具有M细胞靶向性的多房棘球蚴表位疫苗GILE口服乳酸菌载体的构建

Construction of a vector of epitope vaccine GILE oral Lactococcuslactis for Echinococcus multilocularis with M-cell targeting

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。

Objectivee 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-CILE.The expression system for Lactococcus lactis was established by electroporating into Lactobacillus 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-CILE was expressed.The surface display of SAM-GILE was identified via whole-cell ELISA.The M-cell targeting of LLplSAM-CILE was identified by immunofluorescence.Results Double experiments of enzyme digestion and gene sequence showed the successful construction of Lactococcus lactis expression plasmid pNZ8148-SAM-CILE.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.Wholecell ELISA detection showed that SAM-GILE could be displayed on the surface of LL-plSAM-GILE.Immunofluorescence 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 CILE oral Lactococcus lactis for Echinococcus multilocularis with M-cell targeting was successfully constructed.