铜绿假单胞菌waaL突变体构建及糖基化系统的建立  

Construction of waaL Deletion Mutant of Pseudomonas aeru⁃ginosa and Establishment of Glycosylation System

作　　者：徐涵 潘超 黄竞 冯尔玲 朱力 王恒樑 XU Han;PAN Chao;HUANG Jing;FENG Er-Ling;ZHU Li;WANG Heng-Liang(Beijing Institute of Biotechnology,Beijing 100071,China)

机构地区：[1]军事医学研究院生物工程研究所,北京100071

出　　处：《生物技术通讯》2020年第6期634-640,共7页Letters in Biotechnology

基　　金：国家自然科学基金青年基金(31700802)。

摘　　要：目的:构建铜绿假单胞菌PAO1的waaL基因缺失株,并通过在缺失株中建立糖基化系统,合成糖蛋白。方法:采用同源双交换法敲除铜绿假单胞菌PAO1株脂多糖合成途径中的O-抗原连接酶基因waaL,并利用PCR和脂多糖银染对敲除株的基因型和表型进行验证;随后导入可共表达糖基转移酶PglL和重组霍乱毒素B亚单位(rCTB)的糖基工程载体,经IPTG诱导后利用免疫印迹检测糖蛋白的表达情况。结果:PCR和脂多糖银染鉴定表明敲除了PAO1的waaL基因,缺失株和野生株的生长状态及糖代谢情况几乎无差异;在缺失株中导入糖基工程载体并诱导表达,免疫印迹结果表明PAO1的O-抗原多糖能够在PglL催化下转移到底物蛋白rCTB,从而得到了PAO1多糖-蛋白结合物(糖蛋白)。结论:构建了铜绿假单胞菌PAO1的waaL缺失突变体,并在突变株内建立了糖基化系统,合成了一种PAO1多糖-蛋白结合物,为下一步糖蛋白纯化以及制备铜绿假单胞菌多糖结合疫苗奠定了基础。Objective:To construct an O-antigen ligase gene(waaL)deletion mutant of Pseudomonas aeruginosa PAO1 strain,and express glycoprotein by establishing a glycosylation system in the strain.Methods:The homolo⁃gous double exchange method was used to knock out the waaL gene of PAO1 strain,and the mutant was verified by PCR and lipopolysaccharide silver staining.Then the glycosyl-engineering vector,co-expressing of glycosyltrans⁃ferase(PglL)and recombinant cholera toxin B subunit(rCTB),was introduced into the mutant strain.After induced by IPTG,the glycoprotein was detected through immunoblotting.Results:The waaL deletion mutant was successful⁃ly constructed and there was almost no difference in growth and glucose metabolism between mutant and wild strain.After introducing the glycosyl-engineering vector into the mutant and being induced with IPTG,immunoblot⁃ting result showed that the PAO1 polysaccharide(OPS)could be connected to rCTB under the catalysis of PglL.and the P.aeruginosa polysaccharide-protein conjugate(glycoprotein)was obtained.Conclusion:We successfully constructed a P.aeruginosa waaL deletion mutant strain and detected the glycoprotein expression by introducing a glycosylation system in it.A P.aeruginosa polysaccharide-protein conjugate was synthesized,which laid a founda⁃tion for the next step of glycoprotein purification and preparation of P.aeruginosa polysaccharide conjugate vaccine.

关 键 词：铜绿假单胞菌 waaL缺失突变体 多糖结合疫苗 糖蛋白 

分 类 号：Q78[生物学—分子生物学]