生物法合成肠炎沙门菌糖蛋白  

Biosynthesis of Salmonella enteritidis O antigen-based glycoproteins

作　　者：李梦如 刘恩 张文芋 罗洪艳 李沛[1] LI Mengru;LIU En;ZHANG Wenyu;LUO Hongyan;LI Pei(College of Veterinary Medicine,Southwest University,Chongqing 400700,China)

机构地区：[1]西南大学动物医学院,重庆400700

出　　处：《生物工程学报》2022年第6期2377-2388,共12页Chinese Journal of Biotechnology

基　　金：重庆市自然科学基金(cstc2019jcyj-msxm X0532);国家自然科学基金(31902340)。

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waa L基因缺失株SEΔwaa L,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,r EPA)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;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。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;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.

关 键 词：生物法 糖蛋白 肠炎沙门菌 CRISPR/Cas9方法 PglL 

分 类 号：S852.61[农业科学—基础兽医学]