摘要
目的 构建具有M细胞靶向性的多房棘球蚴表位疫苗GILE 口服乳酸菌载体.方法 本研究在前期构建的多表位疫苗GILE的基础上,通过添加SAM基因序列,设计、合成SAM-GILE序列,构建乳酸菌表达质粒pNZ8148-SAM-GILE,并电转至乳酸乳球菌NZ9000中建立乳酸菌表达体系.用酶切与基因测序双重实验验证乳酸菌表达质粒pNZ8148-SAM-GILE是否构建成功,用PCR验证乳酸菌表达质粒pNZ8148-SAM-GILE是否成功电转至乳酸乳球菌NZ9000,用Western Blot实验检测重组蛋白SAM-GILE是否表达,用全细胞ELISA法检测SAM-GILE的表面展示情况,用免疫荧光染色法鉴定LL-plSAM-GILE的M细胞靶向性.结果 酶切与基因测序双重实验结果显示,乳酸菌表达质粒pNZ8148-SAM-GILE构建成功.经PCR验证,pNZ8148-SAM-GILE成功电转至乳酸乳球菌NZ9000.经Western Blot验证,通过Nisin诱导,成功表达约45 KD的重组蛋白.经全细胞ELISA检测,SAM-GILE可展示于LL-plSAM-GILE表面.经免疫荧光染色验证,重组抗原与M细胞高度重合,表明重组乳酸菌疫苗LL-plSAM-GILE具有M细胞靶向性.结论 成功构建具有M细胞靶向性的多房棘球蚴表位疫苗GILE 口服乳酸菌载体.
Abstract
Objective To construct a vector of epitope vaccine GILE oral Lactococcus lactis for Echinococcus multilocularis with M-cell targeting.Methods The SAM-GILE sequence was designed and synthesized by adding the SAM gene to the existing multi-epitope vaccine GILE to construct Lactococcus lactis expression plasmid pNZ8148-SAM-GILE.The expression system for Lactococcus lactis was established by electroporating into Lacto-bacillus lactis NZ9000.Double experiments of enzyme digestion and gene sequencing were used to verify whether Lactococcus lactis expression plasmid pNZ8148-SAM-GILE was constructed successfully.PCR were used to verify whether Lactococcus lactis expression plasmid pNZ8148-SAM-GILE was electroporated into Lactococcus lactis NZ9000 successfully.Western Blot were used to detect whether the recombinant protein SAM-GILE was ex-pressed.The surface display of SAM-GILE was identified via whole-cell ELISA.The M-cell targeting of LL-plSAM-GILE was identified by immunofluorescence.Results Double experiments of enzyme digestion and gene se-quence showed the successful construction of Lactococcus lactis expression plasmid pNZ8148-SAM-GILE.PCR showed that pNZ8148-SAM-GILE was successfully electroporated into Lactococcus lactis NZ9000.Western Blot showed that the recombinant protein was expressed successfully at approximately 45 KD induced by Nisin.Whole-cell ELISA detection showed that SAM-GILE could be displayed on the surface of LL-plSAM-GILE.Immunofluo-rescence verified that the recombinant antigen was highly consistent with M cell,indicating that the recombinant Lactococcus lactis vaccine LL-plSAM-GILE had M-cell targeting.Conclusion A vector of epitope vaccine GILE oral Lactococcus lactis for Echinococcus multilocularis with M-cell targeting was successfully constructed.
基金项目
国家自然科学基金地区项目(81860299)
国家自然科学基金地区项目(32360192)
青海省科技厅应用基础研究项目(2024-ZJ-909)
维普
万方数据