建立糖链长度可调节的大肠杆菌N-连接糖基化系统

发布时间：2018-01-05 07:18

本文关键词：建立糖链长度可调节的大肠杆菌N-连接糖基化系统　出处：《山东大学》2010年硕士论文　论文类型：学位论文

【摘要】： 空肠弯曲菌Campylobacter jejuni为侵染人类消化道粘膜的致病菌,是引起细菌性腹泻的主要诱因之一。1999年,Szymanski等人首次在C. jejuni中发现了编码N-糖基化途径的pgl基因簇。原核N-连接糖基化系统的发现,尤其是原核寡糖基转移酶(OST)Pg1B的发现,为利用原核表达系统合成N-连接糖蛋白提供了一条新的方法。 C. jejuni中的N-连接糖基化途径同E. coli中Wzy-dependent途径进行的O-PS合成过程极为相似,首先在胞质内由各种糖基转移酶向包埋于质膜的undecaprenol上依次添加不同的单糖形成寡糖前体。寡糖前体经翻转酶翻转到周质空间后,或由细菌寡糖基转移酶(OST) Pg1B将寡糖前体转移到蛋白底物中,或经多聚化为O-PS后被O-抗原连接酶WaaL连接到类脂A-核心寡糖上形成LPS。这种相似性为将两个途径相结合,以O-PS代替C. jejuni中的七糖前体提供了理论基础,而Pg1B相对宽松的底物特异性将这一设想变成了现实。本文就利用细菌OST Pg1B建立E. coli N-连接糖基化系统、一步生物法生产糖-蛋白缀合物疫苗、以及PglB的性质等三方面展开研究。敲除E. coli O86:K61:B7中waaL基因得到重组菌B712,使连接在脂载体上的O-PS在周质空间内积累。将来源于C. jejuni NCTC 11168的pg1B和acrA克隆到B712后,Pg1B能够催化积累在周质空间内的O-PS向AcrA的转糖基反应,获得带有O86：B7O-PS的AcrA (AcrA-O-PS)。本文利用细菌OST成功地建立了E.coli N-连接糖基化系统,为以下工作的展开创造了平台。在O-PS合成过程中,O-抗原长度决定蛋白Wzz在O-PS聚合时负责控制链长,不同的E. coli菌株由于Wzz不同而具有链长不等的O-PS。本文用于建立E.coli N-连接糖基化系统的O86：B7具有较短链长的O-PS,同为O86血清型的另一菌株O86：H2则具有中等链长的O-PS。将WzzB7替换为WZZH2后,显著地增加了AcrA-O-PS中O-PS的糖链长度。因此,糖链长度可调节的E.coli N-连接糖基化系统得以成功构建。利用E.coli N-连接糖基化系统,可以将细菌O-PS以N-糖链的形式连接到蛋白上,这种糖-蛋白缀合物可以用作预防细菌感染的疫苗。为此,将E. coliN-连接糖基化系统中的蛋白底物由模式蛋白AcrA替换为广泛使用的疫苗载体蛋白破伤风毒素C片段TTc和白喉毒素无毒突变体CRM197,并向两者中引入PglB的糖基化位点,对一步生物法合成糖-蛋白缀合物进行了初步探索。利用TMHMM软件预测Pg1B的N-端具有11个穿膜a-螺旋。本文还构建了缺失第1个、第1-5个、第1-8个和第1-11个穿膜α-螺旋的截短Pg1B突变体(tPg1Bs)P1、P5、P8和P11,并利用E. coliN-连接糖基化系统对上述截短Pg1B突变体进行转糖基活性检测,发现这些截短Pg1B突变体均失去了转糖基活性,说明N-端结构域对Pg1B的转糖基活性至关重要。总之,糖链长度可以调节的E. coliN-连接糖基化系统的建立为生产糖-蛋白缀合物疫苗开辟了新的途径。研究表明将这些细菌O-PS共价连接到载体蛋白以后得到的糖-蛋白缀合物能够有效地激发机体产生TD免疫应答,保护人体免受细菌感染。目前普遍应用于生产的两步化学法合成糖-蛋白缀合物疫苗具有多次发酵纯化、成本高昂等缺点。而本文建立的一步生物法合成糖-蛋白缀合物疫苗仅需一次发酵、纯化就能够得到结构一致的缀合物；且可作为生产糖-蛋白缀合物疫苗的平台,通过进一步的基因工程改造,可以获得连接有不同细菌来源的多糖-蛋白缀合物疫苗,具有较高的应用价值。
[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.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

【共引文献】