马流产沙门氏菌新疆分离株鉴定免疫及其FliC蛋白增强SeM蛋白及gD蛋白抗体应答的研究

发布时间：2018-02-22 18:36

本文关键词： 马流产沙门氏菌鞭毛蛋白Flic 表达纯化 SeM蛋白 gD蛋白联合免疫　出处：《新疆农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：马流产沙门氏菌可引起妊娠母马流产,幼驹败血症,下痢,公马睾丸炎,髯甲脓肿。该病可给马养殖业造成严重经济损失。本研究首先对新疆地区养马较为集中的昭苏县、富蕴县、特克斯县、和静县、吉木乃县的养殖场(户)的2768份马血清样品进行马流产沙门氏菌血清学调查。间接ELISA法检测结果显示,这5县马养殖场(户)中马流产沙门氏菌病抗体平均阳性率为28.68%；雌性马匹的抗体阳性率(36.9%)略高于雄性马匹(29.7%),但差异不显著；吉木乃和和静县马匹抗体阳性率相对较低,为3.24%-9.78%,特克斯县和昭苏县抗体阳性率相对较高,为27.17%～43.91%；从年龄统计结果来看,各年龄段的的马匹均有一定的抗体阳性率。本研究从流产马匹的病料中分离纯化,并经生化和PCR鉴定得到马流产沙门氏菌新疆分离株。本研究进一步利用分离菌株的基因组,经PCR扩增得到了马流产沙门氏菌鞭毛蛋白基因FliC2,大小为1427 bp,并构建重组质粒pET28a-FliC2,在大肠杆菌BL21(DE3)感受态细胞中经IPTG进行诱导表达,经SDS-PAGE电泳分析该重组蛋白大小约为52KDa,Western-blotting分析显示该蛋白具有反应原性。将纯化后的融合蛋白F1iC2免疫小鼠,检测结果显示,免疫小鼠中产生了较高水平的抗体,抗体效价可达1：218 700,且以IgG1亚型为主。将纯化后的融合蛋白免疫马匹,免疫马血清中同样产生了较高水平的特异性抗体,抗体效价可达1：72 900。本研究进一步利用PCR扩增了截短的马流产沙门氏菌鞭毛蛋白基因FliCl,大小为840 bp,并构建重组质粒pGEX-FliC1。诱导表达的融合蛋白FliCl-GST经SDS-PAGE电泳分析大小为56 KDa。将纯化后的FliC1蛋白和本实验室已表达的gD蛋白联合免疫马匹,结果表明,两种蛋白联合免疫组马血清中抗体水平高于这两种蛋白单独免疫组,抗体效价可达1：218 700。检测结果还表明,FliC1蛋白可促进gD蛋白免疫水平的升高。且产生的抗体以亚型IgG1为主。对抗体持续期的检测表明抗体维持较高水平可达170 d。为比较FliC两个不同大小片段免疫效果的差异,本研究将FliC1和FliC2分别与SeM蛋白联合免疫C57BL/6小鼠,对免疫小鼠抗体的效价、抗体亚型以及攻毒保护力评估不同大小鞭毛蛋白对SeM蛋白免疫效果的影响。结果显示,FliC2与SeM联合免疫组产生的抗体效价更高,抗体主要以IgG1为主；对小鼠的攻毒保护率最高,可达80%。该结果表明,鞭毛蛋白FliC2对SeM辅助免疫效果比FliC1更好。该结果对我们更好地利用FliC蛋白进行相关研究奠定实验基础。
[Abstract]:Salmonella equine abortion can cause miscarriage of pregnant mares, septicemia, dysentery, orchitis of male horses, and abscess of bearded armour. This disease can cause serious economic losses to horse breeding. In Fuyun County, Tex County, Hejing County, Jimonai County, 2768 horse serum samples were collected from farms (households) for serological investigation of Salmonella abortus. Indirect ELISA assay showed that, The average positive rate of salmonellosis antibodies in horse farms (households) in these five counties was 28.68; the positive rate of antibodies in female horses was 36.9%, which was slightly higher than that in male horses (29.7g), but the difference was not significant. The positive rate of horse antibodies in Jimenai and Hejing counties was relatively low. The positive rate of antibody was relatively high in Tex County and Zhaosu County (27.17%, 43.91%). According to the results of age statistics, there was a positive rate of antibody in horses of all ages. The isolates of Salmonella equi from Xinjiang were identified by biochemical and PCR methods. The flagellin gene FliC2 of Salmonella equina was amplified by PCR, and its size was 1427 bp.The recombinant plasmid pET28a-FliC2 was constructed and expressed by IPTG in E. coli BL21DE3. The recombinant protein was analyzed by SDS-PAGE electrophoresis to be about 52K Dawei Western-blotting. The purified fusion protein F1iC2 was immunized by immunizing mice. The results showed that a high level of antibody was produced in the immunized mice. The titer of the antibody reached 1: 218,700, and the IgG1 subtype was the main subtype. The purified fusion protein was used to immunize the horse, and a higher level of specific antibody was also produced in the serum of the immunized horse. The antibody titer can reach 1: 72 900.In this study, the flagellin gene of Salmonella equi was amplified by PCR, the size was 840 BP, and the recombinant plasmid pGEX-FliC1 was constructed. The induced expression of the fusion protein FliCl-GST was 56 by SDS-PAGE electrophoresis. The purified FliC1 protein was combined with the gd protein expressed in our laboratory to immunize horses. The results showed that the level of antibody in serum of the two kinds of protein combined immunized group was higher than that of the two kinds of protein alone immunization group. The titer of antibody reached 1: 218,700. The results also showed that FliC1 protein could promote the increase of immunological level of GD protein, and the antibody produced was mainly subtype IgG1. The detection of antibody duration showed that the antibody maintained a high level up to 170d. The difference of immune effect between two different size fragments of FliC, In this study, FliC1 and FliC2 were combined with SeM protein to immunize C57BL / 6 mice. The effects of flagellins of different sizes on the immune response of SeM protein were evaluated. The results showed that the antibody titers of FliC2 and SeM combined immunized group were higher, the main antibody was IgG1, and the protective rate of attacking virus to mice was the highest. The results showed that the flagellin FliC2 was more effective than FliC1 in assisting SeM immunization. The results laid an experimental foundation for us to make better use of FliC protein to carry on the related research.
【学位授予单位】：新疆农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S852.61

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