肉毒毒素重组基因工程疫苗研究

发布时间：2018-06-07 18:42

本文选题：肉毒毒素 + 重链受体结合区Hc　；参考：《中国人民解放军军事医学科学院》2017年硕士论文

【摘要】：肉毒毒素又被称为肉毒神经毒素,是由厌氧的革兰氏阳性菌肉毒梭菌产生的外毒素,是已知毒性最强的蛋白,根据其抗原性不同可以分为7种类型(BoNT/A-BoNT/G),A、B、E和F型可以导致人类中毒,其中A型肉毒毒素中毒致死率最高。各型肉毒毒素分子结构比较相似,分子量大小约150 kDa,由二硫键连接一个100 kDa的重链(HC)和一个50 kDa的轻链(LC)组成。肉毒毒素重链羧基端(HC)负责与细胞受体的结合,重链氨基端(HN)介导L链的跨膜转运;轻链具有锌离子内肽酶活性,能够将可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体(SNARE)切割而抑制神经递质的传递,最终会导致骨骼肌麻痹,呼吸衰竭而死亡。肉毒毒素生产工艺简单,稳定性良好,具有极强的致死性,是一种潜在的生物武器,极容易被制备成生物战剂应用。此外,近些年来由于肉毒毒素导致的中毒事件也时有发生,因此,对于预防和治疗肉毒毒素中毒的研究具有十分重要的意义。接种疫苗是一种有效预防肉毒毒素中毒的策略。鉴于早期研制的类毒素疫苗存在较多的缺陷,所以一直未能得到十分广泛的应用,因此研究新型肉毒毒素疫苗成为一种迫切的需要。破伤风毒素(TeNT)和肉毒毒素在结构上有较多的相似之处,所以借助于破伤风毒素Hc片段亚单位疫苗研究成功的策略来研制肉毒毒素亚单位疫苗。肉毒毒素受体结合区Hc能介导毒素特异性结合到神经肌肉接点处的神经末梢,此外,Hc片段不存在锌肽链内切酶活性,没有毒性,免疫动物后能刺激产生保护性免疫反应。基于这一重要发现,研究人员开始以肉毒毒素受体结合区Hc为靶标,开展新型肉毒毒素重组亚单位疫苗和核酸疫苗的研究。DNA疫苗是将编码抗原的基因克隆到真核表达载体中作为疫苗,免疫动物后该载体会在体内表达抗原,继而刺激机体产生免疫反应和保护作用。DNA疫苗具有很多优点:生产工艺简单、产品易纯化、便于储存,在接种者体内表达抗原,并且能够产生体液免疫和细胞免疫反应,然而,DNA免疫原性较低,不足以提供足够的保护作用,距离真正的人类临床应用还有一定的差距,需要进一步提高与放大y|DNA的免疫效果。树突状细胞(DCs)是体内已知最强的专职抗原递呈细胞,在抗原递呈和启动机体免疫应答中具有十分重要的作用。DEC205是表达于树突状细胞表面的内吞性受体,能够高效地介导抗原的摄取、加工处理以及递呈。为了增强肉毒毒素DNA疫苗的免疫原性,本研究开展了A型肉毒毒素受体结合区Hc抗原靶向DCs的DNA疫苗研究工作。将编码抗DEC205的抗体基因(scDEC)和编码A型肉毒毒素受体结合区基因(AHc)融合,并克隆到真核表达载体pVAX1中,构建了一个DCs靶向的DNA疫苗(pVAX1-scDEC-AHc),同时还设置了非靶向的疫苗载体(pVAX1-SAHc)和阴性对照(pVAX1)。免疫小鼠后证明该DCs靶向的DNA疫苗确实能够增强抗原特异性体液和细胞免疫反应;相比于非靶向DNA疫苗,其抗体滴度增强了2.5倍,细胞免疫反应也有较大程度的增强。抗体亚型测定的结果表明该DNA疫苗免疫小鼠后产生了以IgG1为主的抗体,即免疫反应以Th2型免疫应答途径为主。保护性试验结果证明pVAX1-scDEC-AHc疫苗免疫后能够刺激机体产生强的保护性免疫反应,30μg剂量免疫两次之后即能对103 LD50的A型肉毒毒素产生完全保护作用,而pVAX1-SAHc疫苗仅为12.5%的保护效率;三次免疫之后对104 LD50的A型肉毒毒素具有87.5%的保护效率,pVAX1-SAHc疫苗仅为12.5%的保护效率,这些结果表明pVAX1-scDEC-AHc DNA疫苗相对于pVAX1-SAHc疫苗具有更高的保护效率。通过脾细胞流式分析和免疫注射部位肌肉组织免疫组化实验研究了该DNA疫苗的DCs靶向效应,结果表明pVAX1-scDEC-AHc疫苗免疫后能够在免疫注射部位募集更多的DCs,促进其成熟,并且能够高效地促进免疫器官中的DCs进行增殖和成熟,这些结果提示该DNA疫苗能够增强免疫反应可能要归功于这种DCs靶向作用。本研究工作还对原核表达的可溶性B型肉毒毒素重组亚单位疫苗的免疫原性进行了研究。本实验将B型肉毒毒素受体结合区Hc段基因克隆到pTIG-Trx原核表达载体,利用大肠杆菌BL21(DE3)菌株实现了BHc的可溶性表达,破菌上清用HisTrapTM HP蛋白纯化柱和Capto TM MMC层析柱进行纯化,纯化到了纯度较高的带His标签的和无His标签的BHc抗原。免疫小鼠研究该大肠杆菌表达的重组BHc的免疫原性和保护效果,结果表明1μg该重组BHc单剂量免疫小鼠就能对103 LD50的B型肉毒毒素产生完全保护作用,两次免疫则可以对104 LD50的B型肉毒毒素产生完全保护作用。测定BHc-His免疫的小鼠血清中特异性抗体滴度平均可以达到1:110000,血清中和效价可以达到3.33 IU/ml;BHc免疫的小鼠血清中特异性抗体滴度平均可以达到1:70000,血清中和效价可以达到2.33 IU/ml;而酵母表达的mBHc免疫组仅为1:40000和1.25 IU/ml;这些结果表明大肠杆菌表达的重组BHc-His抗原具有良好的免疫原性和保护效果。此外,对于没有His标签的BHc抗原的免疫原性研究结果表明其同样具有很好的保护效果,可以作为候选的B型肉毒毒素重组亚单位疫苗进行下一步研究与开发。总结以上研究结果,在A型肉毒毒素Hc靶向DCs的DNA疫苗研究中证明了通过DEC205受体将抗原靶向到DCs的策略确实能够增强融合抗原免疫后的体液和细胞免疫反应,为增强DNA疫苗的免疫原性提供了一个新思路,并为其他型肉毒毒素或者其他病原体DNA疫苗的研究提供了新的技术平台。B型肉毒毒素重组亚单位疫苗的研究结果表明大肠杆菌表达的重组BHc抗原具有良好的免疫原性和保护效果,可以作为一个候选单价疫苗,并与原核表达的A、E和F型重组Hc抗原配制四价肉毒毒素亚单位疫苗。
[Abstract]:Botulinum toxin, also known as botulinum neurotoxin, is an exotoxin produced by the anaerobic gram positive bacteria Clostridium botulinum. It is the most toxic protein known. According to its antigenicity, it can be divided into 7 types (BoNT/A-BoNT/G). A, B, E and F can lead to human poisoning. Among them, the fatality rate of botulinum toxin type A is the highest. The molecular structure is similar, the molecular weight is about 150 kDa, which is composed of two sulfur bonds connecting a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC). The heavy chain carboxyl terminal (HC) of botulinum toxin is responsible for the binding to the cell receptor, the heavy chain amino terminal (HN) mediates the trans membrane transport of the L chain, and the light chain has the activity of zinc ion endopeptidase, and the soluble N- B can be used. Kmaleimide sensitive factor attachment protein receptor (SNARE) cuts and inhibits the transmission of neurotransmitters, eventually leading to skeletal muscle paralysis and respiratory failure. Botulinum toxin production process is simple, stable and highly lethal. It is a potential biological weapon and is very easy to be prepared into biological warfare agent. In addition, close to the application of biological warfare agent. The poisoning caused by botulinum toxin has also occurred in recent years, so the study on the prevention and treatment of botulinum poisoning is of great significance. Vaccination is an effective strategy to prevent botulism. Since the early developed toxoid vaccine has many defects, it has not been widely used. Therefore, the study of new botulinum toxin vaccine has become an urgent need. The structure of tetanus toxin (TeNT) and botulinum toxin are more similar in structure, so the subunit vaccine of botulinum toxin Hc fragment is developed with the help of the successful strategy of tetanus toxin subunit vaccine. The botulinum toxin receptor binding zone Hc can mediate The toxin specifically binds to the nerve endings at the neuromuscular junction. In addition, the Hc fragment does not exist in the zine chain endonuclease activity and does not have toxicity, and the immune animal can stimulate the protective immune response. Based on this important discovery, the researchers began to use the botulinum toxin receptor binding area Hc as a target to carry out the recombinant subunit of new botulinum toxin. The.DNA vaccine of vaccine and nucleic acid vaccine is to clone the gene encoding the antigen into the eukaryotic expression vector as a vaccine. After immunizing animals, the antigen is expressed in the body, and then the immune response and protective effect of the.DNA vaccine have many advantages: simple production, easy purification, easy storage, and inoculation. The body expresses antigen in the body and produces humoral and cellular immune responses. However, DNA has low immunogenicity and is not sufficient to provide sufficient protection. There is still a gap between the real human clinical application and the immune effect of y|DNA. Dendritic cells (DCs) are the strongest specific antigens known in the body. .DEC205 is an endocytic receptor expressed on the surface of dendritic cells, which is an endocytic receptor expressed on the surface of dendritic cells. It can efficiently mediate the uptake of antigen, processing and presenting. In order to enhance the immunogenicity of botulinum toxin DNA vaccine, the A type botulinum toxin receptor junction was developed in this study. The DNA vaccine of Hc antigen targeting DCs was studied. The antibody gene encoding anti DEC205 (scDEC) and the encoded A type botulinum binding region gene (AHc) were fused and cloned into the eukaryotic expression vector pVAX1, and a DCs targeted DNA vaccine (pVAX1-scDEC-AHc) was constructed, and a non targeted vaccine carrier (pVAX1-SAHc) and Yin were also set up. PVAX1. The immunized mice proved that the DCs targeted DNA vaccine was able to enhance the antigen specific humoral and cellular immune responses; the antibody titer increased by 2.5 times compared to the non targeted DNA vaccine and the cellular immune response was also greatly enhanced. The antibody subtype results showed that the DNA vaccine was produced after the vaccine was immune to mice. The IgG1 based antibody, that is, the immune response is dominated by the Th2 type immune response pathway. The protective test results show that the pVAX1-scDEC-AHc vaccine can stimulate the body to produce a strong protective immune response. After two doses of 30 u g, the vaccine can produce complete protective effect on 103 LD50 A botulinum toxin, while the pVAX1-SAHc vaccine is only 12.. 5% protection efficiency; after three immunization, 104 LD50 of botulinum toxin type A has 87.5% protection efficiency, and pVAX1-SAHc vaccine is only 12.5% protection efficiency. These results show that pVAX1-scDEC-AHc DNA vaccine has higher protective efficiency compared with pVAX1-SAHc vaccine. The DCs targeting effect of the DNA vaccine was studied by the histochemical experiment. The results showed that the pVAX1-scDEC-AHc vaccine could raise more DCs at the immune injection site, promote its maturation, and effectively promote the proliferation and maturation of DCs in the immune organs. These results suggest that the immune response of the vaccine may be attributed to the enhancement of the immune response to the vaccine. In this study, the immunogenicity of the recombinant subunit vaccine of the prokaryotic expression of soluble B botulinum toxin was also studied. This experiment cloned the Hc segment gene of the B type botulinum toxin receptor binding region to the pTIG-Trx prokaryotic expression vector, and used the strain of Escherichia coli BL21 (DE3) to realize the soluble expression of BHc, breaking the bacteria. Purified with HisTrapTM HP protein purification column and Capto TM MMC chromatography column, the purified His labeled and His labeled BHc antigen was purified. The immunogenicity and protective effect of the recombinant BHc expressed by the Escherichia coli were studied. The results showed that the 1 micron BHc single dose immune mice could be immune to the 103 LD50. The toxic toxin produced a complete protective effect. The two immunization could fully protect the 104 LD50 botulinum toxin type B. The serum specific antibody titer in the serum of mice immunized with BHc-His could reach 1:110000, the neutralization titer of serum could reach 3.33 IU/ml, and the specific antibody titer in the serum of BHc immunized mice could reach the average. To 1:70000, the neutralization titer of serum could reach 2.33 IU/ml, while the mBHc immune group expressed by yeast was only 1:40000 and 1.25 IU/ml. These results showed that the recombinant BHc-His antigen expressed in Escherichia coli had good immunogenicity and protective effect. In addition, the immunogenicity of the BHc antigen without the His label showed that it had the same effect. A good protective effect can be studied and developed as a candidate recombinant subunit vaccine for botulinum toxin type B. The above results are summarized. In the DNA vaccine study of the A botulinum toxin Hc target to DCs, it is proved that the strategy of targeting the antigen to DCs by the DEC205 receptor can indeed enhance the fluid and finer after the fusion antigen immunization. Cellular immune response provides a new idea to enhance the immunogenicity of DNA vaccine, and provides a new technical platform for the study of other botulinum toxin or other pathogen DNA vaccines. The results of the recombinant subunit vaccine of botulinum toxin type.B show that the recombinant BHc antigen expressed in Escherichia coli has good immunogenicity and protection. The results can be used as a candidate monovalent vaccine, and the tetravalent botulinum toxin subunit vaccine is formulated with the recombinant A antigen expressed in prokaryotic expression of E, F and F.
【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】：2017
【分类号】：R392

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