【作者】 周必英；

【导师】 陈雅棠； 李文桂；

【作者基本信息】 重庆医科大学 ， 内科学， 2010， 博士

【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CD₄⁺和CD₈⁺ 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CD₄⁺和CD₈⁺ 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CD₄⁺ 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,CD₈⁺ 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CD₄⁺ 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,CD₈⁺ 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CD₄⁺和CD₈⁺ 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。更多还原

【Abstract】 ObjectiveEg95 and EgA31 coding gene was respectively amplified by RT-PCR from Echinococcus granulosus （Eg） protoscoleces.The Eg95-EgA31 fusion gene was obtained with Gene SOEing,then cloned into Escherichia coli-Bifidobacteria shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Eg95-EgA31.This recombinant plasmid was electroporated into Bifidobacteria bifidum （Bb） and E.coli BL2（DE3） to construct recombinant Bb-Eg95-EgA31 vaccine,with the purpose of exploring the expression of Eg95-EgA31 fusion gene in E.coli BL2（DE3）,discussing the dynamics of immune responses induced by the recombinant Bb vaccine in BALB/c mice,and meanwhile investigating the protection against challenge with protoscoleces and immune mechanism in mice after immunization with the recombinant Bb vaccine.This could provide a safe and effective vaccine for the control of cystic echinococcosis（CE）.Methods1. The total RNA was extracted from hydatid cyst protoscoleces shattered by ultrasound-breaking,then Eg95 and EgA31 coding gene was respectively amplified by RT-PCR from the total RNA.The Eg95-EgA31 fusion gene was obtained with Gene SOEing,then cloned into Escherichia coli-Bifidobacteria shuttle expression vector pGEX-1λT to construct recombinant plasmid pGEX-Eg95-EgA31.This recombinant plasmid was electroporated into Bifidobacteria bifidum （Bb） and E.coli BL2（DE3） to construct recombinant Bb-Eg95-EgA31 vaccine.The plasmid pGEX-Eg95-EgA31 was transformed into BL21（DE3） by electroporation and the Eg95-EgA31 fusion gene was expressed in the presence of isopropyl-β-D-thiogalactopyranosid（IPTG）.The recombinant Eg95-EgA31 fusion protein was analyzed and identified by SDS-PAGE and Western blot.2. To observe the dynamics of humoral and cellular immune responses in mice after immunization with recombinant Bb-Eg95-EgA31 vaccine.BALB/c mice were immunized orally or intranasally by the vaccine.On week 0,2,4,6,8,10,12,14,16,18 and 20 after vaccination,the level of IgG,IgG subclass and IgE in sera was determined by ELISA.The level of IFN-γ,IL-12,TNF-αand IL-10 in splenocyte culture supernatant was measured by ELISA.The level of proliferation in splenocytes was tested by MTT.The percentage of CD₄⁺ and CD₈⁺ T cells in splenocytes was determined by flow cytometry（FCM）.3. To explore protection against challenge with protoscoleces and immune mechanism in mice after immunization with the recombinant Bb vaccine.BALB/c mice were immunized subcutaneously, intramuscularly,orally or intranasally by the vaccine,blank vector,Bb or MRS served as control groups,respectively.All mice were challenged with 50 protoscoleces in the 8th week after immunization and killed in the 25th week after challenge.The weight of hydatid cyst was measured and the reduction rate of the weight was calculated.The level of IgG,IgG subclass and IgE in sera was determined by ELISA.The level of IFN-γ,IL-12,TNF-αand IL-10 in splenocyte culture supernatant was measured by ELISA.The level of proliferation in splenocytes was tested by MTT.The percentage of CD₄⁺ and CD₈⁺ T cells in splenocytes was determined by FCM.The apoptotic rate of splenocytes was determined by Annexin V-FITC staining method.Results1. Eg95（471bp） and EgA31（500bp） coding genes,and Eg95-EgA31 fusion gene were successfully amplified by agarose gel electrophoresis analysis.The recombinant plasmid pGEX-Eg95-EgA31 was successfully constructed by restriction analysis.The recombinant Bb-Eg95-EgA31 vaccine of Echinococcus granulosus was successfully constructed by PCR.SDS-PAGE showed that the recombinant Eg95-EgA31 fusion protein was successfully expressed in E.coli BL2（DE3） in the presence of IPTG,and the relative molecular weight of the protein was approximately 62.5kDa.The expression level the protein reached highest at 3～5h after induction,and the expression efficiency of the protein was 18% of the total bacterial proteins.Western blot showed that the expressed fusion protein showed specific antigenicity.2. Dynamic observation showed as follows:Compared with 0 week non immunization group,in the oral immunization group,the level of IgG,IgG1,IgG2a,IgG2b,IgG3 and IgE significantly increased in the 8～10th week,4～8th week,2～20th week,2～20th week,6～12th week,and 10th week after immunization,respectively,and peaked in the 8th,6th,2th,6th,8th and 10th week after immunization,respectively.The level of IFN-γ,IL-12,TNF-αand IL-10 significantly increased in the 2～16th week,2～12th week,2～6th week and 4～12th week after immunization,respectively,and peaked in the 4th,2th,4th and 6th week after immunization,respectively.The level of splenocyte proliferation significantly increased in the 4～10th week after immunization,and peaked in the 6th week after immunization.The percentage of CD₄⁺ T in splenocytes significantly increased in the 4～10th week after immunization,and peaked in the 6th week after immunization.The percentage of CD₈⁺ T in splenocytes had no obvious change.In the intranasal immunization group,the level of IgG,IgG1,IgG2a,IgG2b,IgG3 and IgE significantly increased in the 4～10th week,2～12th week,4～20th week,2～20th week,4～12th week and 10～12th week after immunization,respectively,and peaked in the 10th,8th,6th,10th,8th and 10th week after immunization,respectively.The level of IFN-γ,IL-12,TNF-αand IL-10 significantly increased in the 2～8th week,2～12th week,2～8th week and 6～16th week after immunization,respectively,and peaked in the 2th,2th,4th and 8th week after immunization,respectively.The level of splenocyte proliferation significantly increased in the 4～8th week after immunization,and peaked in the 6th week after immunization.The percentage of CD₄⁺ T in splenocytes significantly increased in the 4～8th week after immunization,and peaked in the 6th week after immunization.The percentage of CD₈⁺ T in splenocytes had no obvious change.3. Protective experiments found that the hydatid cyst weight was reduced by 45.33%,41.33%,70.67% and 62.67% in the subcutaneous,intramuscular,oral or intranasal immunization group,respectively.Compared with the control group,in the immunization groups,the level of IgG,IgG2a,IgG2b and IgG1 significantly increased while the level of IgG3 and IgE significantly decreased.The level of IFN-γ,IL-12 and TNF-αsignificantly increased while the level of IL-10 significantly decreased.The level of splenocyte proliferation significantly increased.The percentage of CD4+ and CD₈⁺ T of splenocytes were significantly increased.The apoptotic rate of splenocytes significantly decreased.Conclusion1. Eg95 and EgA31 coding genes were successfully cloned by RT-PCR.2. The Eg95-EgA31 fusion gene was successfully amplified by Gene SOEing.3. The recombinant plasmid pGEX-Eg95-EgA31 was successfully constructed.4. The recombinant Bb-Eg95-EgA31 vaccine was successfully constructed.5. BL21（pGEX-Eg95-EgA31） could express recombinant Eg95-EgA31 fusion protein in the presence of IPTG.The expression efficiency was 18%,and the expressed fusion protein showed specific antigenicity.6. The recombinant Bb-Eg95-EgA31 vaccine could induce effective immune responses in mice.7. The recombinant Bb-Eg95-EgA31 vaccine could induce protective immune responses in mice,which could be responsible for protection against the challenge with Eg protoscoleces. Intranasal inoculation and oral vaccination are two better immune routes,and the former is better than the latter.更多还原