Construction And Immune Efficacy Of Regulated Programmed Lysis RASV Delivering Staphylococcus Aureus Proteantigen

Staphylococcus aureus(S.aureus)is a common zoonotic conditional pathogen,which is widely distributed in nature.It mainly relies on antibiotics for the treatment of S.aureus.However,the abuse of antibiotics has led to the continuous emergence of drug-resistant strains,making the efficacy of antibiotic therapy increasingly worse.New ideas are urgently needed to solve this problem.Over the years,scientists have been carrying out a lot of exploration on the development of the S.aureus vaccine both at home and abroad.A variety of S.aureus vaccines have started clinical trials,while none of which successfully marketed at present in the world.Based on the immune escape mechanism and the failure of vaccine development,scientists generally believe that the previous vaccine design emphasized too much on humoral immunity and did not pay enough attention on the role of cellular immunity against S.aureus.How to stimulate high level of cellular immune response is an important factor to be considered.Studies have shown that attenuated Salmonella as a delivery vehicle can not only stimulate the body to produce a humoral immune response,but also stimulate a cellular immune response.In this study,S.aureus proteantigens,Esx A-Esx B and Hla_H35L were expressed in delayed lysis recombinant attenuated Salmonella typhimurium,respectively.Furthermore,we also studied their immune protective efficacy,orally administrated,against blood infection caused by S.aureus.The results are shown as follows:(1)Recombinant attenuated Salmonella ?11802(pYA3681-esxA-esxB)and?11802(p YA3681-hla_H35L)were successfully constructed.The recombinant proteins were expressed successfully verified by western blotting;(2)The recombinant strains wouldn't grow on the LB medium plates without arabinose,and they can only be subcultured to the second generation in arabinose-free LB borth when transferred from the medium plates containing arabinose.The recombinant strain load was detected in the intestines,stomach,mesenteric lymph nodes,spleen and Peyer's patches on day 1,7,14,28 after mouse being orally administrated,and the results showed that the recombinant strains could only constantly exist within 28 days.The recombinant plasmids could be inherited stably at least 50 generations with the recombinant strains subculturing,verified by PCR.(3)After orally administrated,high-level antigen-specific IgG was detected in mouse serum on the 14^th day after primary and boost immunization,respectively,and high-level antigen-specific s Ig A was detected in mouse intestinal contents on the 7^th day after the boost immunization.(4)On the 14^th day after challenged by tail vein injecting with S.aureus USA300,all mice were sacrificed to observed pathological changes of kidney.The results showed that orally administrated with?11802(p YA3681-esxA-esxB)could reduce ratio of renal abscess area compared to other groups,and S.aureus USA300 load also reduced in the kidney.The renal lesions alleviated significantlyIn a word,mice orally administrated with delayed lysis RASV expressing S.aureus proteantigen showed certain resistance to S.aureus blood infection,which provided a new idea for the development of S.aureus oral live vector vaccine.