A型肉毒毒素多表位串联体的设计、表达及免疫原性分析

发布时间：2018-01-11 12:17

本文关键词：A型肉毒毒素多表位串联体的设计、表达及免疫原性分析　出处：《中国人民解放军军事医学科学院》2007年硕士论文　论文类型：学位论文

【摘要】： 肉毒神经毒素(BoNT)是自然界所发现的生物毒素(包括化学毒剂)中毒性最强的物质。由于BoNT毒性强并且极容易被恐怖分子利用，国际社会已将其列为重要的毒素战剂和最具威胁的生物恐怖剂之一。针对BoNT感染引起的肉毒中毒尚无较好的治疗手段，到目前为止也没有实用型的抗毒素问世，因而其疫苗的研究就显得更为重要。虽然类毒素、特别是其重链重组亚单位疫苗具有很好的保护作用，但均存在着不可忽视的潜在的副作用。多表位疫苗的提出为BoNT疫苗的研发提供了新思路，一方面多表位疫苗能够集中优势的中和表位，去除对保护性免疫不利的结构或潜在的毒性片段；另一方面能优化组合BoNT各种型别的中和性表位，为研制多价疫苗奠定基础，以解决目前国际上多以单一型别BoNT为主研究重组基因工程疫苗的弊端。因此开展BoNT多表位疫苗的探索性研究具有十分重要的医学和社会意义。首先，本研究基于保守性高的BoNT／A的氨基酸序列，根据BioSun和LaserGene软件包中的表位分析相关参数，辅以对BoNT／A蛋白的二级结构的分析，综合预测了BoNT／A的B细胞表位。结果表明，BoNT／A轻链的142-150、284-292区段，重链的440-450、465-480、538-549、699-710、751-760、1087-1095、1224-1231、1263-1270区段是B细胞优势表位区的可能性较大。然后，遴选预测得到的B细胞表位和文献报道的表位，并加入适当的辅助性元件：通用T细胞表位和Linker，设计了多表位串联体A、B。根据大肠杆菌密码子偏好性对其基因进行优化的基础上，经重叠PCR方法合成串联体A、B的基因全长。并构建了克隆测序质粒pMD18-T-A、pMD18-T-B进行序列分析鉴定，结果表明，重叠PCR合成的基因序列均与密码子优化后的基因序列完全一致。然后用BamHⅠ、HindⅢ分别双酶切pMD18-T-A、pMD18-T-B载体和表达载体pQE-30，并连接获得重组表达质粒pQE-30A，pQE-30B。经过双酶切和PCR鉴定正确后，转化到E．coli M15[pREP4]感受态细胞中，构建相应的表达工程菌。用IPTG进行诱导表达，发现这两种重组蛋白都以包涵体形式存在，表达量分别达到全菌蛋白的70％、54％。SDS-PAGE分析显示，与预期蛋白的大小相符，预期蛋白的分子量分别为22.4×10~3、24.9×10~3。Western印迹和间接ELISA结果显示重组蛋白串联体A、B均可被其特异性马血清抗毒素识别，进一步验证了串联体A、B基因在工程菌中获得正确表达，，并具有良好的抗原性。Ni-NTA法纯化后分别获得纯度大于92.2％、99％的重组串联体A、B蛋白，然后采用透析复性法对其进行复性。接着，将串联体A、B分别与等体积的弗氏佐剂混合后免疫Balb／C小鼠，20μg／只，采用背部皮下多点注射的免疫途经。并以PBS作为对照。四免后血清效价达到10~3～10~4，说明设计表达的重组蛋白具有一定的免疫原性。四次免疫后一周，以2、5、10、20、40×LD_(50) BoNT／A腹腔攻击小鼠，观察小鼠存活天数。动物保护性试验发现，免疫组小鼠未表现出比对照组小鼠更好的抵抗天然毒素攻击的能力，并且ELISA及Western印迹试验表明，其免疫小鼠血清不能结合天然的BoNT／A。针对上述试验结果，在串联体A的基础上调整了设计方案，重新优化构建了三个串联体：串联体A′、C以及合成的表位串联体肽(串联体肽)。其中，串联体A′是用8M脲直接溶解串联体A重组蛋白的包涵体得到，即纯化后未经复性的串联体A；串联体C则是将串联体A的Th表位由C端移至N端。串联体肽是由文献报道的两个中和表位通过GGS相连构成。以串联体A′，C和串联体肽免疫Balb／C小鼠。攻毒试验表明新构建的三个串联体可以抵抗3×LD_(50)天然BoNT／A的攻击。该保护作用还不及文献报道的单个表位合成肽免疫效果，据此推测串联体A、B无保护作用的主要原因可能是选用的LinkerGGS没有起到有效的分隔作用。同时认为Th的位置对表位设计影响不大。本研究虽然未能获得具有良好保护性的串联体，但发现了一些问题，做了一些有意义的尝试和探索，为今后BoNT多表位疫苗的进一步研究，提供了参考和借鉴。
[Abstract]:Botulinum neurotoxin (BoNT) is a biological toxin found in nature (including chemical agents) the most toxic substances. Due to the toxicity of BoNT is strong and easy to be used by terrorists, the international community has been classified as important agents and toxins of biological agents of the most threatening for BoNT infection caused by botulism. There is no better treatment, so far there is no practical antitoxin available, thus to study the vaccine becomes more and more important. Although the toxoid, especially in the heavy chain of recombinant subunit vaccine has good protective effect, but there are noticeable potential side effects. The multi table provides a new idea is proposed for vaccine BoNT vaccine, a multi epitope vaccine can focus on the advantages of neutralizing epitopes, remove the structure of protective immunity against or potentially toxic fragments; on the other hand can Optimization of BoNT type neutralizing epitopes, laying a foundation for the development of multivalent vaccines, in order to solve the drawbacks of the current international more than a single type of BoNT based on recombinant gene engineering vaccine. Therefore to carry out exploratory study of BoNT multi epitope vaccine has important medical and social significance.
First of all, this study is based on the amino acid sequence of conserved high BoNT / A, according to the BioSun and LaserGene software package in the epitope analysis of the relevant parameters, analysis of two level structure of BoNT / A protein with the comprehensive prediction BoNT / A B cell epitopes. The results showed that the BoNT / A light chain 142-150284-292 section 440-450465-480538-549699-710751-7601087-10951224-12311263-1270 section is the possibility of heavy chain B cell epitope region greatly.
Then, the selection of the predicted B cell epitope and reported epitopes, and adding proper auxiliary components: General T cell epitope and Linker, designed a multi epitope tandem A, B. based on the optimized gene of Escherichia coli codon preference, synthesized by overlapping PCR the series of A gene, and constructed the full-length B. Cloning and sequencing of plasmid pMD18-T-A, pMD18-T-B sequences were analyzed and identified. The results show that the synthesis of PCR gene sequence of overlapping gene sequences were completely consistent with codon optimized. Then by BamH I, Hind III respectively digested pMD18-T-A, pMD18-T-B vector and expression vector pQE-30. And connect the recombinant plasmid pQE-30A pQE-30B. by double enzyme digestion and PCR after correct identification, transformed into E.coli M15[pREP4] competent cells, expressing engineering bacteria. The corresponding gene was induced by IPTG, found that two The recombinant protein existed in the form of inclusion body, respectively, and the protein expression of 70%, 54%.SDS-PAGE analysis showed that the protein with the expected molecular size match, expected protein were 22.4 * 10~3,24.9 * 10~3.Western blot and indirect ELISA showed that the recombinant protein A could be B concatemer, the specificity of horse serum antitoxin the identification, further validation of the A series, the B gene was correctly expressed in engineering bacteria, and has good antigenicity by.Ni-NTA after purification were obtained with purity of more than 92.2% series A, 99% of the recombinant B protein, then adopt renaturation on the dialysis renaturation method.
Then, the concatemer A, B respectively with equal volume of Freund's adjuvant immunized Balb / C mice, 20 g / only, the immune subcutaneous multi-point injection way. Taking PBS as the control group. After four free serum titer of 10~3 ~ 10~4, illustrate the design expression of recombinant protein with immune some of the original. One week after the four immunization, 2,5,10,20,40 * LD_ (50) BoNT / A intraperitoneal attack mice, mice survival time. Animal protection test, immunized mice showed no ability to resist the attack of natural toxins than control mice better, and ELISA and Western blotting assay showed that the serum of immunized mice cannot be combined with natural BoNT / A.
According to the above experimental results, based on A series body adjusts the design scheme, re optimization of constructed three concatemer: series A 'C, and the synthetic epitope tandem peptide (tandem peptide). Among them, A series' is inclusion body series A recombinant protein by direct dissolution of 8M urea, namely after purification without A refolding series; series C is a body from the C terminal to N terminal Th A. The series table series body is composed of peptide reported two epitopes by GGS linked together.
To concatenate A ', C and Balb / C tandem peptide immunization in mice. The challenge tests showed that the newly constructed three concatemer can resist 3 * LD_ (50) BoNT / A natural attack. The protective effect is less than the reported single epitope peptide immune effect, presumably in series A the protective effect of B, the main reason may be the selection of LinkerGGS did not play a separate role effectively. At the same time that the small table position of Th design. Although this study failed to get good protection of the series, but found some problems, do some meaningful exploration, for further research the future of BoNT multi epitope vaccines, to provide reference.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392

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