鼠傷寒沙門氏菌鞭毛蛋白刺激小鼠細(xì)胞產(chǎn)生的免疫應(yīng)答及鞭毛蛋白佐劑效應(yīng)的初步研究
發(fā)布時(shí)間：2019-04-02 20:55
【摘要】： 細(xì)菌鞭毛有獨(dú)特的結(jié)構(gòu)和功能。具有很強(qiáng)的抗原性(H抗原),有利于細(xì)菌入侵。鞭毛蛋白可刺激機(jī)體產(chǎn)生前炎性因子,在連接天然免疫應(yīng)答和獲得性免疫應(yīng)答中起重要作用。新城疫(Newcastle disease,ND)是危害養(yǎng)禽業(yè)的主要傳染病之一,是國際獸疫局(OIE)規(guī)定的法定通報(bào)疾病。有報(bào)道證實(shí),位于NDV囊膜上的F蛋白與病毒的致病性有關(guān),參與病毒穿透、細(xì)胞融合,除此之外還具有良好的免疫原性,是NDV主要的保護(hù)性抗原。 本研究以含有新城疫病毒F48E8株全長(zhǎng)的融合蛋白(F)基因的真核表達(dá)質(zhì)粒pVAX1-F為模板,擴(kuò)增出594 bp大小的F基因的部分片段。經(jīng)克隆篩選和測(cè)序,構(gòu)建成原核表達(dá)質(zhì)粒pGEX-6P-1-F。重組質(zhì)粒轉(zhuǎn)化表達(dá)菌BL21,獲得重組菌BL21(pGEX-6P-1-F)。經(jīng)IPTG誘導(dǎo)表達(dá)和SDS-PAGE檢測(cè)分析表明,F基因在原核表達(dá)體系中高效表達(dá),表達(dá)量占菌體總蛋白的23%。Western blot結(jié)果顯示,在相對(duì)分子質(zhì)量46 KD位置有特異性條帶。運(yùn)用M-肉湯培養(yǎng)鼠傷寒沙門氏菌SL7207獲得細(xì)菌的鞭毛蛋白。經(jīng)SDS-PAGE分析表明,獲得較純的鞭毛蛋白。Western blot結(jié)果顯示,在相對(duì)分子質(zhì)量51KD位置有特異性條帶。 使用濃度為0.5μg/ml、1μg/ml的去內(nèi)毒素鞭毛蛋白刺激C57BL/6小鼠腹腔巨噬細(xì)胞(Macrophage,MΦ)2、4、6h。用細(xì)胞總RNA進(jìn)行RT-PCR擴(kuò)增,檢測(cè)β-actin、IL-1β、TNF-α、IL-6基因表達(dá)。1.5%瓊脂糖凝膠電泳驗(yàn)證鞭毛蛋白在2h時(shí)已能刺激細(xì)胞表達(dá)IL-1β、TNF-α、IL-6基因;0.5μg/ml濃度的鞭毛蛋白已能刺激細(xì)胞產(chǎn)生應(yīng)答,轉(zhuǎn)錄IL-1β、TNF-α、IL-6。 使用10μg/ml濃度的鞭毛蛋白刺激小鼠腹腔MΦ2 h、12 h,以FACS檢測(cè)細(xì)胞表面F4/80~+-FITC/CD80-biotin,F4/80~+-FITC/CD86-biotin的表達(dá)量。結(jié)果顯示,與2 h相比12 h時(shí)細(xì)胞表達(dá)的CD80,CD86分子明顯升高。以10μg/只劑量腹腔注射BALB/c小鼠,分別作用12 h、24 h。分離小鼠脾臟低浮密度細(xì)胞(Low-density cell,LDC),FACS檢測(cè)細(xì)胞表面CD11c-FITC/CD40-biotin、CD11c-FITC/CD80-biotin、CD11c-FITC/CD86-biotin的表達(dá)量。結(jié)果表明,LDC表面CD40、CD86分子在12 h組、24 h組表達(dá)量均呈現(xiàn)上升,12 h組較24 h組高。CD80分子未出現(xiàn)明顯的變化。 選取4h和0.5μg/ml濃度的鞭毛蛋白、F蛋白、鞭毛蛋白+F蛋白刺激小鼠腹腔MΦ。經(jīng)1.5%瓊脂糖凝膠電泳驗(yàn)證,鞭毛蛋白組和鞭毛蛋白+F蛋白組均能刺激細(xì)胞產(chǎn)生明顯的前炎性因子應(yīng)答,而F蛋白刺激細(xì)胞產(chǎn)生的前炎性因子應(yīng)答較弱。 分別使用PBS、鞭毛蛋白、F蛋白、F蛋白+鞭毛蛋白、F蛋白+弗氏佐劑以皮下注射方式免疫小鼠,F蛋白的免疫劑量為80μg/只,鞭毛蛋白的免疫劑量為10μg/只。三免后可測(cè)得F蛋白+鞭毛蛋白組和F蛋白+弗氏佐劑對(duì)F蛋白的抗體效價(jià)分別為1:4266和1:7680。對(duì)各組小鼠脾臟細(xì)胞進(jìn)行ELISPOT以檢測(cè)抗原特異IFN-γ和IL-4分泌細(xì)胞,結(jié)果顯示鞭毛蛋白+F蛋白組出現(xiàn)較高量的IFN-γ分泌細(xì)胞,同時(shí)也出現(xiàn)IL-4分泌細(xì)胞。而在F蛋白+弗氏佐劑組中F蛋白誘導(dǎo)的免疫應(yīng)答處于平衡狀態(tài),IFN-γ分泌細(xì)胞數(shù)與IL-4分泌細(xì)胞數(shù)相當(dāng)。
[Abstract]:Bacterial flagellum has unique structure and function. It has strong antigenicity (H antigen), which is beneficial to bacterial invasion. Flagellin stimulates the production of proinflammatory factors and plays an important role in linking innate and acquired immune responses. Newcastle disease (Newcastle disease,ND) is one of the main infectious diseases endangering poultry industry. It is a statutory notifiable disease prescribed by the OIE (International Bureau of OIE) (OIE). It has been reported that the F protein located on the envelope of NDV is associated with the pathogenicity of the virus, which is involved in virus penetration and cell fusion. In addition, it has good immunogenicity and is the main protective antigen of NDV. In this study, the eukaryotic expression plasmid pVAX1-F containing the full-length fusion protein (F) gene of Newcastle disease virus F48E8 strain was used as template, and a partial fragment of the F gene with the size of 594 bp was amplified. After cloning, screening and sequencing, the prokaryotic expression plasmid pGEX-6P-1-F. was constructed. The recombinant strain BL21 (pGEX-6P-1-F) was obtained by transforming the recombinant plasmid into BL21,. The results of IPTG-induced expression and SDS-PAGE analysis showed that F gene was highly expressed in prokaryotic expression system. The 23%.Western blot results showed that F gene had a specific band at the position of 46 KD relative molecular weight. Bacterial flagellin was obtained from Salmonella typhimurium SL7207 cultured in M-broth. The purified flagellin was obtained by SDS-PAGE analysis. Western blot analysis showed that there was a specific band at the 51KD site of relative molecular weight. The peritoneal macrophages (Macrophage,M 桅) of C57BL/6 mice were stimulated with decotoxin flagellin 0.5 渭 g / ml, 1 渭 g / ml for 2, 4, 6 h. The expression of 尾-actin,IL-1 尾, TNF- 偽 and IL-6 genes was detected by RT-PCR amplification with total RNA. 1.5% agarose gel electrophoresis showed that flagellin could stimulate the expression of IL-1 尾, TNF- 偽 and IL-6 genes at 2 h. 0.5 渭 g / ml of flagellin can stimulate cell response and transcribe IL-1 尾, TNF- 偽, IL-6.. After the mice were stimulated with 10 渭 g / ml flagellin for 2 h and 12 h, the expression of F4, 80 ~-FITC/CD80-biotin,F4/80~-FITC/CD86-biotin on the cell surface was detected by FACS. The results showed that the expression of CD80,CD86 at 12 h was significantly higher than that at 2 h. BALB/c mice were injected intraperitoneally at a dose of 10 渭 g per mouse for 12 h and 24 h, respectively. Low floating density cells (Low-density cell,LDC), FACS) were isolated from the spleen of mice to detect the expression of CD11c-FITC/CD40-biotin,CD11c-FITC/CD80-biotin,CD11c-FITC/CD86-biotin on the surface of the cells. The results showed that the expression of CD40,CD86 on the surface of LDC increased at 12 h and 24 h groups, and was higher in 12 h group than in 24 h group. There was no significant change of CD 80 molecule in the 12 h group compared with the 24 h group. Four hours and 0.5 渭 g / ml concentrations of flagellin, F protein and flagellin F protein were used to stimulate mouse peritoneal M 桅. The results of 1.5% agarose gel electrophoresis showed that both the flagellin group and the flagellin F protein group could stimulate the cells to produce obvious proinflammatory factor response, while the F protein stimulated cell proinflammatory factor response was weaker than that of the F protein group. The mice were immunized subcutaneously with PBS, flagellin, F protein, F protein flagellin and F protein Freund's adjuvant. The immune dose of F protein was 80 渭 g / mouse, and that of flagellin was 10 渭 g / mouse. The antibody titers of F protein flagellin group and Freund's adjuvant against F protein were 1? 4266 and 1? 768 respectively. ELISPOT was used to detect antigen-specific IFN-緯 and IL-4-secreting cells in spleen cells of mice in each group. The results showed that a higher amount of IFN- 緯-secreting cells and IL-4-secreting cells were observed in flagellin F protein group. In F protein Freund's adjuvant group, the immune response induced by F protein was in balance, and the number of IFN- 緯-secreting cells was equal to that of IL-4-secreting cells.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
本文編號(hào)：2452913
[Abstract]:Bacterial flagellum has unique structure and function. It has strong antigenicity (H antigen), which is beneficial to bacterial invasion. Flagellin stimulates the production of proinflammatory factors and plays an important role in linking innate and acquired immune responses. Newcastle disease (Newcastle disease,ND) is one of the main infectious diseases endangering poultry industry. It is a statutory notifiable disease prescribed by the OIE (International Bureau of OIE) (OIE). It has been reported that the F protein located on the envelope of NDV is associated with the pathogenicity of the virus, which is involved in virus penetration and cell fusion. In addition, it has good immunogenicity and is the main protective antigen of NDV. In this study, the eukaryotic expression plasmid pVAX1-F containing the full-length fusion protein (F) gene of Newcastle disease virus F48E8 strain was used as template, and a partial fragment of the F gene with the size of 594 bp was amplified. After cloning, screening and sequencing, the prokaryotic expression plasmid pGEX-6P-1-F. was constructed. The recombinant strain BL21 (pGEX-6P-1-F) was obtained by transforming the recombinant plasmid into BL21,. The results of IPTG-induced expression and SDS-PAGE analysis showed that F gene was highly expressed in prokaryotic expression system. The 23%.Western blot results showed that F gene had a specific band at the position of 46 KD relative molecular weight. Bacterial flagellin was obtained from Salmonella typhimurium SL7207 cultured in M-broth. The purified flagellin was obtained by SDS-PAGE analysis. Western blot analysis showed that there was a specific band at the 51KD site of relative molecular weight. The peritoneal macrophages (Macrophage,M 桅) of C57BL/6 mice were stimulated with decotoxin flagellin 0.5 渭 g / ml, 1 渭 g / ml for 2, 4, 6 h. The expression of 尾-actin,IL-1 尾, TNF- 偽 and IL-6 genes was detected by RT-PCR amplification with total RNA. 1.5% agarose gel electrophoresis showed that flagellin could stimulate the expression of IL-1 尾, TNF- 偽 and IL-6 genes at 2 h. 0.5 渭 g / ml of flagellin can stimulate cell response and transcribe IL-1 尾, TNF- 偽, IL-6.. After the mice were stimulated with 10 渭 g / ml flagellin for 2 h and 12 h, the expression of F4, 80 ~-FITC/CD80-biotin,F4/80~-FITC/CD86-biotin on the cell surface was detected by FACS. The results showed that the expression of CD80,CD86 at 12 h was significantly higher than that at 2 h. BALB/c mice were injected intraperitoneally at a dose of 10 渭 g per mouse for 12 h and 24 h, respectively. Low floating density cells (Low-density cell,LDC), FACS) were isolated from the spleen of mice to detect the expression of CD11c-FITC/CD40-biotin,CD11c-FITC/CD80-biotin,CD11c-FITC/CD86-biotin on the surface of the cells. The results showed that the expression of CD40,CD86 on the surface of LDC increased at 12 h and 24 h groups, and was higher in 12 h group than in 24 h group. There was no significant change of CD 80 molecule in the 12 h group compared with the 24 h group. Four hours and 0.5 渭 g / ml concentrations of flagellin, F protein and flagellin F protein were used to stimulate mouse peritoneal M 桅. The results of 1.5% agarose gel electrophoresis showed that both the flagellin group and the flagellin F protein group could stimulate the cells to produce obvious proinflammatory factor response, while the F protein stimulated cell proinflammatory factor response was weaker than that of the F protein group. The mice were immunized subcutaneously with PBS, flagellin, F protein, F protein flagellin and F protein Freund's adjuvant. The immune dose of F protein was 80 渭 g / mouse, and that of flagellin was 10 渭 g / mouse. The antibody titers of F protein flagellin group and Freund's adjuvant against F protein were 1? 4266 and 1? 768 respectively. ELISPOT was used to detect antigen-specific IFN-緯 and IL-4-secreting cells in spleen cells of mice in each group. The results showed that a higher amount of IFN- 緯-secreting cells and IL-4-secreting cells were observed in flagellin F protein group. In F protein Freund's adjuvant group, the immune response induced by F protein was in balance, and the number of IFN- 緯-secreting cells was equal to that of IL-4-secreting cells.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392
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