Study On The Construction Of Double Adhesive ETEC Through Cell Surface Display Of FanC-FaeG And Its Biological Characteristics

Enterotoxigenic Escherichia coli(ETEC)is one of the main pathogens causing animal diarrhea,which has brought great losses to animal husbandry.It is characterized by a wide range of transmission and strong pathogenicity.The harm of ETEC comes from the colonization function of adhesin and the toxic effect of enterotoxin.In this study,the ETEC was used as the host for the construction of recombinant strains,and another type of adhesin protein was displayed on the ETEC surface by the Lpp'Omp A surface display system.The strain with double adhesive antigen FanC-FaeG on the cell surface was constructed to provide the bacterial basis for the corresponding ETEC vaccine or related adhesive antigen.Research contents and related results are as follows:Firstly,the reporter plasmid of constructed p QE-30-GFP was constructed.By comparing the relative fluorescence intensity of different recombinant bacteria and the time required for color development,the well-expressed host bacteria BE311 containing fan C gene was screened.In order to increase the activity of adhesive gene expression in host bacteria,the E.coli K88 adhesive protein gene fae G was amplified,and the surface plasmid p QE-30-Lpp'Omp A-Fae G-TC was constructed by over-lap PCR.Then,the constructed plasmid was transformed into host bacterium BE311.Through the screening of positive recombination and the identification of specific labelled Fae G-TC expression products by the biarsenical-tetracysteine system,it was confirmed that the exogenous K88 adhesive gene was successfully displayed on the surface of BE311.The results of growth characteristics and induced expression of recombinant bacteria show that the expression of exogenous proteins did not bring growth pressure to the strain,and Fae G could be expressed stably after IPTG induction.Meanwhile,the maximum amount of expression was reached after 8 h induction.Through hydrophobicity test and IPEC-J2cell adhesion test,the recombinant bacteria B311-fae G showed better in vitro hydrophobicity and specific adhesion to epithelial cells,respectively.In order to further investigate the antagonistic ability of recombinant bacteria against pathogenic E.coli in animal intestines,two specific fluorescent labeled ETEC strains were constructed on the basis of the original laboratory,E.coli k99-m Cherry with red fluorescence and E.coli K88ab-GFP with green fluorescence,respectively.The two labeled combined pathogens were used to challenge rats.Five days after challenge,it was found that the up-regulated expression of inflammatory factor-related genes and proteins in rats was significantly increased.On the basis of the challenge model,this experiment specifically compared the pathogen antagonistic effect of single adhesin Fan C(derived from wild strain BE311)and double adhesive FanC-FaeG(derived from recombinant strain BE311-Fae G)on rats after challenge.E.coli was cultured overnight and the concentration of the suspension was adjusted to 10~9 CFU/m L.After being killed by irradiation,E.coli was added to the drinking water of rats for 5 days,and then the combined labeled strain of 10~9 CFU was used for gastric challenge.The growth performance of rats under different treatments and the number of E.coli labeled in gastrointestinal tract segments and feces were measured respectively.The results showed that there was no significant difference in growth performance between the FanC-FaeG group and the negative control group(P>0.05);The number of E coli in duodenum,jejunum and ileum at 12 h after challenge was ranked:FanC-FaeG grou P<Fan C grou P<positive control;At 24 h,the number of E coli in faeces was ranked:FanC-FaeG grou P<Fan C grou P<positive control.Therefore,compared with the single adhesive group,the double adhesion of recombinant bacteria can significantly reduce the specific adhesion of pathogen in the small intestine,effectively relieve the pathogen invasion of rats after the challenge.In conclusion,this study constructed an ETEC strain with double adhesive FanC-FaeG,which could stably and efficiently express the exogenous K88 adhesin.The constructed strain had a stronger adhesion ability than the wild strain with single adhesin.The recombinant strain could play the role of intestinal protection by preoccupying specific binding sites in the small intestine of rats.The study provides the bacterial strain basis for the development of vaccines or the research of intestinal protection agents.