细粒棘球绦虫重组Bb-Eg95-EgA31疫苗构建及其免疫机制研究

发布时间：2018-04-16 04:04

本文选题：细粒棘球绦虫 + 重组Bb-Eg95-EgA31疫苗　；参考：《重庆医科大学》2010年博士论文

【摘要】： 目的本研究拟从细粒棘球绦虫(Eg)原头节中扩增出Eg95和EgA31抗原编码基因,再通过基因拼接法(Gene SOEing)将两个单基因融合,构建Eg95-EgA31融合基因,并将其定向克隆到大肠杆菌-双歧杆菌穿梭表达载体pGEX-1λT,构建重组质粒pGEX-Eg95-EgA31,将该重组质粒电穿孔转化两歧双歧杆菌(Bb)以及大肠埃希菌BL21(DE3),构建细粒棘球绦虫重组Bb-Eg95-EgA31疫苗,研究Eg95-EgA31融合基因在大肠埃希菌BL21(DE3)中的表达效率,探讨重组Bb-Eg95-EgA31疫苗免疫小鼠后其免疫反应的动态变化和对Eg原头节攻击的保护力及其免疫机制,为囊型棘球蚴病(CE)的防治提供一种安全高效的新型疫苗。方法 1.从细粒棘球蚴包囊中分离原头节,超声粉碎后提取总RNA为模板,通过RT-PCR分别扩增Eg95和EgA31抗原编码基因,然后采用Gene SOEing法剪接Eg95和EgA31,得到Eg95-EgA31融合基因,再将其定向克隆到大肠杆菌-双歧杆菌穿梭表达载体pGEX-1λT中,构建重组质粒pGEX-Eg95-EgA31,将该重组质粒电穿孔转化Bb,构建细粒棘球绦虫重组Bb-Eg95-EgA31疫苗;将该重组质粒电穿孔转化大肠埃希菌BL21(DE3),经异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达后用SDS-PAGE和Western blot对表达产物进行分析和鉴定。 2.为了研究重组Bb-Eg95-EgA31疫苗免疫小鼠后不同时间点小鼠体内体液免疫和细胞免疫的变化,将重组Bb-Eg95-EgA31疫苗口服灌胃或鼻腔粘膜接种免疫BALB/c小鼠,免疫后0、2、4、6、8、10、12、14、16、18和20w用ELISA法检测血清IgG、IgG1、IgG2a、IgG2b、IgG3和IgE水平及脾淋巴细胞培养上清液中IFN-γ、IL-12、TNF-α和IL-10水平,MTT法检测脾淋巴细胞的增殖,流式细胞术(FCM)检测脾CD_4~+和CD_8~+ T细胞百分率。 3.为了研究重组Bb-Eg95-EgA31疫苗对Eg原头节攻击的保护力及其免疫机制,将重组Bb-Eg95-EgA31疫苗皮下注射、肌肉注射、鼻腔粘膜接种或口服免疫BALB/c小鼠,免疫后8周,用50个Eg原头节经腹腔注射攻击,以空质粒、Bb或MRS作对照,25周后处死小鼠,分离细粒棘球蚴包囊并称重,计算囊重减少率,ELISA法检测血清IgG、IgG1、IgG2a、IgG2b、IgG3和IgE水平及脾淋巴细胞培养上清液中IFN-γ、IL-12、TNF-α和IL-10水平,MTT法检测脾淋巴细胞的增殖,FCM检测脾CD_4~+和CD_8~+ T细胞百分率,Annexin V-FITC染色法检测脾细胞凋亡发生率。结果 1.琼脂糖凝胶电泳证实Eg95(471bp)、EgA31(500bp)抗原编码基因和Eg95-EgA31融合基因(1016bp)扩增成功;双酶切证实重组质粒pGEX- Eg95-EgA31构建成功;PCR证实重组Bb-Eg95-EgA31疫苗构建成功;SDS-PAGE证实重组质粒pGEX-Eg95-EgA31在大肠埃希菌BL21(DE3)中经IPTG诱导后能够表达分子量为62.5KDa左右的重组Eg95-EgA31融合蛋白,IPTG诱导3～5h重组蛋白表达量最高,占菌体总蛋白的18%,Western blot证实该融合蛋白具有特异的抗原性。 2.动态观察表明:与0周未免疫小鼠相比,口服免疫组小鼠血清IgG、IgG2a、IgG2b、IgG1、IgG3和IgE水平分别在免疫后8～10周、2～20周、2～20周、4～8周、6～12周和10周显著升高,分别在免疫后8、2、6、6、8和10周达最高水平;脾淋巴细胞培养上清液IFN-γ、IL-12、TNF-α和IL-10水平分别在免疫后2～16周、2～12周、2～6周和4～12周显著升高,分别在免疫后4、2、4和6周达最高水平;脾淋巴细胞增殖水平在免疫后4～10周显著升高,在免疫后6周达最高水平;脾CD_4~+ T细胞在免疫后4～10周显著升高,在免疫后6周达最高水平,CD_8~+ T细胞无明显变化。鼻腔粘膜接种组小鼠血清IgG、IgG2a、IgG2b、IgG1、IgG3和IgE水平分别在免疫后4～10周、4～20周、2～20周、2～12周、4～12周和10～12周显著升高,分别在免疫后10、6、10、8、8和10周达最高水平;脾淋巴细胞培养上清液IFN-γ、IL-12、TNF-α和IL-10水平分别在免疫后2～8周、2～12周、2～8周和6～16周显著升高,分别在免疫后2、2、4和8周达最高水平;脾淋巴细胞增殖水平在免疫后4～8周显著升高,在免疫后6周达最高水平;脾CD_4~+ T细胞在免疫后4～8周显著升高,在免疫后6周达最高水平,CD_8~+ T细胞无明显变化。 3.疫苗免疫加用Eg原头节攻击后发现,皮下注射组、肌肉注射组、鼻腔粘膜接种组和口服免疫组的囊重减少率分别为45.33%、41.33%、70.67%和62.67%;与对照组相比,免疫组小鼠血清IgG、IgG2a、IgG2b和IgG1水平显著升高,IgG3和IgE水平显著降低;脾IFN-γ、IL-12和TNF-α水平显著升高,IL-10水平显著降低;脾淋巴细胞显著增殖;脾CD_4~+和CD_8~+ T细胞显著增加;脾细胞凋亡发生率显著降低。鼻腔粘膜接种和口服灌胃是两种较好的免疫途径,且前者优于后者。结论 1.通过RT-PCR成功扩增出Eg95和EgA31抗原编码基因。 2.通过Gene SOEing法成功扩增出Eg95-EgA31融合基因。 3.成功构建了细粒棘球绦虫重组质粒pGEX-Eg95-EgA31。 4.成功构建了细粒棘球绦虫重组Bb-Eg95-EgA31疫苗。 5.细粒棘球绦虫重组质粒pGEX-Eg95-EgA31能在BL21(DE3)中经IPTG诱导表达,表达效率为18%,且表达的重组Eg95-EgA31融合蛋白具有特异的抗原性。 6.重组Bb-Eg95-EgA31疫苗可诱导小鼠产生有效的免疫应答反应。 7.重组Bb-Eg95-EgA31疫苗可诱导小鼠产生有效的保护性免疫应答,从而对抗Eg原头节的攻击。其诱导的保护力以鼻腔粘膜接种和口服免疫组最强,而鼻腔粘膜接种组优于口服免疫组。
[Abstract]:objective
This study from Echinococcus granulosus (Eg) amplified Eg95 and EgA31 antigen encoding gene protoscolex, by gene splicing method (Gene SOEing) two single fusion gene construct Eg95-EgA31 fusion gene, and cloned into e.coli-bifidobacterium shuttle expression vector pGEX-1 to construct recombinant lambda T. Plasmid pGEX-Eg95-EgA31, the recombinant plasmid was electroporated into Bifidobacterium bifidum (Bb) and Escherichia coli BL21 (DE3), construction of recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus, study of Eg95-EgA31 fusion gene in Escherichia coli BL21 (DE3) expression efficiency in the study of immune recombinant Bb-Eg95-EgA31 vaccine in mice after the dynamic changes of the immune response and of Eg protoscoleces attack protection and immune mechanism for cystic echinococcosis (CE) provides a new vaccine safe and effective prevention and treatment.
Method
1. from the hydatid cysts of Echinococcus granulosus protoscolex in separation, ultrasonic crushing after extracting total RNA as template, Eg95 and EgA31 antigen encoding gene were amplified by RT-PCR, then the Gene SOEing Eg95 and EgA31 Eg95-EgA31 splicing, fusion gene, and then cloned into Escherichia coli Bifidobacterium shuttle expression vector pGEX-1 T. The recombinant plasmid pGEX-Eg95-EgA31, the recombinant plasmid was electroporated into Bb to construct recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus; the recombinant plasmid was transformed into Escherichia coli BL21 (DE3), the isopropyl sulfide generation of beta galactosidase (-D- IPTG) were analyzed and identified by SDS-PAGE and Western on the expression of product expression induced by blot after.
2. for the change of humoral immunity and cellular immunity in mice at different time points of the study in mice immunized with recombinant Bb-Eg95-EgA31 vaccine, recombinant Bb-Eg95-EgA31 vaccine oral or nasal vaccination of BALB/c mice after immunization of 0,2,4,6,8,10,12,14,16,18 and 20W by ELISA assay of serum IgG, IgG1, IgG2a, IgG2b, IgG3 and IgE level and spleen lymphocyte culture the supernatant of IFN- gamma, IL-12, TNF- and IL-10 levels were detected by MTT, spleen lymphocyte proliferation, flow cytometry (FCM) and CD_8~+ T detection of spleen CD_4~+ cell percentage.
3. in order to protect the force of the recombinant Bb-Eg95-EgA31 vaccine of Eg protoscoleces attack and immune mechanism of recombinant Bb-Eg95-EgA31 vaccine, the subcutaneous injection, intramuscular injection, intranasal inoculation or oral immunization of BALB/c mice, 8 weeks after immunization with 50 Eg protoscoleces by intraperitoneal injection attacks, the empty plasmid, Bb or MRS as control, 25 weeks after the mice were killed, the separation of the hydatid cysts of Echinococcus granulosus and weighing, calculation of the cyst weight reduction rate, serum IgG, ELISA IgG1, IgG2a, IgG2b, IgG3 and IgE level and spleen lymphocyte culture supernatant of IFN- gamma, IL-12, TNF- and IL-10 levels were detected by MTT, spleen lymphocyte proliferation, spleen CD_4~+ and FCM detection CD_8~+ T cell percentage Annexin V-FITC staining incidence rate of detection of apoptosis of spleen cells.
Result
1. agarose gel electrophoresis confirmed that Eg95 (471bp), EgA31 (500bp) antigen encoding gene and Eg95-EgA31 fusion gene (1016bp) amplification; double enzyme digestion confirmed that the recombinant plasmid pGEX- Eg95-EgA31 was constructed successfully; PCR confirmed that the recombinant Bb-Eg95-EgA31 vaccine was successfully constructed; SDS-PAGE confirmed that the recombinant plasmid pGEX-Eg95-EgA31 in Escherichia coli BL21 (DE3) by recombinant Eg95-EgA31 IPTG after induction of expression of molecular weight is about 62.5KDa fusion protein induced by IPTG 3 ~ 5h recombinant protein expression was the highest, 18% of the total bacterial protein, Western blot confirmed that the fusion protein had specific antigenicity.
2. dynamic observation showed that compared with 0 weeks of immunization, oral immunization of mice serum IgG, IgG2a, IgG2b, IgG1, IgG3 and IgE level respectively in 8~10 weeks after immunization, 2~20 weeks, 2~20 weeks, 4~8 weeks, 6~12 weeks and 10 weeks were significantly increased, respectively at 8,2,6,6,8 and 10 weeks after immunization and reached the highest level spleen lymphocyte supernatant; IFN- gamma, IL-12, TNF- and IL-10 respectively in the alpha level after immunization for 2~16 weeks, 2~12 weeks, 2~6 weeks and 4~12 weeks were significantly increased, respectively at 4,2,4 and 6 weeks after immunization and reached the highest level; the proliferation of spleen lymphocyte in immune level increased significantly after 4~10 weeks, reached the highest level in 6 weeks of immunization after CD_4~+ T cells in immune; spleen increased significantly after 4~10 weeks, reached the highest level in 6 weeks after immunization, CD_8~+ T cells did not change significantly. Intranasal inoculation of mice serum IgG, IgG2a, IgG2b, IgG1, IgG3 and IgE respectively in 4~10 weeks after immunization, 4~20 weeks, 2 ~ 20 weeks, 2~12 weeks, 4~12 weeks and 10~12 weeks were significantly increased, respectively at 10,6,10,8,8 and 10 weeks after immunization and reached the highest level; spleen lymphocyte culture supernatants of IFN- gamma, IL-12, TNF- and IL-10 respectively in the alpha level after immunization for 2~8 weeks, 2~12 weeks, 2~8 weeks and 6~16 weeks were significantly increased, respectively at 2,2,4 after immunization 8 weeks and reached the highest level; the proliferation of spleen lymphocyte in immune level increased significantly after 4~8 weeks, reached the highest level in 6 weeks after immunization; spleen CD_4~+ T cells in immunity increased significantly after 4~8 weeks, reached the highest level in 6 weeks after immunization, no significant change in CD_8~+ T cells.
The 3. plus vaccine by Eg EM protoscoleces, subcutaneous injection group, intramuscular injection group, intranasal inoculation group and oral immunization group sac weight reduction rate were 45.33%, 41.33%, 70.67% and 62.67%; compared with the control group, the sera of mice immunized by IgG, IgG2a, IgG2b and IgG1 increased significantly, IgG3 and the level of IgE decreased significantly; the spleen IFN- gamma, IL-12 and TNF- levels were significantly increased, IL-10 level decreased significantly; spleen lymphocyte proliferation; spleen CD_4~+ and CD_8~+ of T cells was significantly increased; spleen cell apoptosis rate decreased significantly. Intranasal inoculation and oral gavage is two better immune pathway, and the former is better than the latter.
conclusion
1. the Eg95 and EgA31 antigen coding genes were successfully amplified by RT-PCR.
2. the Eg95-EgA31 fusion gene was successfully amplified by Gene SOEing method.
3. the recombinant plasmid pGEX-Eg95-EgA31. of Echinococcus granulosus was successfully constructed.
4. the recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus was successfully constructed.
5. the recombinant plasmid pGEX-Eg95-EgA31 of Echinococcus granulosus can be induced by IPTG in BL21 (DE3), and the expression efficiency is 18%, and the expressed recombinant Eg95-EgA31 fusion protein has specific antigenicity.
6. the recombinant Bb-Eg95-EgA31 vaccine can induce effective immune response in mice.
7. recombinant Bb-Eg95-EgA31 vaccine can produce potent protective immune response in mice induced by Eg, to counter attack. The protoscolices induced protection to intranasal inoculation and oral immunization group was the strongest, and intranasal inoculation group than oral immunization group.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

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