本文關(guān)鍵詞：建立糖鏈長度可調(diào)節(jié)的大腸桿菌N-連接糖基化系統(tǒng)　出處：《山東大學》2010年碩士論文　論文類型：學位論文

  更多相關(guān)文章： 寡糖基轉(zhuǎn)移酶PglB N-連接糖基化 O-多糖 糖鏈長度 糖-蛋白綴合物疫苗

【摘要】： 空腸彎曲菌Campylobacter jejuni為侵染人類消化道粘膜的致病菌,是引起細菌性腹瀉的主要誘因之一。1999年,Szymanski等人首次在C. jejuni中發(fā)現(xiàn)了編碼N-糖基化途徑的pgl基因簇。原核N-連接糖基化系統(tǒng)的發(fā)現(xiàn),尤其是原核寡糖基轉(zhuǎn)移酶(OST)Pg1B的發(fā)現(xiàn),為利用原核表達系統(tǒng)合成N-連接糖蛋白提供了一條新的方法。 C. jejuni中的N-連接糖基化途徑同E. coli中Wzy-dependent途徑進行的O-PS合成過程極為相似,首先在胞質(zhì)內(nèi)由各種糖基轉(zhuǎn)移酶向包埋于質(zhì)膜的undecaprenol上依次添加不同的單糖形成寡糖前體。寡糖前體經(jīng)翻轉(zhuǎn)酶翻轉(zhuǎn)到周質(zhì)空間后,或由細菌寡糖基轉(zhuǎn)移酶(OST) Pg1B將寡糖前體轉(zhuǎn)移到蛋白底物中,或經(jīng)多聚化為O-PS后被O-抗原連接酶WaaL連接到類脂A-核心寡糖上形成LPS。這種相似性為將兩個途徑相結(jié)合,以O(shè)-PS代替C. jejuni中的七糖前體提供了理論基礎(chǔ),而Pg1B相對寬松的底物特異性將這一設(shè)想變成了現(xiàn)實。本文就利用細菌OST Pg1B建立E. coli N-連接糖基化系統(tǒng)、一步生物法生產(chǎn)糖-蛋白綴合物疫苗、以及PglB的性質(zhì)等三方面展開研究。 敲除E. coli O86:K61:B7中waaL基因得到重組菌B712,使連接在脂載體上的O-PS在周質(zhì)空間內(nèi)積累。將來源于C. jejuni NCTC 11168的pg1B和acrA克隆到B712后,Pg1B能夠催化積累在周質(zhì)空間內(nèi)的O-PS向AcrA的轉(zhuǎn)糖基反應,獲得帶有O86：B7O-PS的AcrA (AcrA-O-PS)。本文利用細菌OST成功地建立了E.coli N-連接糖基化系統(tǒng),為以下工作的展開創(chuàng)造了平臺。 在O-PS合成過程中,O-抗原長度決定蛋白Wzz在O-PS聚合時負責控制鏈長,不同的E. coli菌株由于Wzz不同而具有鏈長不等的O-PS。本文用于建立E.coli N-連接糖基化系統(tǒng)的O86：B7具有較短鏈長的O-PS,同為O86血清型的另一菌株O86：H2則具有中等鏈長的O-PS。將WzzB7替換為WZZH2后,顯著地增加了AcrA-O-PS中O-PS的糖鏈長度。因此,糖鏈長度可調(diào)節(jié)的E.coli N-連接糖基化系統(tǒng)得以成功構(gòu)建。 利用E.coli N-連接糖基化系統(tǒng),可以將細菌O-PS以N-糖鏈的形式連接到蛋白上,這種糖-蛋白綴合物可以用作預防細菌感染的疫苗。為此,將E. coliN-連接糖基化系統(tǒng)中的蛋白底物由模式蛋白AcrA替換為廣泛使用的疫苗載體蛋白破傷風毒素C片段TTc和白喉毒素無毒突變體CRM197,并向兩者中引入PglB的糖基化位點,對一步生物法合成糖-蛋白綴合物進行了初步探索。 利用TMHMM軟件預測Pg1B的N-端具有11個穿膜a-螺旋。本文還構(gòu)建了缺失第1個、第1-5個、第1-8個和第1-11個穿膜α-螺旋的截短Pg1B突變體(tPg1Bs)P1、P5、P8和P11,并利用E. coliN-連接糖基化系統(tǒng)對上述截短Pg1B突變體進行轉(zhuǎn)糖基活性檢測,發(fā)現(xiàn)這些截短Pg1B突變體均失去了轉(zhuǎn)糖基活性,說明N-端結(jié)構(gòu)域?qū)g1B的轉(zhuǎn)糖基活性至關(guān)重要。 總之,糖鏈長度可以調(diào)節(jié)的E. coliN-連接糖基化系統(tǒng)的建立為生產(chǎn)糖-蛋白綴合物疫苗開辟了新的途徑。研究表明將這些細菌O-PS共價連接到載體蛋白以后得到的糖-蛋白綴合物能夠有效地激發(fā)機體產(chǎn)生TD免疫應答,保護人體免受細菌感染。目前普遍應用于生產(chǎn)的兩步化學法合成糖-蛋白綴合物疫苗具有多次發(fā)酵純化、成本高昂等缺點。而本文建立的一步生物法合成糖-蛋白綴合物疫苗僅需一次發(fā)酵、純化就能夠得到結(jié)構(gòu)一致的綴合物；且可作為生產(chǎn)糖-蛋白綴合物疫苗的平臺,通過進一步的基因工程改造,可以獲得連接有不同細菌來源的多糖-蛋白綴合物疫苗,具有較高的應用價值。
[Abstract]:Campylobacter of Campylobacter jejuni jejuni pathogens infecting human gastrointestinal mucosa, is a major cause of bacterial diarrhea in.1999 years, Szymanski et al first found the PGL gene cluster encoding N- glycosylation pathway in C. jejuni. Prokaryotic N- linked glycosylation system is especially prokaryotic oligosaccharides transferase (OST) Pg1B, provides a new method for the synthesis of N- by prokaryotic expression system with glycoprotein.
O-PS C. jejuni N- in the process of synthesis of glycosyl connected with E. pathway of Wzy-dependent pathway in coli is very similar to the first in the cytoplasm by various glycosyltransferases to embedded in the plasma membrane of undecaprenol by adding different monosaccharide oligosaccharide precursors. The formation of oligosaccharide precursor by flip flip to the periplasmic enzyme space, or by bacteria oligosaccharyl transferase (OST) Pg1B will be transferred to the oligosaccharide precursor protein substrates, or by poly O-PS after O- antigen ligase WaaL attached to lipid A- core oligosaccharide on the formation of LPS. this similarity for two ways combined with O-PS instead of C. in jejuni seven sugar provides a theoretical basis for precursor and substrate specificity of Pg1B relatively easy to put this idea into reality. This is to use the OST Pg1B E. coli to establish bacterial N- linked glycosylation system, a biological production of sugar protein conjugate vaccine, and PglB The nature of the three aspects of the study.
Knockdown of waaL E. coli in O86:K61:B7 gene recombinant B712, which is connected to the carrier in the lipid accumulation of O-PS in the periplasmic space. Come from C. jejuni NCTC 11168 pg1B and acrA was cloned into B712, Pg1B can catalyze accumulation in the periplasmic space inside the O-PS to the transglycosylation reaction of AcrA, obtain with O86:B7O-PS AcrA (AcrA-O-PS). The bacterial OST was successfully established E.coli N- linked glycosylation system into the following work to create a platform.
In the process of O-PS synthesis, O- antigen protein Wzz in O-PS depends on the length of the polymerization is responsible for the control of chain length, E. Wzz because of different coli strains with the different chain length ranging from O-PS., this paper used to establish the E.coli N- linked glycosylation system O86:B7 with shorter chain length O-PS, the same as the other O86 blood type a strain O86:H2 with medium chain length O-PS. will replace WzzB7 WZZH2, significantly increased the sugar chain length in AcrA-O-PS O-PS. Therefore, the sugar chain length adjustable E.coli N- linked glycosylation system was successfully constructed.
The use of E.coli N- linked glycosylation system, can be connected to the bacterial O-PS proteins in N- sugar chain in the form of this kind of carbohydrate protein conjugates can be used as a vaccine to prevent bacterial infection. Therefore, the E. coliN- linked glycosylation system in protein substrates by model protein AcrA vaccine carrier protein replacement for tetanus toxin C fragment TTc and nontoxic diphtheria toxin mutant CRM197 widely used, glycosylation sites and the introduction of PglB to the two, conducted a preliminary exploration on the synthesis of sugar - step biological protein conjugates.
TMHMM software was used to predict the Pg1B end N- has 11 transmembrane helix a-. This paper also constructed a deletion of first, 1-5, 1-8 and truncated Pg1B mutant 1-11 transmembrane alpha helix (tPg1Bs) P1, P5, P8 and P11 linked glycosylation system using E. and coliN- for transglycosylation activity detection of the truncated Pg1B mutant, found that these truncated Pg1B mutants lost the transglycosylation activity, indicating that N- terminal domain of Pg1B to critical glycosylation activity.
In short, the establishment of the sugar chain length can be adjusted to E. coliN- linked glycosylation system provides a new way for the production of polysaccharide protein conjugate vaccine. The research showed that these bacteria O-PS covalently attached to carrier protein obtained after sugar protein conjugates can effectively stimulate the body to produce a TD immune response, protect the body from bacterial infection. Now widely applied to the production of two - step method of chemical synthesis of sugar protein conjugate vaccine has repeatedly fermentation purification, high cost and other disadvantages. And the synthesis of sugar - step biological protein conjugate vaccine only once the fermentation, purification can get conjugate structure consistent; and as the production of sugar - protein conjugate vaccine platform, through further genetic modification, can be connected with different sources of bacterial polysaccharide protein conjugate vaccine, has higher application value.

【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2010
【分類號】：R378

【共引文獻】

相關(guān)博士學位論文 前4條

1 關(guān)婉怡;N-乙酰氨基葡萄糖/半乳糖核苷酸及類似物的酶法合成與應用研究[D];山東大學;2011年

2 劉振桐;結(jié)核分枝桿菌H37Rv分泌濾液中糖蛋白的篩選[D];大連醫(yī)科大學;2007年

3 劉現(xiàn)偉;四種微生物多糖合成相關(guān)酶類的生化性質(zhì)與應用研究[D];山東大學;2009年

4 李磊;細菌多糖和糖蛋白生物合成途徑及相關(guān)酶類研究[D];山東大學;2010年

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本文編號：1382114