结核分枝杆菌重组Ag85A、Ag85B蛋白联合母牛分枝杆菌免疫原性的研究
发布时间：2018-10-23 12:06
【摘要】：目的：将Ag85A-pET30a大肠杆菌基因工程菌和Ag85B-pET24b大肠杆菌基因工程菌诱导表达并纯化后获得rAg85A和rAg85B蛋白。研究rAg85A和/或rAg85B蛋白与MV联合免疫的免疫原性。 方法：用IPTG诱导Ag85A-pET30a大肠杆菌基因工程菌和Ag85B-pET24b大肠杆菌基因工程菌表达，通过固化Ni2+树脂纯化后，经SDS-PAGE电泳鉴定目的蛋白的表达及纯化情况。 将60只雌性BALB/c小鼠随机分为下列6组：（1）阴性对照组：PBS组，（2）阳性对照组：BCG组，（3）阳性对照组：MV组，（4）实验组：rAg85A+MV组，（5）实验组：rAg85B+MV组，（6）实验组：rAg85A+rAg85B+MV组。每2周免疫1次，共免疫3次。每周记录小鼠体重变化。第3次免疫结束后第14天杀鼠，取肺脏、肝脏及脾脏称重。通过ELISPOT法检测脾脏分泌IFN-γ的T淋巴细胞斑点数；ELISA法检测第1次免疫前1天、第1次及第2次免疫后第10天、第3次免疫后第14天血清中IgG、IgG1和IgG2a水平；流式细胞术检测全血单个核细胞内分泌IFN-γ的Th1细胞百分比和分泌IL-4的Th2细胞百分比；ELISA法检测脾淋巴细胞培养上清中IFN-γ和IL-4水平。 结果：Ag85A-pET30a大肠杆菌基因工程菌和Ag85B-pET24b大肠杆菌基因工程菌经IPTG诱导成功后，以包涵体的形式表达。经Ni2+柱纯化后获得目的蛋白rAg85A和rAg85B。 第3次免疫结束后第14天，六组小鼠的体重均较免疫开始时显著增加（P＜0.001），且三个实验组小鼠体重均略高于三个对照组；各组间小鼠脾脏重量、肝脏重量及二者的重量指数无显著差别（P0.05），rAg85B+MV组小鼠肺脏指数较其它各组显著增高（P0.05）。 ELISPOT结果显示：三个实验组和两个阳性对照组小鼠的T淋巴细胞斑点数均明显高于阴性对照组（P0.05）； 经过3次免疫后，两个阳性对照组及三个实验组小鼠IgG、IgG1和IgG2a抗体水平较免疫前均显著升高（P0.001）。第3次免疫结束后第14天，三个实验组小鼠血清抗体水平均显著高于三个对照组（P0.001）； 流式细胞术结果显示：rAg85A+rAg85B+MV组小鼠Th1细胞百分比明显高于其余五组（P0.001），三个实验组Th1/Th2比值均明显高于三个对照组（P0.05）； ELISA检测脾淋巴细胞培养上清中IFN-γ和IL-4水平结果显示：两个阳性对照组和三个实验组小鼠脾淋巴细胞培养上清IFN-γ水平均高于PBS组（P0.05），其中rAg85A+rAg85B+MV组较其它五组升高最显著（P0.01）；IL-4的检测结果低于试剂盒的灵敏度，未能检测到。 结论：Ag85A-pET30a大肠杆菌基因工程菌和Ag85B-pET24b大肠杆菌基因工程菌以包涵体形式表达rAg85A和rAg85B蛋白，MV本身具有较好的佐剂作用，，这两种重组蛋白均能够增强MV的免疫原性，与MV联合免疫可诱导机体产生较高水平的Th1型细胞免疫应答。
[Abstract]:Aim: to express and purify rAg85A and rAg85B proteins from Ag85A-pET30a Escherichia coli genetically engineered bacteria and Ag85B-pET24b Escherichia coli genetically engineered bacteria. To study the immunogenicity of rAg85A and / or rAg85B protein combined with MV. Methods: IPTG was used to induce the expression of Ag85A-pET30a Escherichia coli and Ag85B-pET24b Escherichia coli, and the expression and purification of the target protein were identified by SDS-PAGE electrophoresis after the purification by solidified Ni2 resin. Sixty female BALB/c mice were randomly divided into the following six groups: (1) negative control group: PBS group, (2) positive control group: BCG group, (3) positive control group: MV group, (4) experimental group: rAg85A MV group, (5) experimental group: rAg85B MV group, (6) experimental group: rAg85A rAg85B MV group. The mice were immunized once every 2 weeks for 3 times. Changes in body weight were recorded weekly. The rats were killed on the 14th day after the third immunization, and the lungs, liver and spleen were weighed. The number of T lymphocyte spots secreting IFN- 纬 in spleen was detected by ELISPOT method, and the levels of IgG,IgG1 and IgG2a were detected by ELISA method on the first day before the first immunization, on the 10th day after the first and second immunization, on the 14th day after the third immunization. The percentage of Th1 cells secreting IFN- 纬 and the percentage of Th2 cells secreting IL-4 in whole blood mononuclear cells were detected by flow cytometry, and the levels of IFN- 纬 and IL-4 in the culture supernatant of splenic lymphocytes were detected by ELISA method. Results: Ag85A-pET30a Escherichia coli genetically engineered bacteria and Ag85B-pET24b Escherichia coli genetically engineered bacteria were successfully induced by IPTG and expressed as inclusion bodies. Purification of rAg85A and rAg85B. by Ni2 column On the 14th day after the third immunization, the body weight of the mice in the six groups was significantly higher than that at the beginning of the immunization (P < 0.001), and the body weight of the three experimental groups was slightly higher than that of the three control groups, and the spleen weight of the mice in each group was higher than that of the control group. There was no significant difference in liver weight and weight index between the two groups (P0.05), but the lung index in rAg85B MV group was significantly higher than that in other groups (P0.05). ELISPOT results showed that the number of T-lymphocyte spots in three experimental groups and two positive control groups was higher than that in other groups). All of them were significantly higher than those in negative control group (P0.05). After three times of immunization, the levels of IgG,IgG1 and IgG2a antibody in two positive control groups and three experimental groups were significantly higher than those before immunization (P0.001). On the 14th day after the third immunization, the serum antibody levels in the three experimental groups were significantly higher than those in the three control groups (P0.001). The results of flow cytometry showed that the percentage of Th1 cells in the rAg85A rAg85B MV group was significantly higher than that in the other five groups (P0.001), and the Th1/Th2 ratio in the three experimental groups was significantly higher than that in the three control groups (P0.05). The levels of IFN- 纬 and IL-4 in the supernatant of splenic lymphocyte culture were detected by ELISA. The results showed that the levels of IFN- 纬 in spleen lymphocyte culture supernatant of two positive control groups and three experimental groups were higher than those of PBS group (P0.05), and the level of IFN- 纬 in rAg85A rAg85B MV group was higher than that in other groups (P0.05). The elevation was the most significant in the five groups (P0.01). The detection results of IL-4 were lower than the sensitivity of the kit and could not be detected. Conclusion: Ag85A-pET30a Escherichia coli genetically engineered bacteria and Ag85B-pET24b Escherichia coli genetically engineered bacteria express rAg85A and rAg85B proteins in the form of inclusion bodies. MV itself has a good adjuvant effect, and these two recombinant proteins can enhance the immunogenicity of MV. Combined with MV can induce a higher level of Th1 type cellular immune response.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1
本文编号：2289181
[Abstract]:Aim: to express and purify rAg85A and rAg85B proteins from Ag85A-pET30a Escherichia coli genetically engineered bacteria and Ag85B-pET24b Escherichia coli genetically engineered bacteria. To study the immunogenicity of rAg85A and / or rAg85B protein combined with MV. Methods: IPTG was used to induce the expression of Ag85A-pET30a Escherichia coli and Ag85B-pET24b Escherichia coli, and the expression and purification of the target protein were identified by SDS-PAGE electrophoresis after the purification by solidified Ni2 resin. Sixty female BALB/c mice were randomly divided into the following six groups: (1) negative control group: PBS group, (2) positive control group: BCG group, (3) positive control group: MV group, (4) experimental group: rAg85A MV group, (5) experimental group: rAg85B MV group, (6) experimental group: rAg85A rAg85B MV group. The mice were immunized once every 2 weeks for 3 times. Changes in body weight were recorded weekly. The rats were killed on the 14th day after the third immunization, and the lungs, liver and spleen were weighed. The number of T lymphocyte spots secreting IFN- 纬 in spleen was detected by ELISPOT method, and the levels of IgG,IgG1 and IgG2a were detected by ELISA method on the first day before the first immunization, on the 10th day after the first and second immunization, on the 14th day after the third immunization. The percentage of Th1 cells secreting IFN- 纬 and the percentage of Th2 cells secreting IL-4 in whole blood mononuclear cells were detected by flow cytometry, and the levels of IFN- 纬 and IL-4 in the culture supernatant of splenic lymphocytes were detected by ELISA method. Results: Ag85A-pET30a Escherichia coli genetically engineered bacteria and Ag85B-pET24b Escherichia coli genetically engineered bacteria were successfully induced by IPTG and expressed as inclusion bodies. Purification of rAg85A and rAg85B. by Ni2 column On the 14th day after the third immunization, the body weight of the mice in the six groups was significantly higher than that at the beginning of the immunization (P < 0.001), and the body weight of the three experimental groups was slightly higher than that of the three control groups, and the spleen weight of the mice in each group was higher than that of the control group. There was no significant difference in liver weight and weight index between the two groups (P0.05), but the lung index in rAg85B MV group was significantly higher than that in other groups (P0.05). ELISPOT results showed that the number of T-lymphocyte spots in three experimental groups and two positive control groups was higher than that in other groups). All of them were significantly higher than those in negative control group (P0.05). After three times of immunization, the levels of IgG,IgG1 and IgG2a antibody in two positive control groups and three experimental groups were significantly higher than those before immunization (P0.001). On the 14th day after the third immunization, the serum antibody levels in the three experimental groups were significantly higher than those in the three control groups (P0.001). The results of flow cytometry showed that the percentage of Th1 cells in the rAg85A rAg85B MV group was significantly higher than that in the other five groups (P0.001), and the Th1/Th2 ratio in the three experimental groups was significantly higher than that in the three control groups (P0.05). The levels of IFN- 纬 and IL-4 in the supernatant of splenic lymphocyte culture were detected by ELISA. The results showed that the levels of IFN- 纬 in spleen lymphocyte culture supernatant of two positive control groups and three experimental groups were higher than those of PBS group (P0.05), and the level of IFN- 纬 in rAg85A rAg85B MV group was higher than that in other groups (P0.05). The elevation was the most significant in the five groups (P0.01). The detection results of IL-4 were lower than the sensitivity of the kit and could not be detected. Conclusion: Ag85A-pET30a Escherichia coli genetically engineered bacteria and Ag85B-pET24b Escherichia coli genetically engineered bacteria express rAg85A and rAg85B proteins in the form of inclusion bodies. MV itself has a good adjuvant effect, and these two recombinant proteins can enhance the immunogenicity of MV. Combined with MV can induce a higher level of Th1 type cellular immune response.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392.1
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