一株内毒素缺陷型大肠杆菌的构建与鉴定

Construction and characterization of a LPS-defective Escherichia coli strain

为了降低大肠杆菌表达外源蛋白时产生的内毒素(LPS),本研究利用Red重组和Cre-Lox敲除系统相结合的方法,敲除大肠杆菌DH10B-2B中与LPS合成相关的基因kdsD、lpxL、lpxM、pagP、lpxP、eptA,并在其OmpT基因处插入弗朗西斯菌的磷酸酶基因lpxE去除LPS中类脂A的1-磷酸基团,获得改造的E.coli DH10B-2B-Δ6-lpxE。利用鲎试剂检测108 cfu/mL^105 cfu/mL DH10B-2B与DH10B-2B-Δ6-lpxE经超声破碎的裂解液中LPS含量;将9组小鼠每组5只分别腹腔注射PBS及浓度为1010 cfu/mL^107 cfu/mL的两种裂解液,观察小鼠的临床症状并利用ELISA试剂盒检测小鼠脾脏中IL-6及TNF-a的表达水平。结果显示,经鲎试剂检测106 cfu/mL DH10B-2B裂解液中的LPS结果为阳性,LPS含量≥10000 U/mL,而108 cfu/mL DH10B-2B-Δ6-lpxE裂解液LPS结果虽为阳性,但LPS含量≤100 U/mL。将DH10B-2B-Δ6-lpxE裂解液以109 cfu/mL的剂量腹腔注射的小鼠与对照组小鼠均无任何异常,而注射109 cfu/mL DH10B-2B裂解液的小鼠出现精神不振,反应迟钝,眼角有脓性分泌物以及腹泻等症状;IL-6及TNF-a检测结果显示,与DH10B-2B组小鼠相比,DH10B-2B-Δ6-lpxE组小鼠细胞因子的表达水平明显降低。本研究通过对E.coli LPS合成过程中关键基因的改造获得一株LPS缺陷的大肠杆菌DH10B-2B-Δ6-lpxE,其产生LPS的能力明显下降。该菌株既可以用作低LPS重组蛋白表达系统,也可以用于低毒力LPS的提取,为相关免疫增强剂的研究奠定基础。

In order to reduce the level of lipopolysaccharide produced by E.coli when expressing exogenous proteins,the Red recombinant method combined with the Cre-Lox knockout system was used to knock out the LPS synthesis related genes(kdsD,lpxL,lpxM,pagP,lpxP,eptA)in the K-12 derived E.coli strain DH10B-2B,and insert the phosphatase gene lpxE of Francis bacteria at the OmpT gene to remove the 1-phosphate group of lipid A in LPS,and finally we obtained a modified E.coli strain named DH10B-2B-Δ6-lpxE.Endotoxin content in the supernatant obtained by ultrasound cell disruption of 108 cfu/mL-105 cfu/mL DH10B-2B and DH10B-2B-Δ6-lpxE was detected by limulus lysate;5 mice in each of 9 groups were intraperitoneally injected with PBS and two kinds of lysate with concentrations of 1010 cfu/mL-107 cfu/mL,respectively.The clinical symptoms of mice were observed and the expression levels of IL-6 and TNF-αin the spleen of mice were detected with ELISA kit.Results tested positive when the DH10B-2B lysate was tested by limulus lysate at 106 cfu/mL concentration and the endotoxin content was≥10000 U/mL,while it was positive when DH10B-2B-Δ6-lpxE lysate was tested at 108 cfu/mL concentration and the endotoxin content was≤100 U/mL.Compared with the blank control group,the mice intraperitoneally injected with DH10B-2B-Δ6-lpxE lysate with a concentration of 109 cfu/mL only showed no abnormalities,while the mice injected with DH10B-2B lysate with a concentration of 109 cfu/mL showed signs of depression,lags in response,purulent secretion around the eyes,diarrhea,among others.IL-6 and TNF-a detection showed that comparing with DH10B-2B,DH10B-2B-Δ6-lpxE showed lower expression level of cytokines.In this study,a strain of E.coli was obtained by modifying the key genes involved in the synthesis of LPS and showed lower production of endotoxin.The strain can be used as the expression system of recombinant protein of low endotoxin or the extraction of de-toxigenic endotoxin.Therefore,this study lays a foundation for the research of immune booster.