Construction And Preliminary Application Of The High-efficient Preparation System Of Bacterial Ghosts

Bacterial ghosts（BGs）are hollow bacterial cell envelopes originating from Gram-negative bacteria and induced by tightly controlled expression of the protein E from bacteriophageΦX174.By virtue of possessing native extracellular structures as well as devoid of nucleic acid,proteins,and cytoplasm,BGs are proposed to have significant prospects in the field of biomedical research and animal protection.On the one hand,BGs can be used as traditional vaccines to effectively stimulate the mucosal immunity,humoral immunity,and cellular immune responses;on the other hand,the strong loading capacity of BGs is the delivery carriers of drugs,plasmids,antigens and other macromolecular substances.However,the widespread applications of BGs are severely limited by comparatively inefficient bacterial lysis and low production levels.Studies showed that the initiation of bacterial lysis was related to the growth period of bacteria,and the initial concentration of bacterial lysis was limited to OD₆₀₀ value 0.2-0.6,which leaded to the low yield of BGs.In order to solve these key problems,the wild-type lysis gene E（E_W）and mutant lysis gene E/R89Q（E_M）from bacteriophageΦX174 were introduced and characterized in Escherichia coli（E.coli）BL21（DE3）.Results showed that the cell lysis efficiency mediated by protein E_Mwas significantly improved compared to that of protein E_W.In this study,the pLysS plasmid producing T7 lysozyme was implemented.As an inhibitor of T7 RNA polymerase,T7lysozyme can effectively regulate the expression level of T7 RNA polymerase,thus improving the host bacteria lysis efficiency by regulating the high expression of lysis protein E_M.Results showed that the initial concentration of bacterial lysis was significantly increased,and the OD₆₀₀value reached 2.0 while still reaching a cell lysis efficiency of almost 100%.Bacterial immunofluorescence and Western Blot detection results showed that in the presence of pLysS plasmid,the expression of lysis protein E_M was significantly increased,indicating that the large-scale production of BGs can be achieved by regulating the expression of lysis gene E_M.The high quality empty shell BGs with complete structure and lysis channels were observed by SEM and TEM.Salmonella enterica（S.Enterica,SE）is a Gram-negative pathogenic bacteria which frequently causes diarrhea and systemic infections in human and animals.In this study,the simplifiedλRed homologous recombination technology had been used to construct a Salmonella enterica subsp.enterica serovar Pullorum ATCC 9120Δlon::T7 RNA polymerase（T7 RNAP）engineered strain.The high-efficient lysis system was transferred into the engineered SE strain.The lysis kinetics experiments showed that the initial OD₆₀₀ value of bacterial lysis was as high as 2.5,and high quality SE ghosts were observed by SEM and TEM.E.coli Nissle 1917（Ec N）is gram-negative probiotics commonly used as probiotics in the treatment of intestinal chronic inflammation and infection.In this study,the CRISPR/cas9 gene editing technology had been used to construct a Ec NΔtna A::T7 RNAP engineered strain.At the same time,the high-efficient lysis system in this study was introduced into Ec N engineered strain.The lysis kinetics experiments showed that the lysis protein E_M of phageΦX174 had a certain lysis effect in the Ec N engineered strain.To sum up,this study constructed a high-efficient preparation system for BGs,which introduced pLysS plasmid and phageΦX174 lysis protein E to co-express and succeeded in preparating E.coli BL21（DE3）and SE BGs in large quantities.This research provided a novel method to produce high-quality BGs in large quantities.