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
[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
【参考文献】
相关期刊论文 前10条
1 师志海;曹宗喜;邢会杰;张凯韩;曾敏;李守军;;Toll样受体的研究进展[J];黑龙江畜牧兽医;2009年01期
2 张海玲;刘培欣;曹殿军;闫丽辉;孙建宏;冉多良;;NDV F_(48) E_9和La Sota毒株F蛋白B细胞抗原优势表位区段的鉴定和免疫原性比较[J];中国生物工程杂志;2006年09期
3 陈潇;朱迎;胡永秀;;Toll样受体4表达的调节[J];微生物学免疫学进展;2009年01期
4 潘志明,焦新安,黄金林,殷月兰,唐丽华,张辉,张晓明,张小荣,刘秀梵;携带新城疫病毒DNA疫苗的鼠伤寒沙门氏菌及其安全性与免疫效力[J];微生物学报;2005年06期
5 金伯泉;;固有免疫中模式识别受体及其信号转导——当代免疫学中最伟大的发现之一[J];细胞与分子免疫学杂志;2006年01期
6 丁剑冰,魏晓丽,林仁勇,温浩,阿孜古丽·吐尔逊,张亚楼,王国荃;Eg95基因疫苗不同免疫途径的体液免疫应答比较[J];新疆医科大学学报;2003年03期
7 付金玲;李其林;;Toll样受体与病毒感染的研究现状[J];中国皮肤性病学杂志;2009年01期
8 刘兆球,姜世金,常维山;真核表达NDV F基因重组质粒的构建及其在CEF细胞中的转染[J];中国预防兽医学报;2003年06期
9 张晓东;刘怀然;刘培欣;闫丽辉;刘胜旺;孔宪刚;;新城疫病毒La Sota株F蛋白结构与部分功能区的预测[J];中国预防兽医学报;2007年12期
10 张小燕;潘志明;胡青海;董慧;张成全;焦新安;;鸡白细胞介素2分子单克隆抗体的研制[J];中国预防兽医学报;2008年01期
,本文编号:2452913
本文链接:https://www.wllwen.com/yixuelunwen/shiyanyixue/2452913.html