Construction of recombinant attenuated Salmonella typhimurium carrying the lipocalin 2 gene and its effect on the growth of mycobacteria

Abstract:

Objective：To construct attenuated Salmonella typhimurium carrying the lipoprotein 2（Lcn2） gene and to investigate its effect on the growth of mycobacteria. Methods：RNA was extracted from macrophage RAW264.7 cells and reverse transcribed into cDNA. The Lcn2 gene was amplified by PCR using the cDNA as a template and inserted into the multiple cloning sites of the plasmid pBudCE4.1-orim to construct the recombinant plasmid pBudCE4.1-orim-Lcn2. The recombinant plasmid was trans－fected into RAW264.7 cells. The mRNA expression of Lcn2 was measured by RT-qPCR，with the empty plasmid for a control group. Then the recombinant plasmid was transfected into the attenuated Salmonella typhimurium SL7207 to obtain the recom－binant strain SL7207-pBudCE4.1-orim-Lcn2. RAW264.7 cells were infected with the recombinant strain. The protein expres－sion of the Lcn2 gene was measured by Western blot，and its mRNA expression was measured by RT-qPCR，with the empty strain for a control group. Bacterial colonies were counted on the 7H10 plate to observe the effect of the recombinant strain on the survival of bacille Calmette-Guérin（BCG） in RAW264.7 cells，with the empty strain for a control group. Mice were intragastrically given the re－combinant strain to measure the Lcn2 expression in the spleen and the interferon gamma（IFN-γ） level in serum，with the empty strain and normal saline for control groups. Results：The Lcn2 gene was amplified by PCR，and the recombinant plasmid was identified by double-enzyme digestion and DNA sequencing. The protein expression of Lcn2 in RAW264.7 cells infected with the recombinant strain was significantly increased compared with the control group（P=0.000）. The RT-qPCR showed that the mRNA expression of Lcn2 in the recombinant strain group and the recombinant plasmid group was significantly higher than that in the control groups（F=5.281，P=0.000；F=22.570，P=0.000）. The colony number of BCG on the 7H10 plate in the recombinant strain group was significantly reduced compared with the control group（F=11.41，P=0.000）. The expression of Lcn2 in mouse spleen tissues and the level of IFN-γ in serum were significantly higher in the recombinant strain group than in the control groups（F=4.449，P=0.000；F=15.27，P=0.004）. Conclusion：Recombinant attenuated Salmonella typhimurium carrying the Lcn2 gene was successfully constructed. The expression of Lcn2 increased in RAW264.7 cells infected with the recombinant strain，which inhibited the survival of BCG in the cells. The expres－sion of Lcn2 in mouse spleen tissues and the IFN-γ level in serum were elevated after the mice were intragastrically given the recom－binant strain，so that the immune state of mice was beneficial to anti-tuberculosis outcome. This study lays a foundation for further study of the relationship between lipid carrier proteins and Mycobacterium tuberculosis infection and its molecular mechanism.