素方差分析法,當(dāng)P0.05時說明組間存在顯著差異。隨后使用邦弗倫尼法比較實驗組間兩兩的差異,當(dāng)P0.05時說明兩組間存在顯著差異,本實驗所有統(tǒng)計學(xué)數(shù)據(jù)用均數(shù)±標準差表示�！狙芯拷Y(jié)果】通過異丙基硫代-β-D-半乳糖苷(Isopropylthio-β-D-galactoside,IPTG)誘導(dǎo)導(dǎo)入質(zhì)粒pET28a-FliC-6His的BL21E.coli菌種表達rFliC,成功的獲取了高純度的rFliC。經(jīng)化學(xué)連接劑在每摩爾rFliC上添加約5.55摩爾的馬來酰亞胺,用馬來酰亞胺修飾的rFliC制備出了偶聯(lián)比大約為5.84的OVA323-rFliC多肽-蛋白偶聯(lián)產(chǎn)物。ELISA檢測發(fā)現(xiàn),小鼠脾臟細胞體外刺激后OVA323-rFliC免疫組IL-4、IFN-γ的表達量較PBS組和OVA323+rFliC組顯著增高,且差異有統(tǒng)計學(xué)意義;ELISPOT檢測發(fā)現(xiàn),小鼠脾臟細胞體外刺激過程中OVA323-rFliC免疫組特異性分泌IL-4、IFN-γ的細胞數(shù)較PBS組和OVA323+rFliC組顯著增高,且差異有統(tǒng)計學(xué)意義;流式細胞術(shù)分析發(fā)現(xiàn),小鼠脾臟細胞體外刺激后OVA323-rFliC免疫組CD4+IL-4+、CD4+IFNγ+細胞比例(%)較PBS組和OVA323+rFliC組顯著增高,且差異有統(tǒng)計學(xué)意義。【主要結(jié)論】本研究將T細胞表位肽OVA323與rFliC體外偶聯(lián),構(gòu)建OVA323-rFliC偶聯(lián)體,并用偶聯(lián)體免疫小鼠,通過檢測T細胞特異性細胞免疫,證實rFliC可以作為T細胞佐劑,增強T細胞表位肽免疫效果,為rFliC作為自身免疫性疾病治療性疫苗的載體佐劑的研究打好基礎(chǔ)。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R392

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