MM-3 was a live vaccine strain candidate for protecting neonatal piglets from diarrhea.Designed in the 1980s,a high degree of protection from colibacillosis was afforded to piglets in a challengestudy and field trials.However MM-3 had a drawback of carrying the antibiotic resistance gene (chloramphenicolacetyltransferase gene,cat).The introduction of a host-plasmid balanced lethal system into the vaccine wasa good idea to solve the problem.The λ-Red recombination system was adopted in this study to realize thereplacement of cat by aspartate-semialdehyde dehydrogenase gene (asd) in the plasmid pMM085.The newplasmid named pMMASD was introduced into an Escherichia coli strain χ6097 and Salmonella typhimuriumχ4072 where the asd gene had been knocked out in their chromosomes.Cultured in an Erlenmeyer flask,expression levels of two antigens K88ac fimbriae and heat-labile enterotoxin B subunit (LTB) in cell lysatewere similar among MM-3,χ4072(pMMASD) and χ6097(pMMASD).However,χ4072(pMMASD) possessedthe more effective secretion mechanism to transport LTB enterotoxin into culture liquid.The relatively higherstability of pMMASD in Salmonella typhimurium χ4072 than that of pMM085 in MM-3 was determined bothin vitro in the absence of selective pressure,and in vivo following oral inoculation.Oral immunization ofBALB/c mice with χ4072(pMMASD) or χ6097(pMMASD) was sufficient to elicit IgA responses in mucosaltissues as well as systemic IgG antibody responses to the K88 fimbriae,while MM-3 failed to elicit specificantibody responses to K88 fimbriae in mucosal tissues.Among three live strains,only χ4072(pMMASD)could develop strong humoral responses against LTB enterotoxin.The results suggest that χ4072(pMMASD)is expected to be a promising live vaccine strain. MM-3 was a live vaccine strain candidate for protecting neonatal piglets from diarrhea. Designated in the 1980s, a high degree of protection from colibacillosis was afforded to piglets in a challenge study and field trials. Host MM-3 had a drawback of carrying the antibiotic resistance gene (chloramphenicolacetyltransferase gene, cat). The introduction of a host-plasmid balanced lethal system into the vaccine wasa good idea to solve the problem. The λ-Red recombination system was adopted in this study to realize thereplacement of cat by aspartate-semialdehyde dehydrogenase gene (asd) in the plasmid pMM085.The newplasmid named pMMASD was introduced into an Escherichia coli strain χ6097 and Salmonella typhimuriumχ4072 where the asd gene had been knocked out in their chromosomes. Cultured in an Erlenmeyer flask, expression levels of two antigens K88ac fimbriae and heat-labile enterotoxin B subunit (LTB) in cell lysate similar to MM-3, χ4072 (pMMASD) and χ6097 (pMMASD) .owever, χ4072 (pMMA SD) possessedthe more effective secretion mechanism to transport LTB enterotoxin into culture liquid.The relatively higherstability of pMMASD in Salmonella typhimurium χ4072 than that of pMM085 in MM-3 was determined both in vitro in absence of selective pressure, and in vivo following oral inoculation. Oral immunization of BALB / c mice with χ4072 (pMMASD) or χ6097 (pMMASD) was sufficient to elicit IgA responses in mucosal lysates as well as systemic IgG antibody responses to the K88 fimbriae, while MM-3 failed to elicit specific antibody responses to K88 fimbriae in mucosal tissues.Among three live,, only χ4072 (pMMASD) could develop strong humoral responses against LTB enterotoxin. The results suggest that χ4072 (pMMASD) is expected to be a promising live vaccine strain.