为研究M细胞靶向肽co1的免疫增强作用,本研究从肠产毒素性大肠杆菌(ETEC)全基因组中扩增FaeG基因,通过重叠延伸PCR的方法将人工合成的co1靶向肽编码序列连接到FaeG基因的3'端,获得FaeG-co1融合基因.并分别将FaeG和FaeG-co1基因克隆到pET-30b表达载体中,转化于大肠杆菌BL21 (DE3)进行表达.SDS-PAGE和western blot分析显示,FaeG和FaeG-co1基因在大肠杆菌中均获得了高效表达,表达产物主要以包涵体形式存在.分别用两种重组菌表达的包涵体免疫昆明鼠,测定特异性IgG、sIgA抗体水平,并用ETEC强毒株对免疫鼠进行攻毒.结果表明,FaeG-co1重组蛋白免疫小鼠诱导机体产生的特异性IgG和sIgA抗体水平均显著高于FaeG组;FaeG-co1组两次免疫后即可对ETEC致死剂量的攻毒保护率达到100％.这些结果表明M细胞靶向肽对抗原具有免疫增强作用,是一种极具应用前景的免疫增强分子.%To study the immune-enhancement effect of the M cell-targeting ligand col, FaeG gene was amplified from chromosomal DNA of Enterotoxigenic Escherichia coli (ETEC) by PCR, and fused with the synthesized col gene on 3' terminal of the FaeG gene by SOE-PCR. The gene of FaeG or FaeG-col was inserted into pET-30b and transformed into BL21 (DE3) for expression, respectively. The SDS-PAGE and western blot analysis showed that FaeG and FaeG-col gene was highly expressed in E.coli and mainly in the form of inclusion body. Furthermore, Kunming mice were twice immunized with the inclusion body of the recombinant proteins, respectively, and challenged with the ETEC at a dose of 1 LD50. The results showed that the mice in FaeG-col group were completely protected against ETEC challenge, but only 80% protection in FaeG group. In addition, the specific IgG and sIgA antibody titers in FaeG-col group were significantly higher than that of FaeG group. These results suggest that the M cell-targeting peptide of col has immune-enhancing activity to the antigen, which is a promising immune-enhancing molecule.
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