| Fowl Cholera is an acute systemic disease with high morbidity and mortality,causing huge economic losses to the poultry industry.Its pathogen Pasteurella multocida(Pm)is Gram-negative bacteria.Poultry farms used to rely on antibiotics for the prevention and control of fowl cholera in our country.Due to the increasing problem of bacterial resistance,and in the context of the national ban,biological control will play an important role in the prevention of avian cholera.However,the current inactivated vaccines can't generate cross-protection,and there is a risk that the traditional attenuated vaccines will become more virulent.Therefore,it is extremely urgent to develop safe and effective new vaccines.In this thesis,three gene deletion mutant strains without molecular markers were successfully constructed by using NgAgo genetic operating system and using GX-PM as the parent strain,including ?aroA-GX-PM,?hexD-GX-PM and ?hex ABC-GX-PM.We have studied and analyzed the growth and morphological characteristics in vitro,genetic stability,pathogenicity and immune protective efficacy of these three gene deletion mutants.The main research content and results are as follows:1.The construction of gene deletion mutant strainThe recombinant plasmids,which used to knock out aroA,hexD and hex ABC genes,including p SHK5(TS)-NgAgo-aroALR,p SHK5(TS)-NgAgo-hexDLR and p SHK5(TS)--NgAgo-hex ABCLR,were electrotransformed into GX-PM.After many times passaging which induced deletion,three homozygous gene deletion strains were successfully screened out.2.The research on growth and morphological characteristics of gene deletion mutant in vitroIn order to analyze the effect of aroA,hexD and hex ABC gene deletion on the growth characteristics of bacteria in vitro,we performed growth curve determinations on ?aroA-GX-PM,?hexD-GX-PM and ?hex ABC-GX-PM.The results showed that when cultured in vitro,compared with GX-PM wild type strain,the growth rate of ?aroA-GX-PM,?hexD-GX-PM and ?hex ABC-GX-PM mutant strains was lower,and ?aroA-GX-PM mutant strains couln't grow without aromatic amino acids.In order to analyze the effect of capsule transport gene(hexD and hex ABC)deletion on the morphology of the bacterial capsule,we conducted transmission electron microscopy observations on the mutant strains of ?hexD-GX-PM and ?hex ABC-GX-PM.The results showed that when cultured in vitro,the mutant strains of ?hexD-GX-PM and ?hex ABC-GX-PM didn't have normal capsule morphology outside the cell wall of the GX-PM,which indicating that their ability to synthesize capsules was basically lost.3.The research on genetic stability of gene deletion mutant in vitroIn this study,the ?aroA-GX-PM,?hexD-GX-PM and ?hex ABC-GX-PM mutant strains were subcultured on liquid medium for 30 consecutive passages,and the genetic stability of the target gene was detected by PCR to determine whether the target gene was stably missing.The results showed that the mutant strains of ?aroA-GX-PM,?hexD-GX-PM and ?hex ABC-GX-PM didn't undergo back mutations within 30 generations and had good genetic stability.4.The research on pathogenicity of gene deletion mutantIn order to study the pathogenicity of ?aroA-GX-PM,?hexD-GX-PM and ?hex ABC-GX-PM mutant strains,30-day-old healthy broiler chickens were injected intramuscularly with different doses(103,105,107,108,109 CFU)of mutant strains,and the clinical symptoms were recorded for 14 days.The results showed that when the challenging dose in the wild group was 102 CFU,the mortality rate of broilers within 24 hours was 100%(n=4).There was no obvious clinical symptoms of the broiler chickens infected with different doses of ?aroA-GX-PM and ?hex ABC-GX-PM mutant strains.The mortality rate of broiler chickens in the ?hexD-GX-PM mutant challenge dose of 109 and 108 CFU groups was 75% and 25%,respectively.The mortality of the remaining low-dose groups was 0,and the surviving broilers that gradually recovered after clinical symptoms appeared.It shows that compared with the GX-PM,the pathogenicity of ?aroA-GX-PM,?hexD-GX-PM and ?hex ABC-GX-PM mutant strains to chickens is significantly reduced.5.The research on immune efficiency of gene deletion mutantIn this thesis,we researched on the immune efficacy of ?aroA-GX-PM,?hexD-GX-PM and ?hex ABC-GX-PM further,because ?aroA-GX-PM,?hexD-GX-PM,and ?hex ABC-GX-PM mutant strains are significantly less pathogenic to chickens,and have the potential to be used as gene deletion attenuated vaccines.Different doses of ?aroA-GX-PM mutant strains(107,108 and 109 CFU)were used to vaccinate 30-day-old healthy broilers by intramuscular injection.On the 14 th day after immunization,immunized broilers were challenged with a challenge dose of 103 CFU of GX-PM by intramuscular injection.The experimental results manifested that after GX-PM infecting,the mortality rate of broiler chickens in the challenging control group was 100% within 24 hours,while the broiler chickens of different immunization doses of ?aroA-GX-PM mutant strain didn't show apparent clinical symptoms,and the broiler chickens in the ?aroA-GX-PM mutant immunized group didn't die.Similarly,different doses of ?hex ABC-GX-PM mutant strains(107,108 and 109 CFU)were used to vaccinate 30-day-old healthy broilers by intramuscular injection.On the 14 th day after immunization,immunized broilers were challenged with a challenge dose of 103 CFU of GX-PM by intramuscular injection.The experimental results manifested that the immune doses of ?hex ABC-GX-PM mutant were 107,108,and 109 CFU,and theirs group protection rates were 0,0 and 25%.Different doses of ?hexD-GX-PM mutant strains(107 and 108 CFU)were used to vaccinate 30-day-old healthy broilers by intramuscular injection.On the 14 th day after immunization,immunized broilers were challenged with a challenge dose of 103 CFU of GX-PM by intramuscular injection.The experimental results showed that the immune doses of ?hexD-GX-PM mutant were 107 and 108 CFU,and theirs group protection rates were 0 and 25%.It showed that the ?aroA-GX-PM mutant strain can produce higher immunity to chickens,while the ?hex ABC-GX-PM and ?hexD-GX-PM mutant strains basically don't have immune protection to chickens.In order to further study the protective effect of the ?aroA-GX-PM mutant strain on chickens,each chicken in the ?aroA-GX-PM gene deletion strain group was injected intramuscularly with 108 CFU of bacterial for vaccination,and PBS challenge control group and PBS were set at the same time Blank control group(n=10).On the 14 th day after immunization,the ?aroA-GX-PM immunized group and the PBS challenge control group were artificially infected with the GX-PM wild strain with a dose of 20 LD50 by intramuscular injection.The results of immune protection rate showed that the mortality rate of broiler chickens in the PBS challenge control group was 100% within 24 h,while the broiler chickens immunized with the ?aroA-GX-PM mutant strain were all survived,and the immune protection rate was 100%.The chicken serum was collected on the 14 th and 21 st day after immunization,and the Ig G antibody level in the serum was determined.The results showed that: compared with the non-immunized group,higher levels of Ig G antibodies were detected in the serum of the immunized group on the 14 th and 21 st days after immunization,and the difference was significant(P<0.01),indicating that the ?aroA-GX-PM mutant strain It can induce chickens to produce strong humoral immunity and has good immunogenicity.The above results indicate that the ?aroA-GX-PM mutant strain can produce higher immunity to chickens,while the ?hexD-GX-PM and ?hex ABC-GX-PM mutant strains basically have no immune protection to chickens.In summary,in this study,we adopted the NgAgo genetic operating system,successfully constructing three gene deletion mutants without molecular markers which had good genetic stability,named ?aroA-GX-PM,?hexD-GX-PM and ?hex ABC-GX-PM.Through the pathogenicity experiment and immune protection effect experiment on chickens,it was found that ?aroA-GX-PM,?hexD-GX-PM and ?hex ABC-GX-PM had a significant decrease in the pathogenicity to chickens.Among them,the ?aroA-GX-PM mutant strain has good immunogenicity.It could stimulate the body to produce an immune response,creating a 100% immune protection rate for chickens.It could be used as a candidate strain for avian Pasteurella multocida gene deletion attenuated vaccine. |
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