The Effects Of Sigmab Gene Deletion On Pathogenic And Environmental Stress Ability Of Listeria Monocytogenes

Listeria monocytogenes (LM) is a facultative intracellular parasite Gram-positive pathogens with strong existence ability and a wide existence environment. Acute infection caused by LM from the digestive tract of animals and humans had posed a serious threat to livestock and human health. Therefore, LM was classified as one of the four major foodborne pathogens by WHO.As a typical intracellular pathogen, LM can grow and reproduce in professional and non-professional phagocytes, which stimulates the body to produce specific cell immunity. Thus, many researchers hope to transform LM virulence genes to reduce the virulence, and develop as LM attenuated vaccine or an anti-virus, anti-tumor vaccine live vector. However, as a vaccine or live vector vaccine, it must be secure and reliable. Although scholars at home and abroad has been transformed the LM part of the virulence genes, but did not receive expected results.oB factor encoding SigmaB gene, is a subunit of the RNA polymerase holoenzyme of LM. Many studies indicated that the oB factors can regulate the transcription of many virulence genes, Therefore, the aims of this study is to construct SigmaB gene deletion strains by means of gene deletion, provide the theoretical basis for the the LM attenuated vaccine and live vaccine vector. Research methods and main results are as follows:1. Cloning and sequence analysis of the LM Xinjiang wild strain XS5SigmaB operonAccording to GenBank published sequence of SigmaB operon to design primers, PCR amplification LM Xinjiang wild strains XS5, SigmaB operon gene fragment. Using DNAMAN software and online software analysis amplified sequences including the active sites of the advanced structure of genes encoding proteins, respectively. Results SigmaB operon of3000bp fragment was amplified, The sequence contains four genes including rsbV, rsbW, rsbX and SigmaB, and SigmaB gene was801bp encoding a protein with265amino acids.8-264amino acids was RNA polymerase holoenzyme σB factor region.41-110the amino acid Sigma70_r2superfamily, is the most conserved regions of the entire protein. SigmaB gene homology analysis showed that SigmaB gene with a hing homology between different strains of LM is quite conservative gene.2. Construction and identification of LM-XS5ΔSigmaB mutant strainAccording the SigmaB operon sequence to design SOE-PCR primers, ΔSigmaB gene fragment was amplified by SOE-PCR, and then constructed to the vector pMD19-T-ΔSigmaB and shuttle vector pKSV7-ΔSigmaB. Shuttle plasmids was transformed into the LM-XS5cells by the electroporation. Using PCR obtain and identification of recombinant bacteria. The results of PCR showed that1502bp fragment can be amplified from recombinant strain, which is matched with expected length. The sequencing showed that have deletion the SigmaB gene of LM-XS5and named the recombinant strain as LM-XS5-ΔSigmaB. PCR identification results show that the recombinant strain has a good genetic stability through25generations of continuous generation.3. Effects of SigmaB gene deletion on pathogenic and environmental stress ability in LMInfection the macrophages RAW264.7and BALB/C mice after cultured the wild strain and mutant strain were conducted by same way, then contrast pathogenic difference of deletion strain and wild strain were detected by single colony count and Real-time RT-PCR. Results showed that mutant strain’s adhesion rate and invasion rate were decreased, liver and spleen bacteria burden decreased, LD50increased, virulence gene expression reduced compared with wild strain. The results showed that deletion SigmaB gene can reduce LM’s pathogenic. In order to contrast the liver bacteria number of deletion strain and wild strain in high temperature, high osmotic pressure and acid-base conditions were simulated and single colony were counted, and the gene expression levels were detected by real-time RT-PCR. Results showed that viable cell number of deletion strain was significantly reduced in different stress environment and gene expression decreased compared with wild strain. Results indicated the deletionof SigmaB gene can significantly reduce the LM’s ability of environmental stress.In conclusion, LM-XS5-ΔSigmaB was successfully constructed by SOE-PCR technique and homologous recombination methodh in this study. The virulence and environment stress ability of the SigmaB gene deleted strain are significantly reduced compared with the wild strain LM-XS5. This study lay a foundation for the development of LM attenuated vaccine and live vaccine vector.