沙门菌鞭毛蛋白免疫佐剂效应关键位点的研究

A study on the key sites of immune adjuvant effects of Salmonella flagellin

为了研究沙门菌鞭毛蛋白发挥免疫佐剂效应的关键位点,获得新型高效的蛋白免疫佐剂,从实验室保藏的质粒中通过双酶切方法获得了分别编码野生沙门菌鞭毛蛋白、D1保守区N端第88-96位氨基酸突变鞭毛蛋白、D1保守区C端第411位丙氨酸突变鞭毛蛋白、高变区第183-279位缺失鞭毛蛋白SJW61的目的基因,连接入原核表达质粒pET30a＋,并转化入大肠杆菌BL21（DE3）中,以IPTG诱导表达;SDS-PAGE和Western blot鉴定目的蛋白;分别以4种鞭毛蛋白混合卵清白蛋白（OVA）免疫C57BL/6小鼠,二免后收集血清ELISA检测OVA蛋白特异性抗体效价;取脾脏细胞ELISPOT法检测特异性IFN-γ及IL-4分泌细胞。结果显示：4种原核表达的鞭毛蛋白均以胞内可溶形式表达;Western blot分析显示,4种蛋白均具有良好的免疫原性;动物结果表明,4种原核表达的鞭毛蛋白及其突变体均能显著增强OVA蛋白的特异性免疫应答（P〈0.05）,且免疫应答趋向于Th2型（P〈0.05）,其中D1保守区N端第88-96位氨基酸突变鞭毛蛋白佐剂效应显著弱于其余3种鞭毛蛋白（P〈0.05）,高变区缺失鞭毛蛋白SJW61佐剂效应略有降低（P〉0.05）。

To explore the key sites of immune adjuvant effects of Salmonella flagellin and obtain new and highly efficient protein adjuvants,double-enzyme digestion was performed and the target genes were successfully obtained from preserved strains which expressed the wild-type Salmonella typhimurium flagellin,mutation of 88-96 amino acids of fli C residues within the N-terminal conserved D1 domain,alanine mutation at the 411^（th） site within the C-terminal conserved D1 domain and deletion of the 183-279 hypervariable region of flagellin. Then,the genes were inserted into the pET30 a＋ plasmids,and the recombinant plasmids were transformed into E. coli BL21（ DE3） for gene expression. The productions were identified by SDS-PAGE and Western blot. Then,each of the four target proteins mixed with OVA protein was used to immunize C57 BL/6 mice. Their serum was collected after the second immunization,and the OVA specific antibody titers were detected by ELISA. Spleen cells from the rodents were also collected after the second immunization,and the specific IFN-γ-secreting cells and IL-4-secreting cells were examined by ELISPOT. The results showed that the four proteins of flagellins were all expressed in dissoluble form in cells,and the Western blot assay indicated that the four proteins had good immunogenicity. The results of the animal experiments suggested that the four flagellin or flagellin mutants significantly enhanced the specific immune response of OVA protein（ P0. 05）. In conclusion,the four proteins of flagellins had good immunogenicity,and the immune response was likely to the Th2 type（ P 0. 05）. The flagellin with the 88-96 amino acids mutation exhibited significantly lower adjuvant effect when compared with the other three proteins（ P0. 05）. The adjuvant effect of the flagellin SJW61 in the hypervariable region was slightly reduced（ P0. 05）.