【摘要】：目的：將Ag85A-pET30a大腸桿菌基因工程菌和Ag85B-pET24b大腸桿菌基因工程菌誘導(dǎo)表達(dá)并純化后獲得rAg85A和rAg85B蛋白。研究rAg85A和/或rAg85B蛋白與MV聯(lián)合免疫的免疫原性。 方法：用IPTG誘導(dǎo)Ag85A-pET30a大腸桿菌基因工程菌和Ag85B-pET24b大腸桿菌基因工程菌表達(dá)，通過(guò)固化Ni2+樹(shù)脂純化后，經(jīng)SDS-PAGE電泳鑒定目的蛋白的表達(dá)及純化情況。 將60只雌性BALB/c小鼠隨機(jī)分為下列6組：（1）陰性對(duì)照組：PBS組，（2）陽(yáng)性對(duì)照組：BCG組，（3）陽(yáng)性對(duì)照組：MV組，（4）實(shí)驗(yàn)組：rAg85A+MV組，（5）實(shí)驗(yàn)組：rAg85B+MV組，（6）實(shí)驗(yàn)組：rAg85A+rAg85B+MV組。每2周免疫1次，共免疫3次。每周記錄小鼠體重變化。第3次免疫結(jié)束后第14天殺鼠，取肺臟、肝臟及脾臟稱重。通過(guò)ELISPOT法檢測(cè)脾臟分泌IFN-γ的T淋巴細(xì)胞斑點(diǎn)數(shù)；ELISA法檢測(cè)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；流式細(xì)胞術(shù)檢測(cè)全血單個(gè)核細(xì)胞內(nèi)分泌IFN-γ的Th1細(xì)胞百分比和分泌IL-4的Th2細(xì)胞百分比；ELISA法檢測(cè)脾淋巴細(xì)胞培養(yǎng)上清中IFN-γ和IL-4水平。 結(jié)果：Ag85A-pET30a大腸桿菌基因工程菌和Ag85B-pET24b大腸桿菌基因工程菌經(jīng)IPTG誘導(dǎo)成功后，以包涵體的形式表達(dá)。經(jīng)Ni2+柱純化后獲得目的蛋白rAg85A和rAg85B。 第3次免疫結(jié)束后第14天，六組小鼠的體重均較免疫開(kāi)始時(shí)顯著增加（P＜0.001），且三個(gè)實(shí)驗(yàn)組小鼠體重均略高于三個(gè)對(duì)照組；各組間小鼠脾臟重量、肝臟重量及二者的重量指數(shù)無(wú)顯著差別（P0.05），rAg85B+MV組小鼠肺臟指數(shù)較其它各組顯著增高（P0.05）。 ELISPOT結(jié)果顯示：三個(gè)實(shí)驗(yàn)組和兩個(gè)陽(yáng)性對(duì)照組小鼠的T淋巴細(xì)胞斑點(diǎn)數(shù)均明顯高于陰性對(duì)照組（P0.05）； 經(jīng)過(guò)3次免疫后，兩個(gè)陽(yáng)性對(duì)照組及三個(gè)實(shí)驗(yàn)組小鼠IgG、IgG1和IgG2a抗體水平較免疫前均顯著升高（P0.001）。第3次免疫結(jié)束后第14天，三個(gè)實(shí)驗(yàn)組小鼠血清抗體水平均顯著高于三個(gè)對(duì)照組（P0.001）； 流式細(xì)胞術(shù)結(jié)果顯示：rAg85A+rAg85B+MV組小鼠Th1細(xì)胞百分比明顯高于其余五組（P0.001），三個(gè)實(shí)驗(yàn)組Th1/Th2比值均明顯高于三個(gè)對(duì)照組（P0.05）； ELISA檢測(cè)脾淋巴細(xì)胞培養(yǎng)上清中IFN-γ和IL-4水平結(jié)果顯示：兩個(gè)陽(yáng)性對(duì)照組和三個(gè)實(shí)驗(yàn)組小鼠脾淋巴細(xì)胞培養(yǎng)上清IFN-γ水平均高于PBS組（P0.05），其中rAg85A+rAg85B+MV組較其它五組升高最顯著（P0.01）；IL-4的檢測(cè)結(jié)果低于試劑盒的靈敏度，未能檢測(cè)到。 結(jié)論：Ag85A-pET30a大腸桿菌基因工程菌和Ag85B-pET24b大腸桿菌基因工程菌以包涵體形式表達(dá)rAg85A和rAg85B蛋白，MV本身具有較好的佐劑作用，，這兩種重組蛋白均能夠增強(qiáng)MV的免疫原性，與MV聯(lián)合免疫可誘導(dǎo)機(jī)體產(chǎn)生較高水平的Th1型細(xì)胞免疫應(yīng)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- 緯 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- 緯 and the percentage of Th2 cells secreting IL-4 in whole blood mononuclear cells were detected by flow cytometry, and the levels of IFN- 緯 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- 緯 and IL-4 in the supernatant of splenic lymphocyte culture were detected by ELISA. The results showed that the levels of IFN- 緯 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- 緯 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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R392.1

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