铁载体受体蛋白IroN的原核表达及其对肠道外致病性大肠杆菌感染小鼠的免疫保护作用

Prokaryotic Expression of Recombinant Iron Siderophore Receptor IroN and Immune Protective Effect of Expressed Product against Extraintestinal Pathogenic E.coli Infection in Mice

目的原核表达重组铁载体受体蛋白IroN,并探讨其对小鼠肠道外致病性大肠杆菌群(Extraintestinal pathogenic E.coli,ExPEC)感染的免疫保护作用。方法从E.coliJ96基因组DNA中扩增IroN基因,克隆入原核表达载体pET-32a(+)中,构建原核表达质粒pET-32a-IroN,转化E.coliBL21(DE3),IPTG诱导表达。表达的重组IroN蛋白纯化后,经皮下免疫BALB/c小鼠,采用ELISA法检测小鼠血清和尿液中的抗体水平;以E.coliJ96菌株分别经腹腔和尿道途径攻击免疫小鼠,计算保护率并计数细菌数。结果重组IroN蛋白的表达量为32%,纯化后纯度超过85%,且具有良好的反应原性。重组IroN蛋白经皮下免疫小鼠,可诱导小鼠血清产生高水平的特异性IgG抗体,在尿液中未检测到特异性IgA抗体。重组IroN蛋白对腹腔攻击ExPEC感染小鼠的保护率为81.8%(P<0.05);重组IroN蛋白免疫的小鼠经尿道攻击ExPEC后,肾脏分离的菌落数比对照组(PBS免疫)降低了100倍以上(P<0.05),但膀胱和尿液中的菌落数两组差异无统计学意义(P>0.05)。结论已原核表达并纯化了IroN蛋白,其对ExPEC感染小鼠的腹腔和肾脏具有显著的保护作用,但对膀胱感染无保护作用。

Objective To express recombinant iron siderophore receptor IroN in prokaryotic cells and investigate its protective effect against extraintestinal pathogenic Escherichia coli（ExPEC）infection in mice.Methods IroN gene was amplified from the genomic DNA of E.coli J96 and cloned into prokaryotic expression vector pET-32a（＋）.The constructed recombinant plasmid pET-32a-IroN was transformed to E.coli BL21（DE3）for expression under induction of IPTG.The expressed recombinant protein was purified and used for immunization of BALB/c mice by subcutaneous route.The antibody levels in sera and urine of immunized mice were determined by ELISA.The immunized mice were challenged with E.coli J96 strain by intraperitoneal and intraurethral routes respectively,based on which the protective rate was calculated,and the bacteria were counted.Results The recombinant IroN protein,at an expression level of 32%,reached a purity of more than 85% after purification and showed good reactogenicity.Subcutaneous immunization with the recombinant IroN protein induced high titer specific IgG in sera of mice,while no specific IgA was detected in urine.The protective rate of recombinant IroN protein against intraperitoneal challenge with ExPEC was 81.8%（P 〈0.05）.After intraurethral challenge with ExPEC,the number of bacteria isolated from the kidneys of mice immunized with recombinant IroN protein decreased by more than 100 folds（P 〈0.05）,while those in bladder and urine showed no significant difference（P 〉0.05）as compared with those in control groups（immunized with PBS）.Conclusions IroN protein was expressed in prokaryotic cells and purified,which showed significantly protective effect against intraperitoneal and renal infection with ExPEC,but showed no protection against bladder infection.