展呈H5N1亚型禽流感病毒M2e表位鞭毛蛋白的构建及其免疫原性

以重组质粒pET-fliC和pUC57-M2e2为模板,通过重叠PCR将M2e2基因插入fliC的高变区域,构建嵌合鞭毛基因片段fliC/M2e2。将fliC/M2e2片段插入原核表达载体pET30a+,获得重组质粒pET-fliC/M2e2,将其转化鼠伤寒沙门氏菌LB5000,获得重组菌LB5000(fliC/M2e2)。应用P22噬菌体转导技术,将fliC/M2e2基因导入都柏林沙门氏菌SL5928的基因组中,经生化血清学鉴定、动力学测定、透射电镜观察、Western-blot分析,将阳性转导菌命名为SL5928(fliC/M2e2)。提取重组菌鞭毛蛋白fliC/M2e2作用HEK293-mTLR5细胞,能刺激细胞分泌IL-8。将fliC/M2e2通过皮下注射途径免疫C3H/HeJ小鼠,50μg/只。结果显示,二免后第14天,fliC/M2e2鞭毛蛋白免疫组的血清抗体效价与对照组之间呈现显著性差异(P<0.05)。ELISPOT结果表明,在脾细胞中,fliC/M2e2鞭毛蛋白免疫组检测到较高水平的IFN-γ和IL-4分泌细胞,且免疫应答以Th1型为主。结果表明,构建的fliC/M2e2嵌合基因能够在沙门氏菌中表达,重组嵌合鞭毛蛋白fliC/M2e2能激发机体针对M2e表位的体液免疫应答和Th1型细胞免疫应答。

Flagellin displaying the M2e epitope of H5N1 subtype influenza virus and its immunogenicity analysis

The gene encoding two tandem copies of M2e(aa 2-24) derived from the consensus sequence of H5N1 subtype influenza virus was inserted into the hypervariable region of fliC,with pET-fliC and pUC57-M2e2 as the template by using overlap PCR.The chimeric gene was designated as fliC/M2e2.Then the chimeric gene was inserted into pET30a+ and the recombinant plasmid was named as pET-fliC/M2e2.The recombinant plasmid pET-fliC/M2e2 was transformed into the strain LB5000 of Salmonella typhimurium and the recombinant bacteria was named as LB5000(fliC/M2e2).The transduction was conducted between LB5000(fliC/M2e2) and Salmonella dublin strain SL5928 using bacteriophage P22HT int.The positive transductants were analyzed by motility observation,transmission electron microscopy,Western-blot,and followed by biochemical and serological identification.Finally,the positive transductant was named as SL5928(fliC/M2e2).The recombinant chimeric protein fliC/M2e2 was extracted from SL5928(fliC/M2e2) and strongly induced HEK293-mTLR5 cells to secrete IL-8.C3H/HeJ mice were immunized subcutaneously(s.c.) with 50μg of fliC/M2e2.The results showed that on the 14th day post boosting,there was a significant difference between fliC/M2e2-immunized group and control groups(P <0.05).Furthermore,in splenocytes,immune responses against M2e were biased toward Th1 type,the frequency of IFN-γ-secreting cells was much higher than that of IL-4-secreting cells.It demonstrated that when inserted into the hypervariable region of flagellin,the M2e2 gene has no impact on the expression of chimeric flagellins in Salmonella dublin and the chimeric flagellin could be used as a adjuvant to enhance the induction of M2e-specific immunity.