表达融合蛋白CFP21-MPT64和ESAT-6-CFP10的DNA疫苗增强BCG初免小鼠抗结核病的保护性

发布时间：2018-05-21 12:41

  本文选题：结核病 + DNA疫苗　； 参考：《华中科技大学》2011年硕士论文

【摘要】：背景及目的 BCG作为预防TB的唯一疫苗在全球广泛使用。发展中国家每年有超过90%的儿童接种BCG疫苗。但BCG对成人TB的保护效力却存在很大波动,其机制尚不明确。目前,M.tb H37Rv基因组中共鉴定了16个BCG缺失区域即RD区。其中RD1区仅存在于致病性分枝杆菌中,在世界各地的BCG菌株中均缺失,被认为是BCG最初减毒的主要决定因素;RD2区缺失,可以进一步把世界范围内分布的不同BCG亚株分为早期株和晚期株。目前已经明确RD1区丢失的同时对BCG的保护效果也产生了影响。那么,RD2区的缺失会给BCG的保护效力带来什么样的影响呢?是否会是造成BCG对成人TB保护性不稳定的原因呢?针对这些问题,我们分别建立了RD1区所编码的融合抗原ESAT-6-CFP10(rEC)和RD2区所编码的融合抗原CFP21-MPT64(rCM)的原核表达系统和真核表达质粒(我们的前期工作已研究rCM原核表达系统的构建和免疫原性)。首先比较了两种融合抗原在人群中的免疫原性;其次,在C57BL/6小鼠模型中比较研究这两种重组真核质粒作为DNA疫苗,在BCG初免联合DNA疫苗增强策略中的免疫保护性;最后,进一步探讨RD2区缺失对BCG保护效力带来的影响和初免增强策略的优越性。 方法 1.利用PCR法分别扩增获得esat-6和cfp10的基因;用GeneSOEing技术构建新的融合基因esat-6-cfp10,并将其分别克隆入原核表达载体pProEXHTb和真核表达载体pcDNA3.1(-)中,得到重组原核表达质粒pPro610和重组真核质粒pcD610。将表达融合蛋白rCM的重组原核质粒pPro2164酶切,亚克隆入真核表达载体pcDNA3.1(-)中,得到重组真核质粒pcD2164。 2.分别将pPro610和pPro2164转化入E.coli BL21菌株,经IPTG诱导表达,Ni-NTA纯化,尿素梯度透析复性获取rEC和rCM蛋白。rEC蛋白的表达和纯化的过程,分别用SDS-PAGE验证,并用anti-ESAT-6和anti-CFP10 Ab经Western blotting验证。纯化rEC和rCM蛋白的纯度进一步用SDS-PAGE证实,并经BCA法定量。 3.分别将pcD610和pcD2164转化的E.coli DH5α扩增,用Qiagen Plasmid Giga kit提取纯化,并经紫外吸收法定量。 4.对活动性肺结核的密切接触者进行WBIA(the whole blood IFN-γassay)试验,比较两种融合抗原rEC和rCM在人群中的免疫原性。 5.采取BCG(中国株)初免-pcD610或pcD2164增强的方式,免疫C57BL/6小鼠,通过ELISA法检测外周血特异性抗体和体外抗原诱导脾淋巴细胞产生的IFN-γ、qRT-PCR法检测肺脏组织细胞因子和iNOS mRNA表达水平等手段,分析比较不同免疫策略中疫苗在小鼠模型中的免疫原性。 6.对免疫后的小鼠进行M.tb H37Rv毒株攻击实验,通过组织荷菌量、组织病理学改变等指标评价不同免疫策略中疫苗的保护性。 结果 1.基因测序和酶切证实重组原核质粒及重组真核质粒构建成功。 2. SDS-PAGE和Western blotting试验证实融合抗原rEC的原核表达和纯化成功;纯化的rEC和rCM经SDS-PAGE证实。 3. rEC-WBIA刺激TST+健康人群产生了比TST-健康人群更高的IFN-γ(p 0.05 );rCM-WBIA刺激TST+健康人群产生了比TST-健康人群更高的IFN-(γp0.05); rCM-WBIA中TST+健康人群和TST-健康人群产生的IFN-γ水平,均高于rEC-WBIA中的结果,但差异均不具有统计学意义(p0.05)。 4.小鼠外周血抗原特异性IgG抗体分析结果表明针对融合蛋白rEC的特异性抗体反应,pcD610免疫组和BCG初免pcD610增强免疫组明显高于其它组;针对融合蛋白rCM的特异性抗体反应,pcD2164免疫组和BCG初免联合pcD2164增强免疫组最高。 5.抗原特异的小鼠脾淋巴细胞分泌的IFN-γ检测结果为:重组质粒免疫组和BCG与重组质粒联合免疫组的IFN-γ水平明显高于BCG组,且BCG初免联合pcD2164增强免疫组最高。 6.免疫小鼠肺脏组织中,与免疫保护有关的细胞因子IFN-γ、TNF-α和可抑制细菌生长的iNOS mRNA表达,BCG初免DNA增强免疫组都表现出强于或与BCG组相当的水平;相对地,在抑制保护性免疫、利于细菌生长的调节性细胞因子TGF-β和IL-10 mRNA表达中,BCG初免DNA增强免疫组则处于较低的水平。 7. M.tb H37Rv攻击4周后,小鼠组织荷菌量数据表明,疫苗免疫组都有不同程度的下降,其中BCG初免联合DNA增强免疫组比仅接种BCG或DNA疫苗组更低,最低荷菌量出现在BCG初免联合pcD2164增强免疫组中。 8.肺脏组织病理学检查结果与荷菌量一致,采用BCG初免联合DNA增强免疫方案组的病理改变评分明显低于仅接种BCG或DNA疫苗组,且BCG初免联合pcD2164增强免疫组最低。 结论 我们的研究表明pcD610和pcD2164是两种在初免-增强策略中有着良好应用前景的候选DNA疫苗,且支持RD2区缺失会对BCG保护效力产生重要影响的观点。这一发现不但有助于筛选保护性最优的BCG株用于新生儿接种,而且有助于构建合理有效的抗TB新疫苗。
[Abstract]:Background and purpose
BCG is widely used worldwide as the only vaccine to prevent TB. More than 90% children are vaccinated with BCG vaccines every year in developing countries. However, the protective effectiveness of BCG for adult TB is very volatile and its mechanism is not clear. At present, the M.tb H37Rv genome has identified 16 BCG missing regions, that is, RD region. The RD1 region exists only in the pathogenicity branch. The absence of BCG strains all over the world is considered to be the main determinant of the initial detoxification of BCG, and the deletion of RD2 region can further divide the different BCG substrains in the world into early and late strains. At present, the loss of the RD1 region has been identified and the protective effect of BCG has also been affected. Then, the deletion of the RD2 region. What effect will it have on the protective effect of BCG? Is it the cause of BCG's protective instability in adult TB? For these problems, we set up the prokaryotic expression system and eukaryotic expression plasmid of the fusion antigen ESAT-6-CFP10 (rEC) encoded in the RD1 region and the fusion antigen CFP21-MPT64 (rCM) encoded by the RD2 region (I). Our previous work has studied the construction and immunogenicity of the rCM prokaryotic expression system. First, the immunogenicity of the two fusion antigens in the population was compared. Secondly, the two recombinant eukaryotic plasmids were compared and studied in the C57BL/6 mouse model as the DNA vaccine, and the immune protection in the BCG primer immunization combined with the DNA epidemic Miao Zengqiang strategy; finally, further further study was made. To explore the effect of RD2 deletion on BCG protection efficacy and the superiority of initial exemption enhancement strategy.
Method
1. the genes of ESAT-6 and CFP10 were amplified by PCR, and a new fusion gene esat-6-cfp10 was constructed by GeneSOEing technique and was cloned into the prokaryotic expression vector pProEXHTb and the eukaryotic expression vector pcDNA3.1 (-). The Recombinant Prokaryotic expression plasmid pPro610 and the recombinant true nucleate particles pcD610. will be reorganized to express the fusion protein rCM. The prokaryotic plasmid pPro2164 was digested and subcloned into eukaryotic expression vector pcDNA3.1 (-), and the recombinant eukaryotic plasmid pcD2164. was obtained.
2. pPro610 and pPro2164 were transformed into E.coli BL21 strains. The expression and purification process of rEC and rCM protein.REC protein were obtained by IPTG induced expression, Ni-NTA purification and urea gradient dialysis. The purity and purity of the purified protein and the purified protein were further verified by SDS-PAGE. S-PAGE was confirmed and quantified by BCA.
3. the E.coli DH5 alpha transformed by pcD610 and pcD2164 was amplified by Qiagen Plasmid Giga kit and quantified by ultraviolet absorption.
4. the WBIA (the whole blood IFN- assay) test was performed on close contacts of active pulmonary tuberculosis. The immunogenicity of the two fusion antigens rEC and rCM in the population was compared.
5. BCG (Chinese strain) was first immune to -pcD610 or pcD2164 enhancement, and C57BL/6 mice were immunized. IFN- gamma induced by specific antibodies in peripheral blood and in vitro antigen induced spleen lymphocyte were detected by ELISA, and the qRT-PCR method was used to detect the expression level of lung tissue cytokine and iNOS mRNA, and the vaccine in mice in different immunization strategies was analyzed and compared. The immunogenicity in the model.
6. the mice after immunization were attacked by M.tb H37Rv strain, and the protective effects of different immunization strategies were evaluated by tissue load and histopathological changes.
Result
1. gene sequencing and enzyme digestion confirmed that recombinant prokaryotic plasmid and recombinant eukaryotic plasmid were successfully constructed.
2. SDS-PAGE and Western blotting tests confirmed that prokaryotic expression and purification of fusion antigen rEC were successful; purified rEC and rCM were confirmed by SDS-PAGE.
3. rEC-WBIA stimulated TST+ healthy people to produce a higher IFN- gamma (P 0.05) than that of the TST- healthy population; rCM-WBIA stimulated TST+ healthy crowds to produce a higher IFN- (gamma P0.05) than that of the healthy population of TST- (gamma P0.05). Meaning (P0.05).
4. the analysis of antigen specific IgG antibody in peripheral blood of mice showed that the specific antibody response to the fusion protein rEC was significantly higher than that of the other groups in the pcD610 immunization group and the pcD610 enhanced immunization group of BCG, and the highest antibody response to the fusion protein rCM, the pcD2164 immunization group and the BCG initial immunity combined with the pcD2164 enhanced immune group.
The results of IFN- gamma detection in 5. antigen specific mice spleen lymphocytes were that the level of IFN- gamma in the recombinant plasmid immunization group and the combined BCG and recombinant plasmids was significantly higher than that in the BCG group, and the highest BCG immunization combined with the pcD2164 enhanced immunization group.
6. immune protection related cytokine IFN- gamma, TNF- alpha and iNOS mRNA expression that can inhibit bacterial growth in immunized mice, BCG primer DNA enhanced immune groups are stronger than or in the same level as those in the BCG group; relative, the inhibition of protective immunity and the expression of regulatory cytokine, TGF- beta and IL-10 mRNA, is beneficial to the growth of bacteria. In BCG, the initial immunization of DNA was lower in the immunization group.
After 4 weeks of 7. M.tb H37Rv attack, the data of mouse tissue bearing bacteria showed that the immunization group had different degrees of decline, of which the primary BCG immunization group was lower than the BCG or DNA vaccine group, and the lowest amount of bacteria was found in the BCG primer immunization group and the pcD2164 enhanced immune group.
8. the pathological results of lung histopathology were the same as those of the charged bacteria. The score of pathological changes in the immunization group with BCG first immunization combined with DNA was significantly lower than that of the only BCG or DNA vaccine group, and the lowest BCG immunization group and the pcD2164 enhanced immunization group were the lowest.
conclusion
Our study shows that pcD610 and pcD2164 are the two candidate DNA vaccines that have good prospects in the early immune enhancement strategy, and support the view that the absence of RD2 region will have an important impact on the effectiveness of BCG protection. This discovery not only helps to screen the best protective BCG plant for inoculation of newborn infants, but also helps to build a reasonable and effective method. Anti TB new vaccine.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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