Construction Of Listeria Monocytogenes LLS\ LLO Deletion Strains And Immune Protection Of Mice

Listeria monocytogenes is a food-borne gram-positive pathogen that can cross three major human barriers.LLS)is a bacteriocin that regulates the intestinal microbial flora of the host and causes hemolysis reaction of the cells,helping LM to cross the blood-intestinal barrier.Listeria hemolysino O?LLO?is the main pathogenic factor of LM,and the cytolytic phagocytic membrane helps LM to escape from the phagocytic body and enter the cytosol and increase in value.In our laboratory has been successfully build and maintain LM90SB2?lls based building LM90SB2?lls-?llo missing strains,immune protective experiment,to explore important virulence factor LM90SB2 and its lack of immunogenicity and artificial gastric juice of tolerance.Methods:1.Reference F2365 strains and primer design,PCR amplification,respectively,the upstream and downstream of hly gene homologous arm,using overlapping PCR technology get?hly fusion segments to connect win min shuttle carrier pKSV7,get a plasmid pKSV7-?hly,sequencing validation after putting the recombinant plasmid electricity into LM90SB2?lls cells,under the double pressures of chloramphenicol and temperature subculture strain to complete the homologous recombination,during using the side of the lateral primers screened successfully short with lack of homologous recombination,And continue in 30°C and the absence of chloramphenicol resistance will extend the gain of chloramphenicol resistance LM90SB2?lls-?llo.2.Determination of LM90SB2 and LM90SB2?lls-?llo growth curve,the use of the health department of kunming male mice as experimental animals by intraperitoneal injection of two strains of bacteria infection test the LD₅₀0 of microbial load,brain of liver and spleen?at the same time filling and uniform manner?and so on some biological characteristics,analysis llsB/hly genes influence on the growth characteristic of LM and virulence.3.The two strains were inoculated simultaneously into the simulated artificial gastric juice with pH=1.5,pH=2.5 and pH=3.5,respectively,and sampled at 37°C in water bath for 1h,2h and 3h.After gradient dilution with 1*PBS,plate counting?3plates for each dilution?was performed.4.The two strains were inoculated to mice and added to the control group,and inoculated to mice on day 14 and day 21.The listeria antibody content in the blood list at 7,14,21 and 28 days was detected by elisa kit.Then,mice in the immune group and control group were intraperitoneally injected with lethal dose of LM90SB2,and the condition of mice was continuously observed for 7 days,and the immune protection rate PI of mice in each group was calculated.Results:1.The lack of successful build genetic stability strains LM90SB2?lls-?llo.2.Determination of growth curve,the growth of LM90SB2 activity slightly higher than the LM90SB2?lls-?llo;LD₅₀0 in mice and the determination of microbial load brain liver and spleen,LM90SB2 LD₅₀0 of 3.65*10^7.23,LM90SB2?lls-?llo nonexistent LD₅₀;LM90SB2?lls-?llo LM90SB2 compared with oral and intraperitoneal injection in a potential microbial load of the liver and spleen were significantly decreased,the extremely significant difference?P<0.01?.3.When the pH=3.5,the survival rate of two strains of bacteria for LM90SB2>LM90SB2?lls-?llo,when pH=2.5 LM90SB2 growth and significantly higher than LM90SB2?lls-?llo?P<0.01?and with the passage of time,the two strains of bacteria growth curve are declining,when pH=1.5,LM90SB2 and LM90SB2?lls-?llo in lh the survival rates are0.024%and 0.019%respectively,in 2 h parent strains still has a 0.0098%survival rate,The survival rate of the missing strain was 0%.4.According to the determination of antibody content,except the parent strain on day 21 was slightly higher than that of the absent strain,the remaining days of the absent strain were slightly higher than that of the absent strain.According to the analysis of the immune protection rate of mice,the immune protection rate of both strains was 75%,and that of mice in the control group was 30%.Conclusion:1.The lack of success to build a stable genetic strains LM90SB2?lls-?llo;2.LM90SB2?lls hly gene does not exist LD₅₀0 after missing;Compared with the parent strain,the bacterial load in the brain,liver and spleen was also significantly reduced?P<0.01?.3.The tested bacteria survival rate in the artificial gastric juice and the pH of gastric juice and action time correlation in the gastric juice,LM90SB2?lls-?llo LM90SB2 compared in pH=1.5,pH=2.5,pH=3.5 1h,2h,3h survival rate decreased in artificial gastric juice;4.LlsB/hly gene absence will not affect the immunogenicity of LM,lack of LM90SB2?lls-?llo of parental strains LM90SB2 attacks have good immune protection effect,It laid a good foundation for LM gene deletion vaccine development.