To reconstruct the heat-labile enterotoxin subunit B (LTB) gene of Escherichia coil in order to increase the outputs of the prokaryotic expression on recombinant LTB (rLTB), and to determine its immune adjuvant activity on mucosa,the nucleotide sequence of the whole length of LTB gene was synthesized according to the preferred codons of E. coli, and the prokaryotic expression plasmid pET32a-rLTB and its expression system in E. coli BL21DE3 were reconstructed. The recombinant plasmid was extracted and the inserted sequence of rLTB gene was determined. Meanwhile, the expression quantity of the reconstructed rLTB was identified by SDS-PAGE and BioRad agarose image analyzing system, and compared with that of the un-reconstructed rLTB. The abilities of the reconstructed rLTB and the un-reconstructed rLTB to bind with bovine GM1 were determined by means of GM1-ELISA assay. By using the recombinant urease subunit B as antigen, the effects of the reconstructed and the un-reconstructed rLTB on the improvement of immune protection of BALB/c mice infected with Helicobacter pylori strain SS1 and the induction of S-IgAs in infected mice were assayed. The experimental results showed that the expression quantity of the reconstructed rLTB approached upto 35.4% of the total bacterial proteins after induction with 1 mmol/L IPTG for pET32a-rLTB-E. coliBL21DE3 and to be 12.6 times higher than that of the un-reconstructed rLTB (2.8 %). In addition, both the abilities of the recombinant reconstructed LTB and the un-reconstructed rLTB to bind with bovine GM1 could be demonstrated by GM1-ELISA. The immune protection rate of the recombinant urease subunit B in the infected mice was 66.7%; and it could reach up to 91.7% with a significantincrease of the specific S-IgA level, when it was immunized with the reconstructed or the un-reconstructed rLTB. It is concluded that the reconstructed LTB gene in the present study shows a remarkable increased outputs of expression of this gene with a strong immune adjuvant activity on mucosa.%目的改建大肠杆菌不耐热肠毒素B亚单位(LTB)基因序列以提高重组LTB(rLTB)原核表达量,确定重组改建LTB(rrLTB)粘膜免疫佐剂活性.方法按大肠杆菌偏爱密码子合成全长LTB基因的核苷酸序列.构建pET32a-rLTB原核重组表达质粒及pET32a-rLTB-E.coliBL21DE3表达系统,提取重组质粒并测定其插入的rLTB基因序列.采用SDS-PAGE和BioRad凝胶成像分析系统鉴定rrLTB表达产量,并与未改建的重组LTB(roLTB)比较.采用GM1-ELISA确定rrLTB和roLTB结合牛GM1的能力.分别以幽门螺杆菌重组尿素酶B亚单位(rUreB),检测rrLTB和roLTB对rUreB对幽门螺杆菌SS1株感染BALB/c小鼠保护作用及产生特异性S-IgA的增强效应.结果pET32a-rLTB-E.coliBL21DE3经1 mmol/LIPTG诱导后,rrLTB表达量约占细菌总蛋白的35.4%,为roLTB(2.8%)的12.6倍.GM1-ELISA结果证实rrLTB和roLTB均能与牛GMi结合.单一rUreB对感染小鼠的免疫保护率为66.7%,但与rrLTB或roLTB合用时,保护率可至91.6%,并可提高感染小鼠特异性S-IgA产生量(P＜0.01).结论改建的LTB基因能明显提高表达量,所表达的rrLTB仍保持较强的粘膜免疫佐剂活性.
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