重组大肠杆菌不耐热肠毒素B亚单位的表达、纯化及黏膜佐剂活性研究

目的　对大肠杆菌不耐热肠毒素B亚单位(LTB)进行基因克隆、重组表达、蛋白纯化和黏膜佐剂活性分析。方法　应用PCR技术从产肠毒素大肠杆菌基因组中扩增出不耐热肠毒素B基因,将此构建于原核表达载体pET 11C,并在大肠杆菌BL21(DE3)中表达,然后采用D(+) immobilizedgalactose亲和层析柱对重组蛋白进行纯化。以纯化后重组LTB为佐剂,与幽门螺杆菌重组尿素酶B亚单位蛋白(rUreB)共同口服和滴鼻免疫BALB c小鼠,分析重组LTB的黏膜佐剂活性。结果　PCR扩增出390 bp LTB基因,与GenBank公布的核苷酸序列的相似性为99.5%。重组LTB蛋白的表达率为6%,以胞周质分泌形式表达,经亲和层析后蛋白纯度大于95%。该重组蛋白保持了与神经节苷脂GM1结合的生物学活性和良好的免疫原性及免疫反应性。动物试验表明,重组LTB与rUreB抗原共同口服和滴鼻免疫小鼠后的特异性血清IgG及鼻腔、气管、胃黏膜和小肠黏膜sIgA水平显著高于单独rUreB抗原免疫组(P<0.05)。结论　所获得的重组LTB具有较好的黏膜佐剂活性,为其在黏膜疫苗中的应用奠定基础。

Expression, purification of recombinant LTB protein of E. Coli and its mucosal immunoadjuvanticity

Objective To investigate the mucosal immunoadjuvanticity of recombinant Escherichia coli heat-labile enterotoxin B subunit (rLTB). Methods The gene coding for LTB was cloned with polymerase chain reaction(PCR) and constructed into an expression vector (pET-11c). The recombinant LTB was expressed in Escherichia coli BL21(DE3) and purified with D(+)-immobilized galactose affinity chromatography. BALB/c mice were administered via the oral or nasal route with recombinant Helicobacter pylori urease B subunit (rUreB) with and without rLTB as a mucosal adjuvant. After four dosages of immunization, serum IgG and IgA antibody levels or sIgA antibody levels in mucosal sites were investigated. Results rLTB was expressed by Escherichia coli as a secretory protein and the purity of rLTB was up to 95% after affinity chromatography. The purified rLTB keeps GM1 binding ability immunogenicity and immunoreactivity, which were the same as native LTB. After immunization with rUreB and rLTB, anti-rUreB serum IgG antibodies and anti-rUreB mucosal sIgA antibodies in the nasal cavity, trachea, stomach and small intestine were significantly increased as compared with that after rUreB immunization alone without rLTB(P<0.05). Only a slight increase of anti-rUreB serum IgA antibodies was observed after rUreB immunized with and without rLTB. Conclusion The rLTB protein was a powerful mucosal adjuvant for mucosal vaccine.