Construction Of A Novel Lactobacillus Expressing FomA Protein In The Outer Membrane Of Fusobacterium Nucleatum And Its Inhibitory Effect On Inflammatory Bowel Disease

Objective Background:Inflammatory bowel disease(IBD)is a group of gastrointestinal diseases,mainly represented by chronic and recurrent inflammation of the gastrointestinal tract.IBD can be subdivided into two subdiseases,crohn’s disease(CD)and ulcerative colitis(UC),fusobacterium nucleatum,(F.nucleatum)is a typical gram-negative anaerobic bacteria,and studies showed that F.nucleatum was closely correlated with the incidence of IBD,with a significant increase in intestinal tissue and feces of patients with IBD.FomA is the main protein of the outer membrane of F.nucleatum,which is an important pathogenic factor and main immunogen of Fusobacterium nucleate.FomA can not only serve as an effective antigen stimulating the body to produce specific immune response,but also may be an effective target for inhibiting F.nucleatum.lactic acid bacteria(LAB)are food-grade bacteria,which are most notable for their safety and can be used as oral mucosal vaccine carriers.fibronectin binding protein A(FnBPA)is an adhesive of Staphylococcus aureus(SA).Invasive recombinant lactic acid bacteria expressing FnBPA can infect cells.To deliver foreign DNA and express protective antigens.In this study,the Lactobacillus plantarum,L.plantarum NC8 strain and invasive Lactobacillus plantarum expressing FnBPA are used as delivery vectors,and the surface membrane protein FomA of Fusobacterium nucleatus is used as protective antigen to construct two new recombinant Lactobacillus plantarum.To explore the molecular immune mechanism and compare the advantages and disadvantages of two mucosal vaccines in protecting intestinal mucosal tissue and inhibiting IBD.The development of a therapy targeting FomA protein of Fusobacterium nucleate may have a positive effect on the prevention and treatment of IBD.Method:1.Firstly,obtain the FomA protein gene of F.nucleatum by consulting the NCBI website and synthesize it.Then,the recombinant plasmids p ET28a-FomA,p SIP409-pgs A’-FomA and p SIP409-FnBPA-pgs A’-FomA were constructed by double enzyme digestion,glue recovery and seamless cloning.Next,plasmids were transferred into the receptive state of E.coli BL21 and the receptive state of Lactobacillus plantarum by heat shock and electric shock,respectively.The NC8 strain carried the eukaryotic plasmid p Valac-GFP.Three recombinant strains BL21-p ET28a-FomA,NC8-p SIP409-pgs A’-FomA and NC8-p SIP409-FnBPA-pgs A’-FomA were constructed.2.First,the FomA protein was purified and the rabbit derived polyclonal antibody against FomA protein was obtained by immunizing rabbits.Then two recombinant Lactobacillus plantarum strains were tested by western blot,immunofluorescence and flow cytometry The expression of FomA protein,and the growth conditions and growth rate of recombinant Lactobacillus plantarum were explored and the optimal feeding amount and induction conditions were determined.3.The control group fed with PBS and NC8 and the experimental group fed with recombinant Lactobacillus plantarum NC8-p SIP409-pgs A’-FomA and NC8-p SIP409-FnBPA-pgs A’-FomA were set up,with 10 mice in each group.Mice were orally immunized at day 1,2,3,12,13,14,24,25 and 26,respectively.Mice feces and serum were collected at 0,12,24 and 36 days,respectively,and stored at﹣80℃.4.After 36 days,mice in the experimental group and the control group were taken,and the lymphocytes in the spleen,mesenteric lymph nodes and Pyle’s collecting lymph nodes in both groups were measured by flow cytometry.The lymphocyte was then stimulated by FomA antigen to detect proliferation,and the serum Ig G and intestinal mucosa SIg A were detected by ELISA.5.To construct an experimental model of severe IBD in mice caused by F.nucleatum infection by feeding an aqueous solution containing 2.5%DSS.Thirty-two mice were randomly divided into four groups.The first group was fed PBS,the second group F.nucleatum,the third group was fed an aqueous solution containing 2.5%DSS,and the fourth group was fed an F.nucleatum and an aqueous solution containing 2.5%DSS.6.32 mice were randomly divided into 4 groups with 8 mice in each group for immune protection experiment.The first group was fed PBS,the second group was fed Lactobacillus plantarum NC8,the third group was fed NC8-p SIP409-pgs A’-FomA,and the fourth group was fed NC8-p SIP409-FnBPA-pgs A’-FomA.Then the intestinal tissues of mice were observed through pathological sections,and the immune cells and cytokines in the intestines and blood were detected and analyzed by flow cytometry and ELISA experimentsResults:1.A full-length 1140bp FomA protein gene fragment of Fusobacterium nucleatus was synthesized,and then the construction of recombinant plasmids p ET28a-FomA,p SIP409-pgs A’-FomA and p SIP409-FnBPA-pgs A’-FomA was verified by nucleic acid electrophoresis and sequencing.Finally,recombinant Escherichia coli BL21-p ET28a-FomA,and recombinant Lactobacillus plantarum NC8-p SIP409-pgs A’-FomA and NC8-p SIP409-FnBPA-pgs A’-FomA were obtained successfully.2.The FomA protein was purified and the rabbit polyclonal antibody that can verify the exogenously expressed FomA protein was obtained.Western blot assay revealed obvious protein expression at 40k Da,immunofluorescence and flow cytometry both detected FomA(green fluorescence)expression of Lactobacillus plantarum.It is proved that the recombinant Lactobacillus plantarum NC8-p SIP409-pgs A’-FomA and NC8-p SIP409-FnBPA-pgs A’-FomA have good effect on exogenous expression of FomA protein,which can be further verified by oral immunization experiment in mice.It was determined that 8h of recombinant Lactobacillus plantarum culture was the best feeding time.At this time,the activity of Lactobacillus plantarum was better and the effect of FomA expression was more ideal.3.Flow cytometry showed that feeding recombinant Lactobacillus plantarum expressing FomA induced the activation of B cells,T cells and dendritic cells(DCs).The number of CD4~+T cells,CD4~+T cells and CD8~+T cells expressing IFNγ,and Th2 cells secreting IL4 and IL10 were significantly increased in the spleen(SP).There were significant increases in Ig A~+B220~+B cells,CD4~+T cells,Treg cells and Th2 cells secreting IL4 and IL10 in mesenteric lymph nodes(MLN).The number of Ig A~+B220~+B cells and DCs expressing MHCII and CD80 were significantly increased in Pyer’s lymphatic assembly(Payer’s patches,PP).A certain number of memory lymphocytes in SP and MLN were found to proliferate under the stimulation of FomA antigen.4.ELISA results showed that feeding recombinant Lactobacillus plantarum promoted the secretion of SIg A from intestinal mucosa and increased the content of Ig G in serum.Immunofluorescence results were consistent with flow cytometry and ELISA results,and the activation of immune cells in the intestinal tracts of mice fed recombinant Lactobacillus plantarum was the most significant.According to our statistical results,it was found that feeding recombinant Lactobacillus plantarum NC8-p SIP409-FnBPA-pgs A’-FomA was more able to induce a significant protective immune response in mice than NC8-p SIP409-pgs A’-FomA.5.An experimental model of severe IBD in mice caused by infection of F.nucleatum was successfully established,with a survival rate of about 25%,severe weight loss,and pathological changes such as bloody stools and obvious colon atrophy in the mice.Feeding recombinant Lactobacillus plantarum expressing FomA protein could alleviate the symptoms of severe IBD caused by F.nucleatum infection in mice.NC8-p SIP409-FnBPA-pgs A’-FomA could significantly relieve the symptoms of mice compared with NC8-p SIP409-pgs A’-FomA.6.HE staining showed that feeding NC8-p SIP409-FnBPA-pgs A’-FomA could protect the homeostasis of small intestine,maintain the morphology of villi and the distribution of goblet cells.It can also protect the colon of mice and prevent serious pathological changes such as the shedding of villi and epithelium,infiltration of a large number of inflammatory cells and changes in intestinal structure and morphology.Feeding NC8-p SIP409-FnBPA-pgs A’-FomA could significantly promote the secretion of Ig G,Ig A,IL6,IL1βand reduce the secretion of TNFαin serum and colon tissue of mice.It can also promote the activation of CD3~+CD4~+T cells,CD3~+CD8~+T cells,NK cells and macrophages in the blood,and significantly increase the number of Treg cells and CD4~+T cells secreting IL17 and IL22 in the gut.Conclusion:1.In this study,two new functional strains of Lactobacillus plantarum NC8-p SIP409-pgs A’-FomA and NC8-p SIP409-FnBPA-pgs A’-FomA based on FnBPA protein were successfully constructed,and the expression of FomA protein was verified through in vitro experiments.2.Both of the two novel lactic acid bacteria can induce protective immunity in mice,and NC8-p SIP409-FnBPA-pgs A’-FomA based on FnBPA protein has a more significant effect.3.The IBD model of mice was used to verify that both of the two novel lactic acid bacteria could inhibit the symptoms of IBD,among which NC8-p SIP409-FnBPA-pgs A’-FomA based on FnBPA protein had a more significant effect.