大肠杆菌LTB增强PRRSV-GP5蛋白免疫原性的研究

发布时间：2018-05-25 15:07

本文选题：PRRSV + GP5　；参考：《甘肃农业大学》2010年硕士论文

【摘要】：【目的】为研究LTB的佐剂特性,在对LTB和GP5进行了表达条件及纯化方法优化的基础上,以LTB为免疫佐剂,GP5为模式抗原进行小鼠及仔猪免疫试验,为研究新型PRRSV疫苗探寻新途径。【方法】对GP5蛋白原核表达的诱导时间、IPTG浓度、表达菌裂解液及表达蛋白纯化条件进行了优化;对LTB蛋白真核表达的诱导时间、甲醇浓度、表达培养基蛋白浓度、表达菌起始浓度及表达蛋白纯化方法进行了优化;将雌性ICR小鼠分为8组,每组10只,分别为PBS阴性对照组、GP5+弗氏佐剂阳性对照组、GP5肌肉注射组、GP5+LTB肌肉注射组、GP5皮下注射组、GP5+LTB皮下注射组、GP5滴鼻组和GP5+LTB滴鼻组,三免后以间接ELISA法检测血清抗体,以MTT法检测淋巴细胞增殖;将1～2日龄仔猪随机分为4组,每组10只,分别为GP5滴鼻组、GP5+LTB滴鼻组、GP5注射组及GP5注射+LTB滴鼻组,二免后以间接ELISA法检测血清抗体。【结果】GP5蛋白表达及纯化最佳条件:1mmol/L的IPTG诱导3.5h,裂解液为pH7.8的8mol/L尿素,洗涤液为pH5.0和pH4.5的8mol/L尿素,洗脱液为pH4.0的8mol/L尿素,表达蛋白能与PRRSV阳性猪血清发生特异性反应,而与阴性血清不反应。LTB蛋白表达及纯化最佳条件:在1×BMMY中培养表达菌至OD600=4,1%(W/W)甲醇诱导96h,55%饱和硫酸铵沉淀后Ni-NTA柱一次亲和层析,D(+)-Immobilized galactose二次亲和层析,纯化后的LTB以五聚体形式存在,具有结合GM1的生物学活性;小鼠三免后GP5+弗氏佐剂组血清抗体水平最高,GP5+LTB免疫组抗体水平显著高于GP5免疫组,肌肉注射免疫组抗体水平明显高于皮下注射组,滴鼻免疫组没有检测到特异性血清抗体;MTT法检测结果表明,GP5+弗氏佐剂组、GP5+LTB肌肉注射和GP5+LTB皮下注射组B淋巴细胞增殖显著,除了GP5+弗氏佐剂组外,其他各免疫组均无明显的脾脏T淋巴细胞增殖反应。仔猪实验表明,LTB具有一定的黏膜免疫增强作用,GP5+LTB免疫组抗体水平高于GP5免疫组,GP5注射+LTB滴鼻免疫组血清抗体水平增幅大于GP5+LTB滴鼻组。【结论】建立了GP5原核表达纯化及LTB真核表达纯化的最优方法;小鼠免疫试验表明,LTB经肌肉和皮下途径免疫后能够有效地增强GP5蛋白的免疫原性,具有良好的体液免疫增强作用;仔猪免疫试验表明,LTB通过滴鼻方式免疫同样可以增强GP5蛋白的免疫原性,具有一定的黏膜佐剂作用。
[Abstract]:[objective] in order to study the adjuvant characteristics of LTB, on the basis of optimizing the expression conditions and purification methods of LTB and GP5, the mice and piglets were immunized with LTB as model antigen, and a new way to study new PRRSV vaccine was explored. [methods] the induction time of prokaryotic expression of GP5 protein, the concentration of IPTG, the lytic solution of expression bacteria and the purification conditions of LTB protein were optimized, and the induction time, methanol concentration and protein concentration of expression medium of LTB protein were optimized. The initial concentration of expression bacteria and the purification method of expressed protein were optimized, and female ICR mice were divided into 8 groups with 10 mice in each group. The PBS negative control group with Freund's adjuvant positive control group with GP5 intramuscular injection group with GP5 LTB intramuscular injection group and the subcutaneous injection group with GP5 LTB subcutaneous injection group with GP5 nose dropping and GP5 LTB nose dropping group were used to detect the serum antibody by indirect ELISA method after three immunizations. Lymphocyte proliferation was detected by MTT, and the piglets were randomly divided into 4 groups, 10 in each group: GP5 nose dropping group (GP5 group), GP5 LTB nasal drip group (GP5 group) and GP5 injection group (LTB nasal drip group). Serum antibodies were detected by indirect ELISA method after two immunizations. [results] the optimal conditions for the expression and purification of GP5 protein were as follows: 1 mmol / L IPTG was induced 3.5 h, the lysate was 8mol/L urea of pH7.8, the washing solution was 8mol/L urea of pH5.0 and pH4.5, and the eluate was 8mol/L urea of pH4.0. The expressed protein could react specifically with PRRSV positive pig serum. However, the optimal conditions for expression and purification of unreacted. LTB protein with negative serum were as follows: the expressed bacteria were cultured in 1 脳 BMMY to OD600 / 4L / W) methanol was induced to precipitate with 55% saturated ammonium sulfate at 96 h, and then Ni-NTA column one-step affinity chromatography (Ni-NTA) -immobilized galactose secondary affinity chromatography was used. The purified LTB existed in the form of pentamer. The antibody level of GP5 Freund's adjuvant group was significantly higher than that of GP5 immunization group, and the antibody level of GP5 Freund's adjuvant group was significantly higher than that of subcutaneous injection group, while the antibody level of GP5 LTB immunized group was significantly higher than that of GP5 immunized group. No specific serum antibody was detected in nasal drip immunization group. The results showed that the proliferation of B lymphocytes was significant in GP5 LTB intramuscular injection group and GP5 LTB subcutaneous injection group, except for GP5 Freund's adjuvant group. There was no obvious proliferation of spleen T lymphocytes in other immune groups. The results showed that LTB had a certain enhancement effect on mucosal immunity. The antibody level of GP5 LTB immunized group was higher than that of GP5 immunization group. The increase of serum antibody level in LTB nasal immunization group was higher than that in GP5 LTB nasal drip group. [conclusion] the optimal method of GP5 prokaryotic expression purification and LTB eukaryotic expression purification was established, and mouse immunological test showed that the immunogenicity of GP5 protein could be effectively enhanced by muscle and subcutaneous immunization. The immune test of piglets showed that LTB could also enhance the immunogenicity of GP5 protein by nasal drip and had a certain mucosal adjuvant effect.
【学位授予单位】：甘肃农业大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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