Construction And Characterization Of Recombinant Listeria Monocytogenes Expressing Mycobacterium Bovis ESAT-6 Protein

Bovine tuberculosis, one of severe zoonoses, is mainly caused by Mycobacterium bovis and remains the most significant issue of public health. Novel vaccines are urgently needed because of the unefficacy of current BCG,the sole clinical vaccine for TB prevention. As cellular immune responses play an important role in the prevention of Mycobacterium bovis infection, one of the main criteria of excellent vaccines is the enhancement of cellular responses. In this study, taking Listeria monocytogenes (Lm) which can induce strong CD8~+T responses as vector, and ESAT-6 protein which is crucial protective antigen secreted from pathogenetic Mycobacterium bovis as target antigen, two different recombinant strains were constructed. And then, the immunological properties of recombinant strains were characterized as vaccine potential. It laid basic foundation for developing novel bovine tuberculosis vaccines.1. Construction of recombinant Listeria monocytogenes expressing Mycobacterium bovis ESAT-6 proteinThe homologous sequences hlya and hlyb and target gene esat-6 were amplified by PCR from genome of Listeria monocytogenes Lm4 strain and plasmid pET-3a(+)-esat-6 respectively. The complex fragment of hlya-esat-6-hlyb was constructed by splicing overlap extention PCR and then inserted into cloning vector pGEM-T after digested by double enzymes. After identified by sequencing, the hlya-esat-6-hlyb fragment was subcloned in shuttle vector pKSV7 to construct recombinant plasmid pKSV7-hlya-esat-6-hlyb. Then this recombinant plasmid was introduced into competent Lm4 and LmΔactA/plcB by electroporation. Under the double pressure of chloramphenicol and temperature selection, two recombinant bacteria strains were obtained and named as Lm(esat-6) and LmΔactA/plcB(esat-6). The target gene esat-6 was proved to be correctly inserted into the desired site in-frame with hly ORF and the recombinant strains were steady verified by two rounds of PCR using different primer pairs.2. Characterization of recombinant Listeria monocytogenes expressing Mycobacterium bovis ESAT-6 proteinThe immunological properties of two recombinant strains Lm(esat-6) and LmΔactA/plcB(esat-6) were characterized. The results of RT-PCR assay indicated that the target gene esat-6 was successfully transcripted in vitro. The hemolysis assay showed that the expressed fusion protein LLO-ESAT-6 in the supernatants of recombinant strains still remained the hemolysis activity of LLO, and it's hemolysis titers reached to 2~4. It was proved that two recombinant bacteria were less virulent compared with that of their respective parent strains through cell invasion assay and LD50 measurement to C57BL/6 mice, and the LmΔactA/plcB(esat-6) was more safe than the other. After intravenous vaccination of C57BL/6 mice with Lm(esat-6) and LmΔactA/plcB(esat-6), there were 177 and 178 specific INF-γ-secreting cells respectively in 1×106 splenocytes, while less IL-4-secreting cells were detected. This indicated that recombinant strains can induce Th1 cell responses. Proliferative responses of splenocytes as well as DTH responses of foot pad were also observed. Taken together, it was showed that the two recombinant Listeria monocytogenes expressing Mycobacterium bovis ESAT-6 protein, Lm(esat-6) and LmΔactA/plcB(esat-6), have the potential of novel TB vaccine for their security and abilities of inducing specific cellular immune responses. Compared between the two strains, there were no significant differences in series of immunobiological properties.