产肠毒素大肠杆菌K88菌毛蛋白与大肠杆菌不耐热肠毒素b亚单位的原核表达及其鼻腔免疫效果

Prokaryotic expressions of K88 fimbrial protein of enterotoxigenic E. coli and E. coli heat-labile enterotoxin b subunit and their immune effects by intranasal route

目的原核表达产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)K88菌毛蛋白与大肠杆菌不耐热肠毒素(Heat-labile enterotoxin,LT)b亚单位(LTb),并评价其鼻腔免疫效果。方法采用PCR技术分别扩增不含信号肽的K88和LTb基因,克隆至表达载体pQE30中,构建重组表达质粒pQE30-K88和pQE30-LTb,转化大肠杆菌M15,IPTG诱导表达。表达的重组K88和LTb蛋白纯化、复性后,进行SDS-PAGE分析。分别用K88单独、K88联合LTb滴鼻免疫BALB/c小鼠,以生理盐水作为对照,每次间隔1周,共4次,末次免疫后10 d,分离血清,并收集鼻腔和小肠黏膜冲洗液,间接ELISA法检测各组血清特异性IgG和黏膜sIgA抗体水平。结果重组表达质粒pQE30-K88和pQE30-LTb经双酶切和测序证实构建正确;表达的重组K88和LTb蛋白相对分子质量分别为28 100和12 500,表达量分别约占菌体总蛋白的35%和40%,主要以包涵体形式存在;纯化复性后的重组蛋白纯度均可达95%以上。K88联合LTb滴鼻免疫组血清特异性IgG及鼻腔、小肠黏膜冲洗液中特异性SIgA水平均较K88单独免疫组和对照组明显增高,且差异有统计学意义(P<0.01)。结论在大肠杆菌中高效表达并纯化了重组K88和LTb蛋白,K88联合LTb滴鼻免疫BALB/c小鼠后,可增强特异性血清抗体反应,且诱导了黏膜免疫应答,为将来开发新型的ETEC基因工程疫苗奠定了基础。

Objective To express the K88 fimbrial protein of enterotoxigenic E.coli（ETEC） and E.coli heat-labile enterotoxin b subunit（LTb） in prokaryotic cells and evaluate their immune effect by intranasal route.Methods K88 and LTB genes,containing no signal peptides,were amplified by PCR and cloned into expression vector pQE30,respectively.The constructed recombinant plasmids pQE30-K88 and pQE30-LTb were transformed to E.coli M15 and induced with IPTG.The expressed recombinant K88 and LTb proteins were purified and re-naturalized,then identified by SDS-PAGE.BALB / c mice were immunized with K88 alone and K88 together with LTb by intranasal drip respectively for 4 times at an interval of 4 weeks,using physiological saline as control.The sera as well as nasal and intestinal mucosa washes of mice 10 d after the last immunization were collected and determined for serum specific IgG and mucosal sIgA by indirect ELISA.Results Restriction analysis and sequencing proved that recombinant plasmids pQE30-K88 and pQE30-LTb were constructed correctly.The expressed recombinant K88 and LTb,with relative molecular masses of about 28 100 and 12 500 respectively,contained about 35% and 40% of total somatic proteins respectively,both of which mainly existed in forms of inclusion bodies and reached purities of more than 95% after purification and re-naturalization.The specific IgG levels in sera as well as specific sIgA levels in nasal and intestinal mucosa washes of mice immunized with K88 plus LTb were significantly higher than those with K88 alone and those in control group（P 0.01）.Conclusion Recombinant K88 and Ltb were highly expressed in E.coli and purified,which enhanced specific serum antibody response and induced mucosal immune response by intranasal drip in BALB / c mice.It laid a foundation of further development of novel recombinant ETEC vaccine.