| Enterotoxigenic Escherichia coli(ETEC)is a gram-negative enteric pathogen that is one of the important causes of diarrhea in humans and animals.For a long time,piglet diarrhea caused by ETEC has been more and more difficult to be cured by antibiotics.Severe drug resistance of antibiotics and strict restrictions imposed by the state on the supplements,suggesting an urgent need for non-resistant,safe and green biological agents to prevent ETEC-induced diarrhea and reduce the huge economic losses.In this study,the recombinant ETEC K99 Lactobacillus casei expression system was proposed to obtain K99 candidate probiotics.After oral immunization,the immunogenicity of the recombinant Lactobacillus casei and the antigen-antibody complex formed with specific sIgA were studied.To explore whether the antigen-antibody complex effectively prevent the pathogen ETEC K99 entry to the intestinal epithelial cells for maintaining the integrity of intestinal epithelial cells in order to provide a theoretical basis for the prevention of ETEC-related diarrheal diseases.In this study,the gene of ETEC K99 was amplified by PCR and cloned into pQE-30 vector.The recombinant pQE-K99/E.coli was obtained.The purified recombinant protein was immunized four times with New Zealand white rabbits to obtain anti-K99 sera for the immunological experiments.The eGFP-tagged K99 genetically engineered E.coli and lactic acid bacteria were constructed.The expression of K99 fusion protein was detected by Western Blot,fluorescence micro-scopy and flow cytometry.The fusion protein surface-displayed on the recombinant Lactobacillus casei was detected by laser scanning confocal microscope.The SPF Balb/c mice were orally immunized with the recombinant Lactobacillus casei for 7 consecutive days.The level of the specific IgG in mouse serum and the specific sIgA in the faeces and intestinal fluid were detected by ELISA.The Mice were challenged by ETEC K99 at 4d,6d,8d and 10 d after immunization.At 24 hours after attack,the intestinal tissues of mice were collected to prepare for frozen slice.Immunohistochemical staining was performed to observe whether the s IgA-ETEC antigen complex formed could effectively prevent from ETEC K99 entering to the intestinal epithelium cell.The obtained recombinant pQE-K99/E.coli was induced by IPTG,and the recombinant protein was purified,which obtained 2.08 mg/mL K99 fusion protein.The polyclonal antibody titer was up to 1: 51200 after the New Zealand white rabbits were immunized with 2 mg recombinant protein.The recombinant plasmid pLA-K99-eGFP from recombinant E.coli was electroporated into Lactobacillus casei.The recombinant Lactobacillus casei expression system was constructed,which the recombinant protein was surface expressed on Lactobacillus casei.The Western Blot detected the 86 kDa fusion protein,indicating that the foreign protein can be reacted with the rabbit K99 antiserum and was of good reactivity.The pLA-K99-eGFP/ L.casei was orally administered to SPF Balb/c mice by gavage.The blood,feces and small intestinal fluid were collected at 1-12 d after immunization.The higher levels of specific IgG and sIgA were detected.Immunohistochemistry results of the frozen slice in mouse PP and intestinal tissue showed that the recombinant bacteria could colonize in the mice intestine and formed the ETEC-sIgA complex after immunization.After changllege,we found that ETEC K99 failed to invade the mouse intestinal epithelial cells.Therefore,pLA-K99-eGFP/L.casei can stimulate the mice to produce a large number of sIgA which effectively prevent pathogens entry to the intestinal epithelial cells,and thereby maintained the intestinal epithelial integrity. |
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