發(fā)布時(shí)間：2018-09-01 15:09

【摘要】：人類免疫缺陷病毒(HIV)感染及其引起的艾滋病,是我國(guó)及全球所面臨的重大公共衛(wèi)生問題,嚴(yán)重危害人類健康。開發(fā)研制安全有效的HIV疫苗以及抗HIV藥物是控制HIV感染及艾滋病流行的重要措施。 HIV-1反式轉(zhuǎn)錄激活因子(trans-activator of transcription, Tat)是HIV-1在感染早期產(chǎn)生的一種重要調(diào)節(jié)蛋白,在病毒復(fù)制和感染致病中起重要作用。在HIV感染細(xì)胞內(nèi),Tat可反式激活病毒基因組轉(zhuǎn)錄的起始和延長(zhǎng),從而啟動(dòng)和促進(jìn)病毒的復(fù)制。此外,分泌和釋放至細(xì)胞外的Tat還具有多樣的胞外活性,在艾滋病的免疫抑制、艾滋病腦病和Kaposi肉瘤形成等病理過程中發(fā)揮重要作用,被稱為“病毒毒素”,也已成為HIV疫苗研究的重要候選抗原之一。 已有研究表明,盡管天然Tat蛋白的序列在HIV-1不同亞型毒株間具有高度的保守性,但其屬非折疊蛋白,結(jié)構(gòu)松散,使得天然Tat作為HIV疫苗的候選抗原,尚存在誘發(fā)的抗體尤其是免疫保護(hù)性抗體滴度低這一關(guān)鍵問題。因此,本研究根據(jù)天然Tat分子結(jié)構(gòu)及其各功能區(qū)特點(diǎn),構(gòu)建并原核表達(dá)了多種HIV-1 Tat突變體重組蛋白,以期提高其構(gòu)象穩(wěn)定性、免疫原性并能誘導(dǎo)出高滴度和持久的保護(hù)性抗體。 本研究分以下三個(gè)部分: 第一部分HIV-1 HXB2株截短的Tat突變體蛋白的原核表達(dá) 在分析Tat蛋白各功能區(qū)(N末端區(qū)(1-21aa)、半胱氨酸富集區(qū)(22-37aa)、核心區(qū)(38-48aa)、堿性氨基酸富集區(qū)(49-59aa)、谷氨酰胺富集區(qū)(60-72aa)以及C末端區(qū)(73-101aa))以及已有的研究工作基礎(chǔ)上,本研究構(gòu)建并原核表達(dá)了9種HIV-1 HXB2株截短的Tat突變體蛋白。具體方法如下:PCR方法分別獲得tat1-21、tat1-37、tat1-61、tat1-72、tat1-86、tat22-86、tat38-61、tat22-101(a)、tat22-101(b)基因片段,用T/A克隆法將各片段克隆至pMD18-T載體進(jìn)行測(cè)序。將測(cè)序正確的序列分別克隆到原核表達(dá)載體pPET-32a或pPEPTIDE2中,構(gòu)建其原核表達(dá)質(zhì)粒:pPEPTIDE2-tat1-21、pET-32a-tat1-37、pET-32a-tat1-61、pPEPTIDE2-tat1-72、pPEPTIDE2-tat1-86、pPEPTIDE2-tat22-86、pPEPTIDE2-tat38-61、pET-32a-tat22-101、pPEPTIDE2-tat22-101。將上述原核表達(dá)質(zhì)粒分別轉(zhuǎn)入E. coli BL21(DE3)中,經(jīng)IPTG誘導(dǎo)表達(dá),獲得9種截短的Tat突變體融合蛋白PEPTIDE2-Tat1-21、PET-32a-Tat1-37、PET-32a-Tat1-61、PEPTIDE2-Tat1-72、PEPTIDE2-Tat1-86、PEPTIDE2-Tat22-86、PEPTIDE2-Tat38-61、PET-32a-Tat22-101和PEPTIDE2-Tat22-101,其相對(duì)分子量分別約為25,400、22,000、23,100、31,300、33,200、26,900、31,100、26,400和32,600;并用Ni-NTA親和層析法純化目的融合蛋白。 Western blot鑒定結(jié)果表明,5種含Tat N末端的Tat突變體重組蛋白( PEPTIDE2-Tat1-21、PET-32a-Tat1-37、PET-32a-Tat1-61、PEPTIDE2-Tat1-72、PEPTIDE2-Tat1-86)均與小鼠抗Tat N末端單克隆抗體呈特異性反應(yīng),而不含N末端的Tat重組蛋白及兩種載體蛋白PET-32a和PEPTIDE2則與該單抗無結(jié)合反應(yīng)。ELISA檢測(cè)顯示,4種Tat N末端缺失的融合蛋白PEPTIDE2-Tat22-86、PEPTIDE2-Tat38-61、PET-32a-Tat22-101和PEPTIDE2-Tat22-101均與兔抗全長(zhǎng)Tat抗血清呈陽性結(jié)合反應(yīng),而兩種載體蛋白PET-32a和PEPTIDE2則無此反應(yīng),表明本研究成功構(gòu)建并表達(dá)出9種截短的Tat突變體融合蛋白。 第二部分Tat突變體重組蛋白的免疫原性分析 用含Tat N末端的PET-32a-Tat1-37、PET-32a-Tat1-48、PET-32a-Tat1-61、PEPTIDE2-Tat1-72、PEPTIDE2-Tat1-86突變體融合蛋白和天然全長(zhǎng)Tat融合蛋白(PET-32a-Tat1-101)分別免疫新西蘭兔,制備兔抗血清,以檢測(cè)各Tat突變體蛋白誘導(dǎo)產(chǎn)生抗Tat N末端抗體的差異;用缺失Tat N末端的突變體PET-32a-Tat22-101免疫新西蘭兔和BALB/c小鼠,制備其兔抗血清及小鼠抗血清,用以觀察PET-32a-Tat22-101誘導(dǎo)產(chǎn)生針對(duì)Tat N末端之外表位的抗體情況。 ELISA檢測(cè)結(jié)果顯示,上述Tat突變體融合蛋白和全長(zhǎng)Tat融合蛋白誘導(dǎo)產(chǎn)生的兔抗血清或小鼠抗血清均與全長(zhǎng)Tat蛋白呈特異性結(jié)合反應(yīng),表明Tat突變體融合蛋白較好地保留了免疫原性。然而,含Tat N末端的Tat突變體融合蛋白(PET-32a-Tat1-37、PET-32a-Tat1-48、PET-32a-Tat1-61、PEPTIDE2-Tat1-72和PEPTIDE2-Tat1-86)誘導(dǎo)產(chǎn)生的兔抗血清與Tat N末端合成肽sTat1-21反應(yīng)的抗體滴度均明顯偏低(抗體滴度為200至3200),而PET-32a-Tat1-101能夠誘導(dǎo)產(chǎn)生較高滴度(25600)的抗N末端抗體,提示抗Tat N末端抗體的產(chǎn)生可能具有一定的構(gòu)象依賴性。其次,Tat N末端缺失的突變體融合蛋白PET-32a-Tat22-101誘導(dǎo)產(chǎn)生的兔抗及鼠抗血清與PEPTIDE2-Tat38-101、PEPTIDE2-Tat49-101和PEPTIDE2-Tat60-101的反應(yīng)性均高于全長(zhǎng)Tat誘導(dǎo)產(chǎn)生的抗血清與它們的反應(yīng),提示Tat N末端免疫優(yōu)勢(shì)表位的存在有可能會(huì)掩蓋Tat其他表位抗體的產(chǎn)生。 第三部分Tat突變體重組蛋白的抗原性分析 用ELISA檢測(cè)觀察5種Tat突變體融合蛋白( PEPTIDE2-Tat1-21、PEPTIDE2-Tat1-72、PEPTIDE2-Tat1-86、PEPTIDE2-Tat22-86和PEPTIDE2-Tat22-101)分別與先前本課題組已制備的8種兔抗Tat血清(抗全長(zhǎng)Tat融合蛋白PET-32a-Tat1-101兔血清,以及抗PET-32a-Tat22-72、抗PET-32a-Tat38-101、抗PET-32a-TatCC、抗PET-32a-TatCN、抗PET-32a-TatΔC、抗PET-32a-TatB41-101C和抗PET-32a-TatB41-101N兔抗血清)的反應(yīng),結(jié)果顯示PEPTIDE2-Tat1-21與抗全長(zhǎng)Tat兔抗血清的反應(yīng)性最強(qiáng),明顯高于與其他兔抗血清的反應(yīng),表明Tat N末端Tat1-21是抗全長(zhǎng)Tat抗體所識(shí)別的重要抗原表位。此外,Tat N末端缺失的PEPTIDE2-Tat22-86和PEPTIDE2-Tat22-101兩種融合蛋白與所檢測(cè)的抗Tat兔抗血清的反應(yīng)存在明顯差異,兩者與多數(shù)兔抗血清均有良好結(jié)合反應(yīng),提示與天然Tat抗原相比,改造后的Tat突變體蛋白所誘導(dǎo)產(chǎn)生的抗Tat抗體的抗原表位譜已發(fā)生了一定改變。 用原核表達(dá)的天然Tat融合蛋白PET-32a-Tat1-101以及Tat突變體融合蛋白(PET-32a-Tat1-48、PEPTIDE2-Tat1-72、PEPTIDE2-Tat1-86、PEPTIDE2-Tat38-61、PEPTIDE2-Tat38-101、PEPTIDE2-Tat49-101、和PEPTIDE2-Tat60-101)分別檢測(cè)其與10例抗Tat抗體陽性艾滋病患者血清的結(jié)合反應(yīng)。ELISA結(jié)果顯示:(1)同一份抗Tat抗體陽性的艾滋病患者血清與不同的Tat突變體蛋白反應(yīng)強(qiáng)度不一,其中與Tat1-86蛋白的反應(yīng)性普遍較強(qiáng);(2)每一種Tat突變體蛋白與不同的抗Tat抗體陽性的艾滋病患者血清反應(yīng)的敏感性不同,顯示出一定的特性和共性,值得進(jìn)一步研究。 總結(jié): 1.本研究成功構(gòu)建9種HIV-1 HXB2株Tat突變體原核表達(dá)質(zhì)粒并經(jīng)E.coli表達(dá),獲得了9種截短的Tat突變體重組蛋白。 2.所構(gòu)建的5種Tat突變體蛋白( PET-32a-Tat1-37、PET-32a-Tat1-61、PEPTIDE-Tat1-72、PEPTIDE-Tat1-86和PET-32a-Tat22-101)均保留了較好的免疫原性,誘導(dǎo)出高滴度的與天然Tat性質(zhì)不同的抗體,特別是與全長(zhǎng)Tat蛋白相比, Tat22-101誘導(dǎo)產(chǎn)生的兔抗血清及小鼠抗血清均與N末端缺失的Tat抗原反應(yīng)性增高,提示Tat N末端免疫優(yōu)勢(shì)表位的存在有可能掩蓋Tat其他表位抗體的產(chǎn)生,實(shí)驗(yàn)結(jié)果不僅對(duì)HIV Tat分子的結(jié)構(gòu)及功能的研究具有重要意義,而且也為研發(fā)有效的新型Tat疫苗奠定了基礎(chǔ)。 3.在所構(gòu)建的含Tat N末端的融合蛋白中,只有全長(zhǎng)Tat融合蛋白能夠誘導(dǎo)產(chǎn)生高滴度的抗Tat N末端抗體,而含Tat N末端的截短的Tat突變體融合蛋白誘導(dǎo)產(chǎn)生抗Tat N末端抗體水平明顯降低,結(jié)果提示抗Tat N末端抗體的產(chǎn)生可能具有一定的構(gòu)象依賴性。 4.初步研究顯示,天然全長(zhǎng)Tat抗原及不同截短的Tat突變體重組蛋白與抗Tat抗體陽性的艾滋病患者血清反應(yīng)的強(qiáng)度和敏感性有所差異,其中Tat1-86與艾滋病患者血清的反應(yīng)性普遍較強(qiáng),初步研究結(jié)果為進(jìn)一步分析和篩選臨床用抗Tat抗體檢測(cè)的抗原候選物提供一定的實(shí)驗(yàn)依據(jù)。
[Abstract]:Human immunodeficiency virus (HIV) infection and AIDS caused by HIV are major public health problems facing China and the world, seriously endangering human health. The development of safe and effective HIV vaccines and anti-HIV drugs is an important measure to control HIV infection and AIDS epidemic.
HIV-1 trans-activator of transcription (Tat) is an important regulatory protein produced by HIV-1 in the early stage of infection and plays an important role in viral replication and pathogenesis. In HIV-infected cells, Tat transactivates the initiation and extension of viral genomic transcription, thereby initiating and promoting viral replication. Tat secreted and released to the extracellular also has a variety of extracellular activities, in the immunosuppression of AIDS, AIDS encephalopathy and Kaposi sarcoma formation and other pathological processes play an important role, known as the "viral toxin", has become an important candidate antigen for HIV vaccine research.
Studies have shown that natural Tat proteins are highly conserved among HIV-1 subtypes, but they are unfolded proteins with loose structure, which makes natural Tat a candidate antigen for HIV vaccines. In order to improve the conformational stability, immunogenicity and induce high titer and persistent protective antibodies, several recombinant HIV-1 Tat mutant proteins were constructed and expressed in prokaryotic cells.
This study is divided into three parts.
Part one prokaryotic expression of HIV-1 HXB2 strain truncated Tat mutant protein
Based on the analysis of the functional regions (N-terminal region (1-21aa), cysteine enrichment region (22-37aa), core region (38-48aa), alkaline amino acid enrichment region (49-59aa), glutamine enrichment region (60-72aa) and C-terminal region (73-101aa) of Tat protein, nine truncated Tat mutants of HIV-1 HXB2 were constructed and expressed in prokaryotic cells. The specific methods are as follows: Tat1-21, tat1-37, tat1-61, tat1-72, tat1-86, tat22-86, tat38-61, tat22-101 (a), tat22-101 (b) gene fragments were obtained by PCR, and the fragments were cloned into pMD18-T vector for sequencing by T/A cloning. The correct sequences were cloned into prokaryotic expression vector pPET-32a or pPEPTIDE2 to construct the prokaryotic expression vector. Nuclear expression plasmids: pPEPTIDE 2-tat 1-21, pET-32a-tat 1-21-21, pET-32a-tat 1-37, pET-32a-tat 1-61, pPEPTIDE 2-tat 1-61, pPEPTIDE 2-tat 1-72, pPEPTIDE 2-tat 1-72, pPEPTIDE 2-tat 1-86, pPEPTIDE 2-tat 22-86, pPEPTIDE 2-tat 22-86, pPEPTIDE 2-tat 38-61, pET-32a-tat 22-101, pPEPTIDE 2-tat 22-tat 21-101, pPEPTIDE2-tat 22-101. These plasmplasmplasmplasmplasmidwere transferred into Expression of 9 truncated Tat mutant fusion eggs White PEPTIDE 2-Tat 1-21, PET-32a-Tat 1-37, PET-32a-Tat 1-37, PET-32a-Tat 1-37, PET-32a-Tat 1-61, PET-32a-Tat 1-61, PEPTIDE 2-Tat 1-72, PEPTIDE 2-Tat 1-72, PEPTIDE 2-Tat 1-86, PEPTIDE 2-Tat 22-Tat 22-86, PEPTIDE 2-Tat 2-Tat 38-61, PET-32a-Tat 22-101 and PEPTTIDE2-Tat 22-101, 200,200,22,200,23,23,23,100,100,100,31,31,300,300,300,300,300,33,33,200,200 Purification of Purpose Fusion by Ni-NTA Affinity Chromatography Protein.
Western blot analysis showed that five Tat mutant recombinant proteins (PEPTIDE2-Tat1-21, PET-32a-Tat1-37, PET-32a-Tat1-61, PEPTIDE2-Tat1-72, PEPTIDE2-Tat1-86) had specific reactions with mouse anti-Tat N terminal monoclonal antibodies, but not with the recombinant Tat protein and two carrier proteins PET-32a and PEPTTIDE2. The results of ELISA showed that four Tat N terminal deleted fusion proteins PEPTIDE2-Tat22-86, PEPTIDE2-Tat38-61, PET-32a-Tat22-101 and PEPTTIDE2-Tat22-101 were positively bound to rabbit anti-full-length Tat antiserum, while two carrier proteins PET-32a and PEPTIDE2-Tat22-101 did not. 9 truncated Tat mutant fusion proteins were obtained.
The second part is the immunogenicity analysis of Tat mutant weight histone.
New Zealand rabbits were immunized with PET-32a-Tat1-37, PET-32a-Tat1-48, PET-32a-Tat1-61, PEPTIDE2-Tat1-72, PEPTIDE2-Tat1-86 mutant fusion protein and natural full-length Tat fusion protein (PET-32a-Tat1-101) to prepare rabbit antisera to detect the difference of anti-Tat N-terminal antibodies induced by Tat mutant proteins. The N-terminal mutant PET-32a-Tat22-101 was used to immunize New Zealand rabbits and BALB/c mice. The rabbit and mouse antisera were prepared to observe the production of antibodies against Tat N-terminal epitopes induced by PET-32a-Tat22-101.
ELISA results showed that the Tat mutant fusion protein and the rabbit antiserum or mouse antiserum induced by the full-length Tat fusion protein showed a specific binding reaction with the long-term Tat protein, indicating that the Tat mutant fusion protein retained immunogenicity. However, the Tat mutant fusion protein containing the terminal of Tat N (PET-32a-Tat 1-37, PET-Tat 1-37, PET-Tat 1-37) had a good immunogenicity. The titers of rabbit antisera induced by 32a-Tat 1-48, PET-32a-Tat 1-61, PEPTIDE 2-Tat 1-72 and PEPTIDE 2-Tat 1-86 were significantly lower than those induced by the synthetic peptide sTat 1-21 (antibody titers were 200 to 3200), while PET-32a-Tat 1-101 could induce the production of high titers (25600) of anti-N-terminal antibodies, suggesting the production of anti-Tat N-terminal antibodies. Secondly, the reactivity of rabbit and mouse antisera induced by Tat N-terminal deleted mutant fusion protein PET-32a-Tat 22-101 to PEPTIDE 2-Tat 38-101, PEPTIDE 2-Tat 49-101 and PEPTIDE 2-Tat 60-101 was higher than that induced by full-length Tat, suggesting that Tat N-terminal immunity is superior. The presence of potential epitopes may obscure the production of Tat epitopes.
The third part is the antigenicity analysis of Tat mutant weight histone.
The five Tat mutant fusion proteins (PEPTIDE2-Tat1-21, PEPTIDE2-Tat1-72, PEPTIDE2-Tat1-86, PEPTIDE2-Tat22-86, PEPTIDE2-Tat22-86 and PEPTTIDE2-Tat22-101) and eight rabbit anti-Tat sera (anti-full-length Tat fusion protein PET-32a-Tat1-101, anti-PET-32a-Tat22-72, anti-PET-32a-Tat38, anti-Tat 101) were detected by ELISA. The reactions of PET-32a-TatCC, anti-PET-32a-TatCN, anti-PET-32a-Tat C, anti-PET-32a-TatB41-101C and anti-PET-32a-TatB41-101N rabbit antiserum showed that PEPTIDE2-Tat1-21 had the strongest reactivity with anti-full-length Tat rabbit antiserum, significantly higher than with other rabbit antisera, suggesting that Tat N terminal Tat1-21 was important for the recognition of anti-full-length Tat antibody. In addition, there was a significant difference between the two fusion proteins, PEPTIDE 2-Tat 22-86 and PEPTIDE 2-Tat 22-101, which were deleted at the terminal of Tat N and the anti-Tat rabbit antisera detected. Both of them had a good binding reaction with most of the rabbit antisera, suggesting that the anti-Tat antibody induced by the modified Tat mutant protein was higher than that induced by the natural Tat antigen. The epitopes of the body have changed.
Natural Tat fusion protein PET-32a-Tat1-101 and Tat mutant fusion protein (PET-32a-Tat1-48, PEPTIDE2-Tat1-72, PEPTIDE2-Tat1-86, PEPTIDE2-Tat38-61, PEPTIDE2-Tat38-101, PEPTIDE2-Tat49-101, and PEPTTIDE2-Tat60-101) were used to detect their binding reactions to 10 anti-Tat antibody positive sera. The results showed that: (1) the serum of the same anti-Tat antibody positive AIDS patients reacted differently with different Tat mutant proteins, and the reactivity with Tat 1-86 protein was generally stronger; (2) The sensitivity of each Tat mutant protein to different anti-Tat antibody positive AIDS patients was different, showing certain characteristics and characteristics. Generality is worth further study.
Summary:
1. The prokaryotic expression plasmids of 9 Tat mutants of HIV-1 HXB2 strains were successfully constructed and expressed by E.coli.
2. The five Tat mutant proteins (PET-32a-Tat 1-37, PET-32a-Tat 1-61, PEPTIDE-Tat 1-72, PEPTIDE-Tat 1-86 and PET-32a-Tat 22-101) retained good immunogenicity and induced high titers of antibodies with different properties from natural Tat, especially the rabbit and mouse antisera induced by Tat 22-101 compared with full-length Tat protein. The increased reactivity of both sera to Tat antigen with N-terminal deletion suggests that the presence of Tat N-terminal immunodominant epitopes may mask the production of antibodies against other epitopes of Tat. The experimental results are not only of great significance to the study of the structure and function of HIV Tat molecules, but also lay a foundation for the development of an effective new type of Tat vaccine.
3. Among the constructed fusion proteins containing Tat N-terminal, only the full-length Tat fusion protein could induce high titer of anti-Tat N-terminal antibody, while the truncated Tat mutant fusion protein containing Tat N-terminal could induce the production of anti-Tat N-terminal antibody significantly decreased, suggesting that the production of anti-Tat N-terminal antibody might have some structure. Image dependency.
4. Preliminary studies showed that the serum reaction intensity and sensitivity of natural full-length Tat antigen and different truncated Tat mutant recombinant proteins were different from those of AIDS patients with positive anti-Tat antibody. Tat 1-86 was generally more responsive to AIDS patients. The preliminary results of this study will further analyze and screen anti-Tat antibodies for clinical use. Detection of antigen candidates provides some experimental evidence.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1

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相關(guān)期刊論文 前1條

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本文編號(hào)：2217569