本文關鍵詞：A型肉毒毒素多表位串聯(lián)體的設計、表達及免疫原性分析　出處：《中國人民解放軍軍事醫(yī)學科學院》2007年碩士論文　論文類型：學位論文

  更多相關文章： 肉毒毒素 表位預測 多表位串聯(lián)體

【摘要】： 肉毒神經(jīng)毒素(BoNT)是自然界所發(fā)現(xiàn)的生物毒素(包括化學毒劑)中毒性最強的物質(zhì)。由于BoNT毒性強并且極容易被恐怖分子利用，國際社會已將其列為重要的毒素戰(zhàn)劑和最具威脅的生物恐怖劑之一。針對BoNT感染引起的肉毒中毒尚無較好的治療手段，到目前為止也沒有實用型的抗毒素問世，因而其疫苗的研究就顯得更為重要。雖然類毒素、特別是其重鏈重組亞單位疫苗具有很好的保護作用，但均存在著不可忽視的潛在的副作用。多表位疫苗的提出為BoNT疫苗的研發(fā)提供了新思路，一方面多表位疫苗能夠集中優(yōu)勢的中和表位，去除對保護性免疫不利的結(jié)構(gòu)或潛在的毒性片段；另一方面能優(yōu)化組合BoNT各種型別的中和性表位，為研制多價疫苗奠定基礎，以解決目前國際上多以單一型別BoNT為主研究重組基因工程疫苗的弊端。因此開展BoNT多表位疫苗的探索性研究具有十分重要的醫(yī)學和社會意義。 首先，本研究基于保守性高的BoNT／A的氨基酸序列，根據(jù)BioSun和LaserGene軟件包中的表位分析相關參數(shù)，輔以對BoNT／A蛋白的二級結(jié)構(gòu)的分析，綜合預測了BoNT／A的B細胞表位。結(jié)果表明，BoNT／A輕鏈的142-150、284-292區(qū)段，重鏈的440-450、465-480、538-549、699-710、751-760、1087-1095、1224-1231、1263-1270區(qū)段是B細胞優(yōu)勢表位區(qū)的可能性較大。 然后，遴選預測得到的B細胞表位和文獻報道的表位，并加入適當?shù)妮o助性元件：通用T細胞表位和Linker，設計了多表位串聯(lián)體A、B。根據(jù)大腸桿菌密碼子偏好性對其基因進行優(yōu)化的基礎上，經(jīng)重疊PCR方法合成串聯(lián)體A、B的基因全長。并構(gòu)建了克隆測序質(zhì)粒pMD18-T-A、pMD18-T-B進行序列分析鑒定，結(jié)果表明，重疊PCR合成的基因序列均與密碼子優(yōu)化后的基因序列完全一致。然后用BamHⅠ、HindⅢ分別雙酶切pMD18-T-A、pMD18-T-B載體和表達載體pQE-30，并連接獲得重組表達質(zhì)粒pQE-30A，pQE-30B。經(jīng)過雙酶切和PCR鑒定正確后，轉(zhuǎn)化到E．coli M15[pREP4]感受態(tài)細胞中，構(gòu)建相應的表達工程菌。用IPTG進行誘導表達，發(fā)現(xiàn)這兩種重組蛋白都以包涵體形式存在，表達量分別達到全菌蛋白的70％、54％。SDS-PAGE分析顯示，與預期蛋白的大小相符，預期蛋白的分子量分別為22.4×10~3、24.9×10~3。Western印跡和間接ELISA結(jié)果顯示重組蛋白串聯(lián)體A、B均可被其特異性馬血清抗毒素識別，進一步驗證了串聯(lián)體A、B基因在工程菌中獲得正確表達，，并具有良好的抗原性。Ni-NTA法純化后分別獲得純度大于92.2％、99％的重組串聯(lián)體A、B蛋白，然后采用透析復性法對其進行復性。 接著，將串聯(lián)體A、B分別與等體積的弗氏佐劑混合后免疫Balb／C小鼠，20μg／只，采用背部皮下多點注射的免疫途經(jīng)。并以PBS作為對照。四免后血清效價達到10~3～10~4，說明設計表達的重組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腹腔攻擊小鼠，觀察小鼠存活天數(shù)。動物保護性試驗發(fā)現(xiàn)，免疫組小鼠未表現(xiàn)出比對照組小鼠更好的抵抗天然毒素攻擊的能力，并且ELISA及Western印跡試驗表明，其免疫小鼠血清不能結(jié)合天然的BoNT／A。 針對上述試驗結(jié)果，在串聯(lián)體A的基礎上調(diào)整了設計方案，重新優(yōu)化構(gòu)建了三個串聯(lián)體：串聯(lián)體A′、C以及合成的表位串聯(lián)體肽(串聯(lián)體肽)。其中，串聯(lián)體A′是用8M脲直接溶解串聯(lián)體A重組蛋白的包涵體得到，即純化后未經(jīng)復性的串聯(lián)體A；串聯(lián)體C則是將串聯(lián)體A的Th表位由C端移至N端。串聯(lián)體肽是由文獻報道的兩個中和表位通過GGS相連構(gòu)成。 以串聯(lián)體A′，C和串聯(lián)體肽免疫Balb／C小鼠。攻毒試驗表明新構(gòu)建的三個串聯(lián)體可以抵抗3×LD_(50)天然BoNT／A的攻擊。該保護作用還不及文獻報道的單個表位合成肽免疫效果，據(jù)此推測串聯(lián)體A、B無保護作用的主要原因可能是選用的LinkerGGS沒有起到有效的分隔作用。同時認為Th的位置對表位設計影響不大。本研究雖然未能獲得具有良好保護性的串聯(lián)體，但發(fā)現(xiàn)了一些問題，做了一些有意義的嘗試和探索，為今后BoNT多表位疫苗的進一步研究，提供了參考和借鑒。
[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.

【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】：2007
【分類號】：R392

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