Construction Of A Recombinant Attenuated Salmonell Typhimurium Carrying Lipocalin 2 Gene And Its Effects On The Growth Of Mycobacteria

Objective:To construct attenuated Salmonella which expresses lipoprotein 2 gene and investigate its effects on the growth of Mycobacteria.Methods:RNA was extracted from RAW264.7 cells and reversed transcript ion into cDNA.Lcn2 gene was amplified by PCR using the cDNA a s a template and inserted into the multiple cloning sites of plasmid p BudCE4.1-orim to construct the recombinant plasmid pBudCE4.1-orim-Lcn2.The recombinant plasmid was transfected into RAW264.7 cells and the mRNA expression of Lcn2 was detected by RT-qPCR.Then the recombinant plasmid was transfected into the attenuated Salmone lla typhimurium SL7207 to obtain the recombinant strain SL7207-pBu dCE4.1-orim-Lcn2.RAW264.7 cells were infected with the recombina nt strain,and the protein expression of Lcn2 gene was detected by Western blot,and its mRNA expression was detected by RT-qPCR.B acterial colonies were counted on the 7H10 plates to observe the effects of the recombinant strain on the survival of BCG in RAW264.7 cells.Mice were orogastrically(o.g)with the recombinant strain to d etect Lcn2 expression in spleen and IFN-? ?IL12 levels in serum.A BCG mouse infection model was established to study the therapeutic effect of recombinant bacteria.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.The mRNA level of Lcn2 in the recombinant strain group and the recombinant plasmid group was significantly higher than that in the control group by RT-qPCR assay respectively(P=0.000,F=5.281;P=0.000,F=22570).The colony number of BCG in the recombinant strain group on the 7H10 plate was significantly reduced compared with the control group(P=0.00,F=11.41).The expression of Lcn2 was increased in mouse spleen tissues and serum IFN-? was elevated in the recombinant strain group(P=0.000,F=4.449;P=0.004,F=15.27).However,there was no significant difference in serum IL-12 level between the recombinant strain group and the control group(P>0.05,F=2.26),nor in the splenic bacterial load(P > 0.05,F = 1.30).Conclusion:Recombinant attenuated Salmonella typhimurium carrying Lcn2 gene was successfully constructed.The expression of Lcn2 increased in RAW264.7 cells infected with recombinant strain,which inhibited the survival of BCG in the cells.The expression of Lcn2 was increased in mouse spleen tissues and serum IFN-? was elevated after the mice were orogastrically with the recombinant strain so that the immune state of mice was beneficial to anti tuberculosis.This study laid a foundation for further study of the relationship between lipid carrier proteins and Mycobacterium tuberculosis infection and its molecular mechanism.