本文選題：肉毒毒素 + 重鏈受體結合區(qū)Hc�。� 參考：《中國人民解放軍軍事醫(yī)學科學院》2017年碩士論文

【摘要】：肉毒毒素又被稱為肉毒神經(jīng)毒素,是由厭氧的革蘭氏陽性菌肉毒梭菌產(chǎn)生的外毒素,是已知毒性最強的蛋白,根據(jù)其抗原性不同可以分為7種類型(BoNT/A-BoNT/G),A、B、E和F型可以導致人類中毒,其中A型肉毒毒素中毒致死率最高。各型肉毒毒素分子結構比較相似,分子量大小約150 kDa,由二硫鍵連接一個100 kDa的重鏈(HC)和一個50 kDa的輕鏈(LC)組成。肉毒毒素重鏈羧基端(HC)負責與細胞受體的結合,重鏈氨基端(HN)介導L鏈的跨膜轉運;輕鏈具有鋅離子內肽酶活性,能夠將可溶性N-乙基馬來酰亞胺敏感因子附著蛋白受體(SNARE)切割而抑制神經(jīng)遞質的傳遞,最終會導致骨骼肌麻痹,呼吸衰竭而死亡。肉毒毒素生產(chǎn)工藝簡單,穩(wěn)定性良好,具有極強的致死性,是一種潛在的生物武器,極容易被制備成生物戰(zhàn)劑應用。此外,近些年來由于肉毒毒素導致的中毒事件也時有發(fā)生,因此,對于預防和治療肉毒毒素中毒的研究具有十分重要的意義。接種疫苗是一種有效預防肉毒毒素中毒的策略。鑒于早期研制的類毒素疫苗存在較多的缺陷,所以一直未能得到十分廣泛的應用,因此研究新型肉毒毒素疫苗成為一種迫切的需要。破傷風毒素(TeNT)和肉毒毒素在結構上有較多的相似之處,所以借助于破傷風毒素Hc片段亞單位疫苗研究成功的策略來研制肉毒毒素亞單位疫苗。肉毒毒素受體結合區(qū)Hc能介導毒素特異性結合到神經(jīng)肌肉接點處的神經(jīng)末梢,此外,Hc片段不存在鋅肽鏈內切酶活性,沒有毒性,免疫動物后能刺激產(chǎn)生保護性免疫反應�；谶@一重要發(fā)現(xiàn),研究人員開始以肉毒毒素受體結合區(qū)Hc為靶標,開展新型肉毒毒素重組亞單位疫苗和核酸疫苗的研究。DNA疫苗是將編碼抗原的基因克隆到真核表達載體中作為疫苗,免疫動物后該載體會在體內表達抗原,繼而刺激機體產(chǎn)生免疫反應和保護作用。DNA疫苗具有很多優(yōu)點:生產(chǎn)工藝簡單、產(chǎn)品易純化、便于儲存,在接種者體內表達抗原,并且能夠產(chǎn)生體液免疫和細胞免疫反應,然而,DNA免疫原性較低,不足以提供足夠的保護作用,距離真正的人類臨床應用還有一定的差距,需要進一步提高與放大y|DNA的免疫效果。樹突狀細胞(DCs)是體內已知最強的專職抗原遞呈細胞,在抗原遞呈和啟動機體免疫應答中具有十分重要的作用。DEC205是表達于樹突狀細胞表面的內吞性受體,能夠高效地介導抗原的攝取、加工處理以及遞呈。為了增強肉毒毒素DNA疫苗的免疫原性,本研究開展了A型肉毒毒素受體結合區(qū)Hc抗原靶向DCs的DNA疫苗研究工作。將編碼抗DEC205的抗體基因(scDEC)和編碼A型肉毒毒素受體結合區(qū)基因(AHc)融合,并克隆到真核表達載體pVAX1中,構建了一個DCs靶向的DNA疫苗(pVAX1-scDEC-AHc),同時還設置了非靶向的疫苗載體(pVAX1-SAHc)和陰性對照(pVAX1)。免疫小鼠后證明該DCs靶向的DNA疫苗確實能夠增強抗原特異性體液和細胞免疫反應;相比于非靶向DNA疫苗,其抗體滴度增強了2.5倍,細胞免疫反應也有較大程度的增強。抗體亞型測定的結果表明該DNA疫苗免疫小鼠后產(chǎn)生了以IgG1為主的抗體,即免疫反應以Th2型免疫應答途徑為主。保護性試驗結果證明pVAX1-scDEC-AHc疫苗免疫后能夠刺激機體產(chǎn)生強的保護性免疫反應,30μg劑量免疫兩次之后即能對103 LD50的A型肉毒毒素產(chǎn)生完全保護作用,而pVAX1-SAHc疫苗僅為12.5%的保護效率;三次免疫之后對104 LD50的A型肉毒毒素具有87.5%的保護效率,pVAX1-SAHc疫苗僅為12.5%的保護效率,這些結果表明pVAX1-scDEC-AHc DNA疫苗相對于pVAX1-SAHc疫苗具有更高的保護效率。通過脾細胞流式分析和免疫注射部位肌肉組織免疫組化實驗研究了該DNA疫苗的DCs靶向效應,結果表明pVAX1-scDEC-AHc疫苗免疫后能夠在免疫注射部位募集更多的DCs,促進其成熟,并且能夠高效地促進免疫器官中的DCs進行增殖和成熟,這些結果提示該DNA疫苗能夠增強免疫反應可能要歸功于這種DCs靶向作用。本研究工作還對原核表達的可溶性B型肉毒毒素重組亞單位疫苗的免疫原性進行了研究。本實驗將B型肉毒毒素受體結合區(qū)Hc段基因克隆到pTIG-Trx原核表達載體,利用大腸桿菌BL21(DE3)菌株實現(xiàn)了BHc的可溶性表達,破菌上清用HisTrapTM HP蛋白純化柱和Capto TM MMC層析柱進行純化,純化到了純度較高的帶His標簽的和無His標簽的BHc抗原。免疫小鼠研究該大腸桿菌表達的重組BHc的免疫原性和保護效果,結果表明1μg該重組BHc單劑量免疫小鼠就能對103 LD50的B型肉毒毒素產(chǎn)生完全保護作用,兩次免疫則可以對104 LD50的B型肉毒毒素產(chǎn)生完全保護作用。測定BHc-His免疫的小鼠血清中特異性抗體滴度平均可以達到1:110000,血清中和效價可以達到3.33 IU/ml;BHc免疫的小鼠血清中特異性抗體滴度平均可以達到1:70000,血清中和效價可以達到2.33 IU/ml;而酵母表達的mBHc免疫組僅為1:40000和1.25 IU/ml;這些結果表明大腸桿菌表達的重組BHc-His抗原具有良好的免疫原性和保護效果。此外,對于沒有His標簽的BHc抗原的免疫原性研究結果表明其同樣具有很好的保護效果,可以作為候選的B型肉毒毒素重組亞單位疫苗進行下一步研究與開發(fā)�？偨Y以上研究結果,在A型肉毒毒素Hc靶向DCs的DNA疫苗研究中證明了通過DEC205受體將抗原靶向到DCs的策略確實能夠增強融合抗原免疫后的體液和細胞免疫反應,為增強DNA疫苗的免疫原性提供了一個新思路,并為其他型肉毒毒素或者其他病原體DNA疫苗的研究提供了新的技術平臺。B型肉毒毒素重組亞單位疫苗的研究結果表明大腸桿菌表達的重組BHc抗原具有良好的免疫原性和保護效果,可以作為一個候選單價疫苗,并與原核表達的A、E和F型重組Hc抗原配制四價肉毒毒素亞單位疫苗。
[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.
【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R392

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