Constraction Of A Novel Recombinant Lactobacillus Delivery System And Its Aplication In S.aureus Mucosal Vaccine

Lactobacillus,a type of gram-positive bacterium,is widely used in food,medicine and other industries.Lactobacillus is not only safe and non-toxic,but also has varieties of probiotic function.It has also been evidenced to be a viable carrier for the delivery vector.Due to the diversity of Lactobacillus species,they have differences in biochemical,physiological and immune characteristics,and it also reflects in interspecific and translation.Therefore,the ability of presenting antigens,and the expression level are different.And the ability of inducing antibodies to animals and stimulating lymphocytes to express cytokines is different too.Although Lactobacillus as a carrier to delivery heterologous antigen has many advantages,its genetic engineering development is slow,as a recipient,we are still facing numerous challenges.S.aureus is a widely distributed zoonotic pathogen,which can cause bacteremia,infective endocarditis,infectious arthritis,skin infection,soft tissue infection,pleurisy and other diseases.In recent years,due to the abuse of antibiotics,drug-resistant strains increase year by year and show a trend of multiple drug resistance,which leads to the increased difficulty in the prevention and treatment of S.aureus.S.aureus infects the body through mucosa.At present,there is no effective vaccine for S.aureus,let alone any report about the mucosal vaccine for it.In this study,we obtained 3 strains which namely Lactobacillus kefiri SXJ29,Lactobacillus casei SXJ30 and Lactobacillus plantarum WXD234 which screened from the library of the laboratory.Using egfp as the reporter gene,different recombinant strains SXJ29/SXJ30/WXD234（pNZ8148-egfp）were constructed.We analyzed the expression products of recombinant bacteria by Western blot and laser scanning confocal microscope,and proved that egfp can be successfully expressed in 3 Lactobacillus strains.Compared with the wild strains,the physiological characteristics of the three strains of recombinant Lactobacillus did not change significantly,and the exogenous plasmids could be inherited stably.The feasibility of Lactobacillus delivery system based on Lactobacillus kefiri SXJ29,Lactobacillus casei SXJ30 and Lactobacillus plantarum WXD234 was preliminarily proved.The lactobacilli delivery system can successfully express S.aureus antigen ferric hydroxamate uptake protein D2（FhuD2）or non-toxic mutationɑ-hemolusin(Hla_H35L).After immunizing mice with recombinant Lactobacillus orally,it can provide different degrees of protection in the pneumonia infection model caused by S.aureus.Among them,immune recombinant bacterium WXD234(pNZ8148-hla_H35L)has the best protective effect,which can improve the survival rate of mice in the pneumonia model caused by S.aureus,alleviate the degree of lung infection,and reduce the colonization of S.aureus in the lungs.This study constructed Lactobacillus delivery system based on Lactobacillus kefiri SXJ29,Lactobacillus casei SXJ30 and Lactobacillus plantarum WXD234.The system as a living bacteria carrier can deliver S.aureus antigen with a strong protective effect,which provides new ideas and theoretical guidance for the development of S.aureus vaccine.