Construction And Partial Biological Characteristics Of Listeria Monocytogenes LlsB Gene Deletion Strain

Listeriosis is a zoonotic disease that can infect livestock,poultry and humans,caused by Listeria monocytogenes?LM?.In humans patients,listeriosis is characterized by febrile gastroenteritis,meningoencephalitis,abortion and sepsis,with a mortality rate of 30%.The process of LM infected to the susceptible host,which depends on the synergy of various virulence factors.Early studies have found that activity of Listeriolysin O?LLO?is responsible for the characteristic hemolytic phenotype of LM,which is encoded by the hly gene located in Listeria virulence island I?LIPI-1?.Virulence Island 3?LIPI-3?is also an additional virulence island in the lineage type I strain.The lls gene cluster encodes a second hemolysin,named Listeria hemolysin S?LLS?,in LIPI-3.LLS is a bacteriocin that regulates the host gut microbiota and causes hemolysis of cells.And it plays an extremely important role in the process which LM invades cells across the blood-intestinal barrier.The llsB gene encodes a specific post-translational modification enzyme in the LLS operon,which is required for colonization of mouse internal organs.The inactivation of the llsB gene is sufficient to reduce LM proliferation in liver and spleen,indicating that the production encoded by llsB gene plays an important role in the biological activity of LM.Object:In this study,We used 4b-type LM90 as test subject,it is isolated from sheep brain tissue of listeriosis in a sheep farm in Xinjiang.Studies were performed to determine whether LM90 has a virulence factor LLS and the biological characteristics of LLS in the process of causing Listeria ovis disease.This study provides a certain research basis for further studying the pathogenesis of LLS and the mechanism of LM across the body's barrier.It's determined whether the llsB gene deletion strain can be used as an alternative strain for Listeria attenuated vaccine.Methods:1.Cloning of LM90 hemolysin S llsB gene and construction of its deletion strainThe LM90 llsB gene was amplified by PCR and sequenced.It lays a foundation for studying the function and mechanism of llsB gene in LM.The upstream and downstream homology arms of the llsB gene were amplified by PCR,and the upstream and downstream homology arms were connected by Overlap PCR to obtain the fusion fragment?llsB lacking the llsB gene.The fusion fragment was ligated with pMD19-T plasmid to construct pMD19-T-?llsB.The recombinant plasmid with the correct sequencing result was double-digested with the temperature-sensitive vector pKSV7.The digested?llsB was ligated to pKSV7 to construct the pKSV7-?llsB recombinant plasmid,which was electroporated into LM90competent cells.Continuous subculture with homologous recombination technology under the pressures of temperature and chloramphenicol.In the passaging process,the lateral side primers were used for PCR detection and screening to obtain the llsB gene deletion strain LM90-?llsB.The pKSV7 was lost at30?without chloramphenicol resistance.The gene deletion strain LM90-?llsB without chloramphenicol resistance was screened,and the Paralateral prime were detected by PCR.2.Study on the in vitro biological characteristics of LM fraction by deletion of llsB virulence geneThe mice were infected by intraperitoneal injection,the effects of parental strain LM90 and the deletion strain LM90-?llsB on the biological characteristics were examined,including growth curve,LD₅₀,brain,liver,spleen-loaded bacteria.To analysis of the growth characteristics and virulence of LM after deletion of llsB gene.The hemolysis test was used to detect the hemolytic titer of the parental strain LM90and the deletion strain LM90-?llsB to determine the effect of the missing llsB gene on the hemolysis ability of LM90.Result:1.In this study,the llsB gene was obtained by PCR amplification,and it was confirmed that the LM90 strain contained the virulence gene llsB.2.The LM90 llsB gene deletion strain LM90-?llsB was successfully constructed and the heredity was good.3.The LD₅₀0 of the parent strain LM90 and the deletion strain LM90-?llsB differed by only 0.33 logarithm order,indicating that the llsB gene deletion had little effect on LM virulence.After 24,48,and 72 hours of infection of the mice by intraperitoneal injection,the liver and spleen carriers of the parent strain LM90 were higher than the deletion strain LM90-?llsB.Statistical analysis showed that the difference in bacterial load was statistically significant?P<0.01?,but no LM was detected in the mouse brain..The LM90-?llsB was twice as high as that of the parental strain LM90,indicating that the llsB gene deletion had a certain effect on the hemolytic ability of LM90.The results showed that the llsB gene played a regulatory role in the LM crossing the blood-intestinal barrier and the virulence.Conclusion:In this study,overlap PCR and homologous recombination techniques were used to successfully construct the LM90 llsB gene deletion strain:LM90-?llsB.It laid a certain research foundation for further studying the role of llsB virulence gene in the process of LM crossing the host's three barriers.By comparing the parental strain LM90 with the deletion strain LM90-?llsB the difference in the results of the growth curve,the median lethal dose(LD₅₀),the amount of bacteria in the liver,spleen,brain bacterial load and hemolysis test.,And the effect of the deletion of llsB gene on the biological characteristics of LM90 was analyzed.it was confirmed that the llsB gene had a certain influence on some biological characteristics of LM90,but the effect was not obvious.It indicated that the llsB gene deletion strain could't be used as a candidate strain for the preparation of attenuated strains of Listeria.