Construction And Immunobiological Properties Of Recombinant Listeria Monocytogenes Expressing Enhanced Green Fluorescent Protein

Listeria monocytogenes is an intracellular bacterial pathogen, which can be propagated in the host cell plasma, so its attenuated strain can be used as live vaccine vector to induce specific cellular immunity. PCR amplificated respectively egfp, the promoter of listeria monocytogenes virulence genes hly and actA,then Phly-egfp and PactA-egfp expressing box were cloned into L. monocytogenes shuttle plasmid pKSV7,resulting the recombinant plasmid pKSV7-Phly-egfp and pKSV7-PactA-egfp,pKSV7-Phly-egfp and pKSV7-PactA-egfp were eleetroporated into actA/plcB mutant competent cells LM1003.The antibiotic resistance screening and PCR identification of candidate strains Lm-PactA-EGFP(LM-1007) and Lm-Phly-EGFP(LM-1010).Under the fluorescence microscope candidate strains confirmed to be able to express EGFP,confocal microscopy confirmed candidate strains in infected mice macrophage J774can also express EGFP.These showed that the promoter Phly and PactA work both in vivo and in vitro.The mouse spleen cells of PBS group、LM1011group and LM1010group by flow cytometry in CD4+、CD8+T cells, CD8+/CD4+T cells rate respectively was66.30±3.97%,128±0.26%和129±0.82%,(The CD8+/CD4+of the LM1011and LM1010was higher than the control group).It is proved that LM1011and LM1010induce effective cellular immunity.15BABL/c mice (H2-Kd),6-8weeks old,were divided into three groups averagely. PBS,107cfu LM-1011(pKSV7-PactA eleetroporated into LM-1003),107cfu LM-1010, were administered s.c.3times; the Last7days after immunization, preparation of spleen lymphocytes.Splenocytes from each experimental group were stimulate by EGFP-special H2-Kd epitope,HYLSTQSAL.The ELISpot kit was used to detect the number of IFN-y-secreting CD8+splenocytes.PBS group、LM1011group、LM1010group respectively are13.50±1.291SFC/4×105,18.75±3.304SFC/4×105、71.25±7.089SFC/4×105(13.50±1.291SFC/4×105vs18.75±3.304SFC/4×105, P>0.05, the differences were not significant;13.50±1.291SFC/4×105vs71.25±7.089SFC/4×105, P<0.01, the differences were statistically significant;18.75±3.304SFC/4×105vs71.25±7.089SFC/4×105, P<0.01, the differences were statistically significant); To determine the cytolytic activity of splenocytes,we used the cytoTox96Non-Radioactive cytotoxicity Assay that is based on the colorimetric detection of the released levels of the LDH enzyme to compare the different E:T ratio the CTL toxicity. CTL cytotoxic of each E:T ratio, the results from this experiment confirmed that no matter the E:T ratio was50:1,10:1or1:1,the released levels of the LDH enzyme from LM1010group was significant higher than the other group.Taken together,the data presented in this paper indicate that a recombinant Lm,which can express and secrete the EGFP protein fused to ActA,can generate a strong and specific immune responses toward EGFP.PCR amplification respectively EGFP gene and ActA30-264aa,then cloned into E.coli expressing plasmid pET-28a,transformed to E.coli BL21(DE3).As demonstrated by SDS-PAGE the induction and expression was succeed. After the purification of EGFP and ActA30-264aa by Ni-NTA column,the protein was verified by SDS-PAGE,and cut the protein gel respectively,for further purification and quantitative.ActA30-264aa+EGFP、 ActA30-264aaand EGFP immune BABL/c mice respectively,preparing spleen lymphocytes.The mouse spleen cells of ActA group^EGFPgroup and ActA+EGFP group by flow cytometry in CD4+CD8+T cells, CD8+/CD4+T cells rate respectively was3.75±6.39%,56.43±0.30%和69.12±5.64%,(The CD8+/CD4+of the ActA+EGFP was higher than the other two group).Splenocytes from each experimental group were stimulate by EGFP-special H2-Kd epitope,HYLSTQSAL.The ELI Spot kit was used to detect the number of IFN-y-secreting CD8+splenocytes. ActA group、EGFPgrop、ActA+EGFP group respectively are11.5±1.732SFC/4×105cells、20.00±2.449SFC/4×105cells,28.75±0.957SFC/4×105cells,(ActA+EGFP vs ActA, P<0.01, the differences were statistically significant, ActA+EGFP vs EGFP, P<0.01, the differences were statistically significant), CTL toxicity.According to the formula to calculate the sepecific CTL cytotoxic of each E:T ratio, the results from this experiment confirmed that no matter the E:T ratio was50:1,10:1or1:1, the released levels of the LDH enzyme from LM ActA+EGFP group was significant higher than the other two groups.In this paper,we present evidence suggesting that Listeria-derived virulence factor,ActA30-64aacan induce specific immune responses toward EGFP.Surface protein ActA30-264aa structural domain has molecular adjuvants features.