肠炎沙门菌已被认为是一种重要的人畜共患病原体,沙门氏菌病的预防和控制一直是一个难题。然而,糖缀合物疫苗似乎是一个有希望的解决方案。糖蛋白通常通过化学交联合成,其特征是程序复杂且成本密集。因此,迫切需要一种低成本稳定的生物合成方法。对于基于SEO-抗原的糖蛋白的生物合成,我们使用CRISPR/Cas9开发了waaL缺失的SE菌株ΔwaaL。基于银染检测脂多糖(LPS)的合成。环状聚合酶延伸克隆(CPEC)用于构建表达糖基转移酶PglL的质粒,重组铜绿假单胞菌外毒素A(rEPA),和霍乱毒素B亚基(CTB)。同时,将PilES45-K73糖基化基序添加到rEPA和CTB的N端和C端,分别。将重组质粒转化为SEΔwaaL。感应后,通过蛋白质印迹法验证糖蛋白的合成,并通过Ni-NTA柱纯化合成的糖蛋白。结果表明,waaL缺失阻断了LPS合成SE,并且rEPA和CTB蛋白在SEΔwaaL中表达。此外,当PglL表达时,rEPA和CTB发生明显的糖基化,糖基化部分为SEO抗原多糖。总之,在SE中删除waaL后,PglL可以将自身的O抗原多糖(OPS)转移到载体蛋白rEPA和CTB,产生OPS-rEPA和OPS-CTB糖蛋白。该结果为SE糖蛋白的生物合成奠定了基础。
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, PilES45-K73 glycosylation 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.