生物法合成肠炎沙门菌糖蛋白

Biosynthesis of Salmonella enteritidis O antigen-based glycoproteins

摘要:

肠炎沙门菌（Salmonella enteritidis）是一种重要的人兽共患病原菌，在对该菌感染的预防与控制上一直存在困难，而糖蛋白疫苗的出现为其预防提供了新的思路。对于糖蛋白的合成，一般采用传统的化学交联方法，该法制备流程烦琐、生产成本高。因此，探索经济且稳定的生物合成方法非常必要。为了实现生物法合成肠炎沙门菌糖蛋白，本研究利用CRISPR/Cas9方法构建肠炎沙门菌waaL基因缺失株SE ΔwaaL，使用银染的方法检测细菌外膜脂多糖（lipopolysaccharide，LPS）的合成情况。使用环形PCR方法构建了表达寡糖转移酶PglL、重组铜绿假单胞菌的外毒素（recombinant Pseudomonas aeruginosa exotoxin A，rEPA）和霍乱毒素B亚单位（cholera toxin B subunit，CTB）的表达质粒，并分别在rEPA的N端和CTB的C端加入了PilE^S45-K73糖基化位点序列。将重组质粒转化到SE ΔwaaL中，诱导表达后通过Western blotting方法对糖蛋白的合成进行验证，并通过镍柱（Ni-NTA）对糖蛋白进行纯化。结果表明，waaL基因的缺失阻断了肠炎沙门菌LPS正常合成，在该缺失株中rEPA和CTB蛋白均可成功表达。此外，在表达寡糖转移酶PglL的情况下，rEPA和CTB发生了明显的糖基化，其糖基化部分为肠炎沙门菌O抗原多糖。本研究结果证明肠炎沙门菌缺失waaL基因后，在寡糖转移酶PglL的作用下可以将自身O抗原多糖链共价连接到载体蛋白rEPA和CTB上，形成糖蛋白，为生物法合成肠炎沙门菌糖蛋白的研究奠定了基础。

Abstract:

Salmonella enteritidis (SE) has been recognized as an important zoonotic pathogen, and the prevention and control of salmonellosis has long been a conundrum. However, glycoconjugate vaccines seem to be a promising solution. Glycoproteins are conventionally synthesized by chemical cross-linking which features complex procedure and cost-intensiveness. Therefore, a stable biosynthesis method at lower cost is in urgent need. For the biosynthesis of SE O-antigen-based glycoproteins, we used CRISPR/Cas9 to develop the waaL-deleted SE strain ∆waaL. The synthesis of lipopolysaccharide (LPS) was detected based on silver staining. Circular polymerase extension cloning (CPEC) was employed to construct the plasmids expressing glycosyltransferase PglL, recombinant Pseudomonas aeruginosa exotoxin A (rEPA), and cholera toxin B subunit (CTB). Meanwhile, PilE^S45-K73glycosylation motif was added to the N-terminal and C-terminal of rEPA and CTB, respectively. The recombinant plasmids were transformed into SE ∆waaL. After induction, the synthesis of glycoprotein was verified by Western blotting and the synthesized glycoprotein was purified by Ni-NTA column. The results showed that waaL deletion blocked the LPS synthesis of SE, and that rEPA and CTB proteins were expressed in SE ∆waaL. In addition, obvious glycosylation occurred to rEPA and CTB when PglL was expressed, and the glycosylated part was SE O antigen polysaccharide. In summary, after waaL deletion in SE, PglL can transfer its own O antigen polysaccharides (OPS) to the carrier proteins rEPA and CTB, resulting in OPS-rEPA and OPS-CTB glycoproteins. The result lays a basis for the biosynthesis of SE glycoprotein.