Abstract
Objective To investigate the inhibitory effect of live-attenuated Listeria monocytogenes （LM）-based vaccines expressing the gene encoding a melanoma differentiation antigen,MART-1,on malignant melanoma and their mechanism.Methods The constructed plasmid pERL3-MART-1 was used to transform live-attenuated LM by electroporation to construct recombinant LM.i.e.△inlB LM-MART-1 and △actA/△inlB LM-MART-1.The half lethal dose （LD50） of attenuated listeria strains was determined by concentration gradient dilution method.C57BL/6 mice were randomly divided into three groups,namely PBS group,△inlB LM-MART-1 group and △actA/△inlB LM-MART-1 group.Mice were inoculated by intraperitoneal injection of O.1 LD50 of each rLM strain or PBS only.One week later,the mice were injected subcutaneously with 1×105 B16F10 cells（a mouse melanoma cell strain）in 200μl of PBS.Reimmunization was performed on day 14 and 21.Subsequently,the growth of tumor and survival of tumor bearing mice were observed.All mice were killed on day 28,and tumor tissue as well as splenocytes were obtained from these mice for the detection of MART-1 gene expression by real-time quantitative PCR and the percentage of CD4^＋CD25^＋T cell by flow cytometry.Results The recombinant △inlB LM-MART-1 and △actA/△inlB LM-MART-1 were constructed successfully.The LD50 of △inlB LM and △actA/△inlB LM was lower than LM-EGDe by 100 and 10 000 times respectively.Compared with PBS,the tumor growth was inhibited with △inlB LM-MART-1 by 46.95%（F=6.3,P〈0.05）,and by 83.96% with △actA/△inlB LM-MART-1（F=37.8,P〈0.01）.The relative expression level of MART-1 in △inlB LM-MART-1 group and △actA/△inlB LM-MART-1 group was 8.988±0.207 and 11.315±0.445 times that in PBS group （both P〈0.05）.The percentage of CD4^＋CD25^＋T cells in splenocytes was （2.52±0.20）%,（1.14±0.13）% and （0.44±0.15）% in PBS group,△ialB LM-MART-1 group and △actA/△inlB LM-MART-1 group,respectively;the differences were statistically significant between the three groups （all P〈O.01）.Conclusions The attenuated LM carrying MART-1 gene has a lower virulence than LM reference strain,and could efficiently suppress the growth of melanoma and lengthen the survival of melanoma-beating mice. Key words: Melanoma,experimental; Listeria monocytogenes; MART-1
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