Preparation Of The Recombinant ETEC K88Lactobacillus Casei Oral Liquid And The Primary Study Of Clinical Effectiveness

The recombinant pLA-K88/L.casei is a bacterial species used as a live oral vaccine to prevent theoutbreak of enterotoxigenic Escherichia coli (ETEC) infections in suckling pigletsWhey protein is notonly a nutritious protein material, but also has many functional roles. We used whey proteinconcentrate as raw material to develop an oral liquid preparation for recombinant L. casei livestockvaccines. To do so, the process of oral liquid production was studied. We tested the immune effects oforal liquid in pigs, and provide the necessary theoretical and technical foundation for the industrialproductionof an oral liquid vaccine for preventing diarrhea in livestock.The bacteriostasis of recombinant L.casei metabolites was studied. The oxford plate assay systemwas used to detect the bacteriostasis of the metabolites on E. coli and S. aureus under treatment ofdifferent pH values, temperatures, and pepsin concentrations. The result shows that the metaboliteswere active within a pH range from2to4, heat stable and resistant to pepsin. We conclude that themetabolites of recombinant L.casei have a bacteriostatic effect on some pathogenic bacteria.The conditions for cultivation of recombinant L. casei, such as medium concentration,inoculation quantity, pH and cultivation methods were optimized. The optimal growth condition wasdetermined to be the following: medium concentration of4%; inoculum ratio of4%; pH of6.6andshaking frequency of80r/min. The number of recombinant L. casei under this culture condition was5.9×109CFU/mL. The stability of recombinant L. casei and number of viable cells during storage weredetected. The results showed that the whey protein medium can effectively extend the preservation timeof recombinant L. casei, and stable expression of exogenous genes.Oral liquid preparations of recombinant L. casei were fed to suckling piglets and pregnant sows.Sow’s milk, as well as blood and stool of immune piglets, were periodically collected. Using theindirect ELISA in serum for IgG and IgA, as well as milk and feces-specific slgA, we found thatrecombinant L. casei administred orally to suckling piglets for5days or10days inducedK88-specificsystemic and mucosal immune responses. After pregnant sows were administered recombinant L. casei,the K88whey antibody levels were also significantly increased. Through clinical observation, whether5days or10days after oral innoculation, recombinant L. casei was very effective in preventing pigletdiarrhea. In addition, oral liquid preparations of recombinant L. casei promoted the growth anddevelopment of piglets and treatment of diarrhea.