The Construction Of Recombinant Lactobacillus Expressing FaeG Of Enterotoxigenous E. Coli Fusion DC-targeting Peptide And The Evaluation Of The Immunological Effectiveness

The K88fimbriae of enterotoxigenic Escherichia coli (ETEC) are strongly immunogenic antigens that can be use to evoke protective immunity.FaeG is the major subunit of K88fimbriae, and it has good immune immunoprotection effect diarrhoea in piglets caused by ETEC. In this study, FaeG gene was amplified from ETEC by PCR, and integration of DC-targeting peptide (DCpep), which consists of36bp, and cloned in a shuttle vector pSIP-409. The expression of FaeG-DC was observed both in E.coli and Lactic acid bacteria. The mice which were inoculated with FaeG-DC gene recombinant Lactic acid bacteria got a good immune protection effect.In this research, FaeG gene sequences published in GenBank was used to design primers with Kpn I and Hind III restriction sites, FaeG gene was amplified from the plasmid of ETEC by PCR, which purchased from the China Veterinary Drug Control. It is full-length846bp, containing a complete open reading frame, and integration of DC-targeting peptide, it is consist of36bp.The PCR production was cloned to pMD18-T vector and transformed into E. coli competent cells DH5α.By analyses of plasmid extraction, PCR, restriction enzyme Kpnl and HindⅢ digestion and sequencing,the results showed that the FaeG gene was cloned successfully, and the DC-targeting peptide was fused well, the BLAST analysis revealed that both nucleotide and amino acid sequences with GenBank released99%homology.FaeG-DC was cloned in a shuttle vector pSIP-409and transfered in to competent cell BL21. Identification results showed that the recombinant plasmid pSIP-409-FaeG-DC was obtained. In the presence of an inducing peptide, Sakacin-P, the FaeG-DC was observed in SDS-PAGE, Western-blot, the result showed that32kDa fusion protein expressed in E.coli BL21and with reactionogenicity well.The recombinant plasmid pSIP-409-FaeG-DC was electroporated into Lb.plantarum NC8. The SDS-PAGE and Western-blot showed that32kDa fusion protein was expressed and with reactionogenicity.Six weeks BALB/c mice were inoculated with FaeG-DC gene recombinant Lactic acid bacteria. Immunization lasted six weeks. The oral route was repeated3times weekly, two weeks later, the groups of mice were boosted twice, continue oral route4weeks.Two weeks later the mice were challenged with ETEC, then the mice were killed one week later. Weighing the mice, bleeding tail vein blood and serum was separated every week. Before and after the mice were challenged with ETEC, detecting IgG in the sera and sIgA in the intestinal contents change of mice by ELISA. The data was analyzed by SPSS17.0statistic software, and analyze the pathologic changes in the colon tissue of the mice by HE staining.The study showed, FaeG-DC gene expressed successfully in lactic acid bacteria. The FaeG-DC gene recombinant Lactic acid bacteria may enhance immunity of mice, which is certificated by animal experiment. This study laid the foundation for the development of the engineering Lactic acid bacteria oral preparations to against diarrhoea in piglets caused by ETEC.