大肠杆菌鞭毛蛋白不同结构域与F4菌毛主要结构亚单位FaeG嵌合基因的构建及其表达蛋白的功能研究

Construction and functional study of chimeric genes of different domains of E.coli flagellin with FaeG,the main structural subunit of F4 fimbriae

为探究大肠杆菌鞭毛蛋白不同结构域与其免疫佐剂活性的相关性,本研究通过重叠延伸PCR技术(SOE-PCR)分别构建了出血性大肠杆菌EDL933(O157􀏑H7)鞭毛蛋白FliCH7基因的全长(aa1~aa585)、N端保守区(aa1~aa174)、N端加中间的高变区(aa1~aa496)与产肠毒素大肠杆菌F4ac+国内标准株C83902(O8􀏑K88􀏑H19)主要亚单位FaeG串联表达的嵌合基因,即FliC-FaeG、FliCN-FaeG和FliCNV-FaeG,分别将其克隆至pET-28a(+)表达载体中经IPTG诱导表达;经SDS-PAGE和western blot分别对表达的融合蛋白进行鉴定。PCR结果显示3个嵌合基因分别约为2600 bp、1300 bp和2300 bp;SDS-PAGE结果显示融合蛋白的大小分别约为:90 ku、48 ku和80 ku,均与预期大小一致。Western blot结果表明3种融合蛋白均能被抗FaeG的单克隆抗体识别;表明融合蛋白中FaeG仍维持其独立的结构和生物学活性。抗FliCH7的抗体可识别FliC-FaeG和FliCNV-FaeG融合蛋白,而FliCN-FaeG融合蛋白不含鞭毛蛋白高变区,所以不能被识别;TLR5受体活性检测结果表明,仅FliC-FaeG融合蛋白能激活TLR5受体,引起IL-8、TNF-α炎性因子的分泌,以上结果表明TLR5受体活性的激活需要完整的鞭毛蛋白结构,本研究以FaeG为模型抗原,构建了大肠杆菌鞭毛蛋白不同结构域与其相结合的融合蛋白,为进一步研究大肠杆菌鞭毛蛋白不同结构域免疫佐剂活性的差异性和深入研究鞭毛蛋白发挥其免疫佐剂活性的机理提供了参考依据。

In order to study the different domains of E. coli flagellin related to its immune adjuvant activity, we genetically fused fae G gene [the main subunit of F4ac fimbriae derived from enterotoxigenic E. coli C83902(O8:K88:H19)] to different domains of hemorrhagic E. coli EDL933(O157:H7) flagellin FliCH7 gene by overlap extension PCR(SOE-PCR), resulting in three chimeric genes, eg. FliC(aa1-aa585)-FaeG, FliCN(aa1-aa174)-FaeG, and FliCNV(aa1-aa496)-FaeG. They were cloned into pET-28 a(+)expression vector and induced by IPTG. The expressed fusion proteins were verified by combined methods of SDS-PAGE and western blot. PCR amplification produced that three chimeric genes with sizes of approximately 2600 bp, 1300 bp, and 2300 bp,respectively. SDS-PAGE results indicated that the proteins with molecular mass of 90 ku, 48 ku, and 80 ku, respectively, consistent with the expected sizes of the three fusion proteins. Further, western blot results showed that all three fusion proteins could be recognized by anti-FaeG monoclonal antibodies, indicating that FaeG still maintains its independent structure and biological activity in the fusion protein. The anti-FliCH7 antibody can recognize FliC-FaeG and FliCNV-FaeG fusion proteins, whereas not the FliCN-FaeG fusion protein due to a lack of the flagellin hypervariable region. TLR5 receptor activity test results showed that only the FliC-FaeG fusion protein can activate the TLR5 receptor, leading to IL-8 and TNF-α inflammatory factors production. The above results indicate that the activation of TLR5 receptor activity requires a complete flagellin structure, In this study, FaeG was used as a model antigen to construct a fusion protein with different domains of E. coli flagellin, thus our results will provide a reference for studying the difference in adjuvanticity of different domains of E. coli flagellin and provide a reference for further study on the mechanism of flagellin activity in immune-adjuvant.