人类免疫缺陷病毒1型Tat不同突变体重组蛋白的构建及其免疫原性和抗原性分析
发布时间：2018-09-01 15:09
【摘要】：人类免疫缺陷病毒(HIV)感染及其引起的艾滋病,是我国及全球所面临的重大公共卫生问题,严重危害人类健康。开发研制安全有效的HIV疫苗以及抗HIV药物是控制HIV感染及艾滋病流行的重要措施。 HIV-1反式转录激活因子(trans-activator of transcription, Tat)是HIV-1在感染早期产生的一种重要调节蛋白,在病毒复制和感染致病中起重要作用。在HIV感染细胞内,Tat可反式激活病毒基因组转录的起始和延长,从而启动和促进病毒的复制。此外,分泌和释放至细胞外的Tat还具有多样的胞外活性,在艾滋病的免疫抑制、艾滋病脑病和Kaposi肉瘤形成等病理过程中发挥重要作用,被称为“病毒毒素”,也已成为HIV疫苗研究的重要候选抗原之一。 已有研究表明,尽管天然Tat蛋白的序列在HIV-1不同亚型毒株间具有高度的保守性,但其属非折叠蛋白,结构松散,使得天然Tat作为HIV疫苗的候选抗原,尚存在诱发的抗体尤其是免疫保护性抗体滴度低这一关键问题。因此,本研究根据天然Tat分子结构及其各功能区特点,构建并原核表达了多种HIV-1 Tat突变体重组蛋白,以期提高其构象稳定性、免疫原性并能诱导出高滴度和持久的保护性抗体。 本研究分以下三个部分: 第一部分HIV-1 HXB2株截短的Tat突变体蛋白的原核表达 在分析Tat蛋白各功能区(N末端区(1-21aa)、半胱氨酸富集区(22-37aa)、核心区(38-48aa)、碱性氨基酸富集区(49-59aa)、谷氨酰胺富集区(60-72aa)以及C末端区(73-101aa))以及已有的研究工作基础上,本研究构建并原核表达了9种HIV-1 HXB2株截短的Tat突变体蛋白。具体方法如下:PCR方法分别获得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克隆法将各片段克隆至pMD18-T载体进行测序。将测序正确的序列分别克隆到原核表达载体pPET-32a或pPEPTIDE2中,构建其原核表达质粒: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。将上述原核表达质粒分别转入E. coli BL21(DE3)中,经IPTG诱导表达,获得9种截短的Tat突变体融合蛋白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,其相对分子量分别约为25,400、22,000、23,100、31,300、33,200、26,900、31,100、26,400和32,600;并用Ni-NTA亲和层析法纯化目的融合蛋白。 Western blot鉴定结果表明,5种含Tat N末端的Tat突变体重组蛋白( PEPTIDE2-Tat1-21、PET-32a-Tat1-37、PET-32a-Tat1-61、PEPTIDE2-Tat1-72、PEPTIDE2-Tat1-86)均与小鼠抗Tat N末端单克隆抗体呈特异性反应,而不含N末端的Tat重组蛋白及两种载体蛋白PET-32a和PEPTIDE2则与该单抗无结合反应。ELISA检测显示,4种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均与兔抗全长Tat抗血清呈阳性结合反应,而两种载体蛋白PET-32a和PEPTIDE2则无此反应,表明本研究成功构建并表达出9种截短的Tat突变体融合蛋白。 第二部分Tat突变体重组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突变体融合蛋白和天然全长Tat融合蛋白(PET-32a-Tat1-101)分别免疫新西兰兔,制备兔抗血清,以检测各Tat突变体蛋白诱导产生抗Tat N末端抗体的差异;用缺失Tat N末端的突变体PET-32a-Tat22-101免疫新西兰兔和BALB/c小鼠,制备其兔抗血清及小鼠抗血清,用以观察PET-32a-Tat22-101诱导产生针对Tat N末端之外表位的抗体情况。 ELISA检测结果显示,上述Tat突变体融合蛋白和全长Tat融合蛋白诱导产生的兔抗血清或小鼠抗血清均与全长Tat蛋白呈特异性结合反应,表明Tat突变体融合蛋白较好地保留了免疫原性。然而,含Tat N末端的Tat突变体融合蛋白(PET-32a-Tat1-37、PET-32a-Tat1-48、PET-32a-Tat1-61、PEPTIDE2-Tat1-72和PEPTIDE2-Tat1-86)诱导产生的兔抗血清与Tat N末端合成肽sTat1-21反应的抗体滴度均明显偏低(抗体滴度为200至3200),而PET-32a-Tat1-101能够诱导产生较高滴度(25600)的抗N末端抗体,提示抗Tat N末端抗体的产生可能具有一定的构象依赖性。其次,Tat N末端缺失的突变体融合蛋白PET-32a-Tat22-101诱导产生的兔抗及鼠抗血清与PEPTIDE2-Tat38-101、PEPTIDE2-Tat49-101和PEPTIDE2-Tat60-101的反应性均高于全长Tat诱导产生的抗血清与它们的反应,提示Tat N末端免疫优势表位的存在有可能会掩盖Tat其他表位抗体的产生。 第三部分Tat突变体重组蛋白的抗原性分析 用ELISA检测观察5种Tat突变体融合蛋白( PEPTIDE2-Tat1-21、PEPTIDE2-Tat1-72、PEPTIDE2-Tat1-86、PEPTIDE2-Tat22-86和PEPTIDE2-Tat22-101)分别与先前本课题组已制备的8种兔抗Tat血清(抗全长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)的反应,结果显示PEPTIDE2-Tat1-21与抗全长Tat兔抗血清的反应性最强,明显高于与其他兔抗血清的反应,表明Tat N末端Tat1-21是抗全长Tat抗体所识别的重要抗原表位。此外,Tat N末端缺失的PEPTIDE2-Tat22-86和PEPTIDE2-Tat22-101两种融合蛋白与所检测的抗Tat兔抗血清的反应存在明显差异,两者与多数兔抗血清均有良好结合反应,提示与天然Tat抗原相比,改造后的Tat突变体蛋白所诱导产生的抗Tat抗体的抗原表位谱已发生了一定改变。 用原核表达的天然Tat融合蛋白PET-32a-Tat1-101以及Tat突变体融合蛋白(PET-32a-Tat1-48、PEPTIDE2-Tat1-72、PEPTIDE2-Tat1-86、PEPTIDE2-Tat38-61、PEPTIDE2-Tat38-101、PEPTIDE2-Tat49-101、和PEPTIDE2-Tat60-101)分别检测其与10例抗Tat抗体阳性艾滋病患者血清的结合反应。ELISA结果显示:(1)同一份抗Tat抗体阳性的艾滋病患者血清与不同的Tat突变体蛋白反应强度不一,其中与Tat1-86蛋白的反应性普遍较强;(2)每一种Tat突变体蛋白与不同的抗Tat抗体阳性的艾滋病患者血清反应的敏感性不同,显示出一定的特性和共性,值得进一步研究。 总结: 1.本研究成功构建9种HIV-1 HXB2株Tat突变体原核表达质粒并经E.coli表达,获得了9种截短的Tat突变体重组蛋白。 2.所构建的5种Tat突变体蛋白( PET-32a-Tat1-37、PET-32a-Tat1-61、PEPTIDE-Tat1-72、PEPTIDE-Tat1-86和PET-32a-Tat22-101)均保留了较好的免疫原性,诱导出高滴度的与天然Tat性质不同的抗体,特别是与全长Tat蛋白相比, Tat22-101诱导产生的兔抗血清及小鼠抗血清均与N末端缺失的Tat抗原反应性增高,提示Tat N末端免疫优势表位的存在有可能掩盖Tat其他表位抗体的产生,实验结果不仅对HIV Tat分子的结构及功能的研究具有重要意义,而且也为研发有效的新型Tat疫苗奠定了基础。 3.在所构建的含Tat N末端的融合蛋白中,只有全长Tat融合蛋白能够诱导产生高滴度的抗Tat N末端抗体,而含Tat N末端的截短的Tat突变体融合蛋白诱导产生抗Tat N末端抗体水平明显降低,结果提示抗Tat N末端抗体的产生可能具有一定的构象依赖性。 4.初步研究显示,天然全长Tat抗原及不同截短的Tat突变体重组蛋白与抗Tat抗体阳性的艾滋病患者血清反应的强度和敏感性有所差异,其中Tat1-86与艾滋病患者血清的反应性普遍较强,初步研究结果为进一步分析和筛选临床用抗Tat抗体检测的抗原候选物提供一定的实验依据。
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
本文编号：2217569
[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.
【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1
【参考文献】
相关期刊论文 前1条
1 ;CTL Responses to Regulatory Proteins Tat and Rev in HIV-1 B’/C Virus-Infected Individuals[J];Biomedical and Environmental Sciences;2008年04期
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