Accurate Editing Of Salmonella Enteritidis T3SS Effect Protein-related Genes Based On CRISPR/Cas9 System

Salmonella enteritidis is one of the host-specific and invasive pathogens.The host includes humans and various animals.The bacteria can not only cause serious economic losses due to the onset and death of poμLtry,but also cause the contaminated poultry products to be carriers of Salmonella enteritidis and also seriously endanger human health.The acute gastroenteritis(food poisoning)caused by Salmonella enteritidis has increased in various countries in the world and has become an important issue in international public health.In recent years,CRISPR/Cas9 novel gene editing technology has attracted much attention.In this study,CRISPR/Cas9 technology was used to map S.enteritidis virulence related genes Sop B,Spi C,and bioinformatics software Effective T3 and BPBAac tool to predict the two effector proteins STY1741 and STM0932.Knockouts were performed to construct corresponding deletion mutants and biological characteristics were studied to explore the feasibility of these deletion mutants as attenuated vaccines.The research content and results are as follows:1.Using bioinformatics software Effective T3 and BPBAac tool to predict Salmonella Enteritidis effector proteins,two effector proteins were obtained,namely STY1741 and STM0932.2.Using p ET-28 a as expression vector,the recombinant expression vector p ET-28a-Spi C was successfμLly constructed.The constructed recombinant expression vector p ET-28a-Spi C was transformed into E.coli BL21(DE3)for induction and soluble analysis.The results showed that most of the expressed proteins Spi C existed in the form of inclusion bodies,and the proteins were obtained by the method of gel recovery and immunized mice.The antibody titers were detected by indirect ELISA.The results showed that high-titer antiserum against protein Spi C was obtained.3.Using the CRISPR/Cas9 technique,the deletion strain ΔSop B,the deletion strainΔSpi C,the deletion strain ΔSTY1741 and the deletion strain ΔSTM0932 were successfully obtained.4.Biological characteristics of S.enteritidis wild-type and deletion strains ΔSop B,ΔSpi C,ΔSTY1741,and ΔSTM0932 were studied.The results showed that the deletion strains ΔSop B,ΔSpi C,ΔSTY1741,and ΔSTM0932 showed no change in biochemical characteristics compared to the wild strain,and the growth rate did not change significantly compared with the wild strain.The deletion strain was stable and inherited,and The virulence of the deletion strain △Sop B,ΔSpi C,ΔSTY1741,ΔSTM0932 was significantly lower than that of the wild strain.