肠炎沙门菌菌影的制备及其作为疫苗载体的特性分析

Preparation and immunological characterization of Salmonella enteritidis ghosts as a vaccine vector

目的制备肠炎沙门菌菌影并对其作为疫苗载体的特性进行研究。方法以带有噬菌体裂解基因E的溶菌质粒为模板,温控溶菌盒EBOX经PCR扩增和酶切后重组到pBBR1MCS2载体中并导入沙门菌,经42℃诱导表达后测定裂解效率;将真核表达载体pEGFP-N1负载沙门菌菌影后转染小鼠巨噬细胞,荧光显微镜观察和流式细胞仪检测GFP的表达情况;用负载重组质粒pVAX1-NspA的沙门菌菌影经口灌胃免疫BALB/c小鼠,ELISA检测诱导抗体产生情况。结果重组菌诱导后的裂解效率达99%。沙门菌菌影负载pEGFP-N1转染小鼠巨噬细胞后可检测到GFP的表达。3次免疫两周后,负载重组质粒pVAX1-NspA沙门菌菌影免疫小鼠特异性血清IgG水平和黏膜IgA水平(A_(450)值)分别为0.60±0.05和0.53±0.06,负载pVAX1沙门菌菌影免疫组分别为0.32±0.07和0.22±0.03,差异有统计学意义(P<0.01)。结论制备的肠炎沙门菌菌影免疫能诱导特异性IgG和IgA抗体产生,可作为疫苗载体介导抗原的表达。

Objective To prepare Salmonella enteritidis ghosts and to assess their immunological characteristics as a vaccine vector.Methods The lysis cassette EBOX containing the lysis gene E and λpL/pR-cI857 was amplified with PCR using the plasmid pBV220-E as a template and then subcloned in the plasmid pBBR1 MCS2 to construct the recombinant plasmid p-EBOX.The resultant plasmid p-EBOX was transformed by electroporation into S.enteritidis.S.enteritidis ghosts were prepared by heat-inducible expression at 42℃.The eukaryotic expression plasmid pEGFP-N1 was loaded into S.enteritidis ghosts and transfected into murine macrophages.The expressed GFP was detected using an indirect immunofluorescent assay and flow cytometry.6-week-old BALB/c mice were inoculated orally with S.enteritidis ghosts loaded with pVAX1-NspA,and antibodies against NspA were detected in serum and vaginal lavage fluid using indirect ELISA.Results The efficiency of induction of S.enteritidis ghosts was 99%.The transfection efficiency of S.enteritidis ghosts loaded with pEGFP-N1 was investigated in murine macrophages.The cells exhibited significant green fluorescence in their cytoplasm under fluorescence microscopy.GFP expression was verified with flow cytometry.Oral immunization with S.enteritidis ghosts loaded with pVAX1-NspA induced significantly higher levels of specific serum IgG and mucosal IgA(0.60±0.05 and 0.53±0.06 at A450,respectively) than those in S.enteritidis ghosts loaded with pVAX1(0.32±0.07 and 0.22±0.03 at A450,respectively)(P<0.01).Conclusion Results suggested that S.enteritidis ghosts have the potential to be a vaccine vector for the induction of humoral and mucosal immune responses and for protection against infectious diseases.