本文選題：結(jié)核病 + DNA疫苗��； 參考：《華中科技大學(xué)》2011年碩士論文

【摘要】：背景及目的 BCG作為預(yù)防TB的唯一疫苗在全球廣泛使用。發(fā)展中國家每年有超過90%的兒童接種BCG疫苗。但BCG對成人TB的保護(hù)效力卻存在很大波動,其機(jī)制尚不明確。目前,M.tb H37Rv基因組中共鑒定了16個BCG缺失區(qū)域即RD區(qū)。其中RD1區(qū)僅存在于致病性分枝桿菌中,在世界各地的BCG菌株中均缺失,被認(rèn)為是BCG最初減毒的主要決定因素;RD2區(qū)缺失,可以進(jìn)一步把世界范圍內(nèi)分布的不同BCG亞株分為早期株和晚期株。目前已經(jīng)明確RD1區(qū)丟失的同時對BCG的保護(hù)效果也產(chǎn)生了影響。那么,RD2區(qū)的缺失會給BCG的保護(hù)效力帶來什么樣的影響呢?是否會是造成BCG對成人TB保護(hù)性不穩(wěn)定的原因呢?針對這些問題,我們分別建立了RD1區(qū)所編碼的融合抗原ESAT-6-CFP10(rEC)和RD2區(qū)所編碼的融合抗原CFP21-MPT64(rCM)的原核表達(dá)系統(tǒng)和真核表達(dá)質(zhì)粒(我們的前期工作已研究rCM原核表達(dá)系統(tǒng)的構(gòu)建和免疫原性)。首先比較了兩種融合抗原在人群中的免疫原性;其次,在C57BL/6小鼠模型中比較研究這兩種重組真核質(zhì)粒作為DNA疫苗,在BCG初免聯(lián)合DNA疫苗增強(qiáng)策略中的免疫保護(hù)性;最后,進(jìn)一步探討RD2區(qū)缺失對BCG保護(hù)效力帶來的影響和初免增強(qiáng)策略的優(yōu)越性。 方法 1.利用PCR法分別擴(kuò)增獲得esat-6和cfp10的基因;用GeneSOEing技術(shù)構(gòu)建新的融合基因esat-6-cfp10,并將其分別克隆入原核表達(dá)載體pProEXHTb和真核表達(dá)載體pcDNA3.1(-)中,得到重組原核表達(dá)質(zhì)粒pPro610和重組真核質(zhì)粒pcD610。將表達(dá)融合蛋白rCM的重組原核質(zhì)粒pPro2164酶切,亞克隆入真核表達(dá)載體pcDNA3.1(-)中,得到重組真核質(zhì)粒pcD2164。 2.分別將pPro610和pPro2164轉(zhuǎn)化入E.coli BL21菌株,經(jīng)IPTG誘導(dǎo)表達(dá),Ni-NTA純化,尿素梯度透析復(fù)性獲取rEC和rCM蛋白。rEC蛋白的表達(dá)和純化的過程,分別用SDS-PAGE驗(yàn)證,并用anti-ESAT-6和anti-CFP10 Ab經(jīng)Western blotting驗(yàn)證。純化rEC和rCM蛋白的純度進(jìn)一步用SDS-PAGE證實(shí),并經(jīng)BCA法定量。 3.分別將pcD610和pcD2164轉(zhuǎn)化的E.coli DH5α擴(kuò)增,用Qiagen Plasmid Giga kit提取純化,并經(jīng)紫外吸收法定量。 4.對活動性肺結(jié)核的密切接觸者進(jìn)行WBIA(the whole blood IFN-γassay)試驗(yàn),比較兩種融合抗原rEC和rCM在人群中的免疫原性。 5.采取BCG(中國株)初免-pcD610或pcD2164增強(qiáng)的方式,免疫C57BL/6小鼠,通過ELISA法檢測外周血特異性抗體和體外抗原誘導(dǎo)脾淋巴細(xì)胞產(chǎn)生的IFN-γ、qRT-PCR法檢測肺臟組織細(xì)胞因子和iNOS mRNA表達(dá)水平等手段,分析比較不同免疫策略中疫苗在小鼠模型中的免疫原性。 6.對免疫后的小鼠進(jìn)行M.tb H37Rv毒株攻擊實(shí)驗(yàn),通過組織荷菌量、組織病理學(xué)改變等指標(biāo)評價(jià)不同免疫策略中疫苗的保護(hù)性。 結(jié)果 1.基因測序和酶切證實(shí)重組原核質(zhì)粒及重組真核質(zhì)粒構(gòu)建成功。 2. SDS-PAGE和Western blotting試驗(yàn)證實(shí)融合抗原rEC的原核表達(dá)和純化成功;純化的rEC和rCM經(jīng)SDS-PAGE證實(shí)。 3. rEC-WBIA刺激TST+健康人群產(chǎn)生了比TST-健康人群更高的IFN-γ(p 0.05 );rCM-WBIA刺激TST+健康人群產(chǎn)生了比TST-健康人群更高的IFN-(γp0.05); rCM-WBIA中TST+健康人群和TST-健康人群產(chǎn)生的IFN-γ水平,均高于rEC-WBIA中的結(jié)果,但差異均不具有統(tǒng)計(jì)學(xué)意義(p0.05)。 4.小鼠外周血抗原特異性IgG抗體分析結(jié)果表明針對融合蛋白rEC的特異性抗體反應(yīng),pcD610免疫組和BCG初免pcD610增強(qiáng)免疫組明顯高于其它組;針對融合蛋白rCM的特異性抗體反應(yīng),pcD2164免疫組和BCG初免聯(lián)合pcD2164增強(qiáng)免疫組最高。 5.抗原特異的小鼠脾淋巴細(xì)胞分泌的IFN-γ檢測結(jié)果為:重組質(zhì)粒免疫組和BCG與重組質(zhì)粒聯(lián)合免疫組的IFN-γ水平明顯高于BCG組,且BCG初免聯(lián)合pcD2164增強(qiáng)免疫組最高。 6.免疫小鼠肺臟組織中,與免疫保護(hù)有關(guān)的細(xì)胞因子IFN-γ、TNF-α和可抑制細(xì)菌生長的iNOS mRNA表達(dá),BCG初免DNA增強(qiáng)免疫組都表現(xiàn)出強(qiáng)于或與BCG組相當(dāng)?shù)乃?相對地,在抑制保護(hù)性免疫、利于細(xì)菌生長的調(diào)節(jié)性細(xì)胞因子TGF-β和IL-10 mRNA表達(dá)中,BCG初免DNA增強(qiáng)免疫組則處于較低的水平。 7. M.tb H37Rv攻擊4周后,小鼠組織荷菌量數(shù)據(jù)表明,疫苗免疫組都有不同程度的下降,其中BCG初免聯(lián)合DNA增強(qiáng)免疫組比僅接種BCG或DNA疫苗組更低,最低荷菌量出現(xiàn)在BCG初免聯(lián)合pcD2164增強(qiáng)免疫組中。 8.肺臟組織病理學(xué)檢查結(jié)果與荷菌量一致,采用BCG初免聯(lián)合DNA增強(qiáng)免疫方案組的病理改變評分明顯低于僅接種BCG或DNA疫苗組,且BCG初免聯(lián)合pcD2164增強(qiáng)免疫組最低。 結(jié)論 我們的研究表明pcD610和pcD2164是兩種在初免-增強(qiáng)策略中有著良好應(yīng)用前景的候選DNA疫苗,且支持RD2區(qū)缺失會對BCG保護(hù)效力產(chǎn)生重要影響的觀點(diǎn)。這一發(fā)現(xiàn)不但有助于篩選保護(hù)性最優(yōu)的BCG株用于新生兒接種,而且有助于構(gòu)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392

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相關(guān)期刊論文 前1條

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本文編號：1919237