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鼠伤寒沙门氏菌鞭毛蛋白刺激小鼠细胞产生的免疫应答及鞭毛蛋白佐剂效应的初步研究

发布时间:2019-04-02 20:55
【摘要】: 细菌鞭毛有独特的结构和功能。具有很强的抗原性(H抗原),有利于细菌入侵。鞭毛蛋白可刺激机体产生前炎性因子,在连接天然免疫应答和获得性免疫应答中起重要作用。新城疫(Newcastle disease,ND)是危害养禽业的主要传染病之一,是国际兽疫局(OIE)规定的法定通报疾病。有报道证实,位于NDV囊膜上的F蛋白与病毒的致病性有关,参与病毒穿透、细胞融合,除此之外还具有良好的免疫原性,是NDV主要的保护性抗原。 本研究以含有新城疫病毒F48E8株全长的融合蛋白(F)基因的真核表达质粒pVAX1-F为模板,扩增出594 bp大小的F基因的部分片段。经克隆筛选和测序,构建成原核表达质粒pGEX-6P-1-F。重组质粒转化表达菌BL21,获得重组菌BL21(pGEX-6P-1-F)。经IPTG诱导表达和SDS-PAGE检测分析表明,F基因在原核表达体系中高效表达,表达量占菌体总蛋白的23%。Western blot结果显示,在相对分子质量46 KD位置有特异性条带。运用M-肉汤培养鼠伤寒沙门氏菌SL7207获得细菌的鞭毛蛋白。经SDS-PAGE分析表明,获得较纯的鞭毛蛋白。Western blot结果显示,在相对分子质量51KD位置有特异性条带。 使用浓度为0.5μg/ml、1μg/ml的去内毒素鞭毛蛋白刺激C57BL/6小鼠腹腔巨噬细胞(Macrophage,MΦ)2、4、6h。用细胞总RNA进行RT-PCR扩增,检测β-actin、IL-1β、TNF-α、IL-6基因表达。1.5%琼脂糖凝胶电泳验证鞭毛蛋白在2h时已能刺激细胞表达IL-1β、TNF-α、IL-6基因;0.5μg/ml浓度的鞭毛蛋白已能刺激细胞产生应答,转录IL-1β、TNF-α、IL-6。 使用10μg/ml浓度的鞭毛蛋白刺激小鼠腹腔MΦ2 h、12 h,以FACS检测细胞表面F4/80~+-FITC/CD80-biotin,F4/80~+-FITC/CD86-biotin的表达量。结果显示,与2 h相比12 h时细胞表达的CD80,CD86分子明显升高。以10μg/只剂量腹腔注射BALB/c小鼠,分别作用12 h、24 h。分离小鼠脾脏低浮密度细胞(Low-density cell,LDC),FACS检测细胞表面CD11c-FITC/CD40-biotin、CD11c-FITC/CD80-biotin、CD11c-FITC/CD86-biotin的表达量。结果表明,LDC表面CD40、CD86分子在12 h组、24 h组表达量均呈现上升,12 h组较24 h组高。CD80分子未出现明显的变化。 选取4h和0.5μg/ml浓度的鞭毛蛋白、F蛋白、鞭毛蛋白+F蛋白刺激小鼠腹腔MΦ。经1.5%琼脂糖凝胶电泳验证,鞭毛蛋白组和鞭毛蛋白+F蛋白组均能刺激细胞产生明显的前炎性因子应答,而F蛋白刺激细胞产生的前炎性因子应答较弱。 分别使用PBS、鞭毛蛋白、F蛋白、F蛋白+鞭毛蛋白、F蛋白+弗氏佐剂以皮下注射方式免疫小鼠,F蛋白的免疫剂量为80μg/只,鞭毛蛋白的免疫剂量为10μg/只。三免后可测得F蛋白+鞭毛蛋白组和F蛋白+弗氏佐剂对F蛋白的抗体效价分别为1:4266和1:7680。对各组小鼠脾脏细胞进行ELISPOT以检测抗原特异IFN-γ和IL-4分泌细胞,结果显示鞭毛蛋白+F蛋白组出现较高量的IFN-γ分泌细胞,同时也出现IL-4分泌细胞。而在F蛋白+弗氏佐剂组中F蛋白诱导的免疫应答处于平衡状态,IFN-γ分泌细胞数与IL-4分泌细胞数相当。
[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- 伪 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- 伪 and IL-6 genes at 2 h. 0.5 渭 g / ml of flagellin can stimulate cell response and transcribe IL-1 尾, TNF- 伪, 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-纬 and IL-4-secreting cells in spleen cells of mice in each group. The results showed that a higher amount of IFN- 纬-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- 纬-secreting cells was equal to that of IL-4-secreting cells.
【学位授予单位】:扬州大学
【学位级别】:硕士
【学位授予年份】:2009
【分类号】:R392

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