Obtainment of a rough-type salmonella enteritidis and its genetic modifications for use as an avian vaccine

Method for generating a mutant strain of Salmonella enteritidis 3934vac, wherein it comprises:

• Construct an integrative vector, where for the construction of the integrative vector, two fragments flanking the waaL gene, one of 520 bp (oligonucleotides A and B) and the other of 504 bp (oligonucleotides C and D), respectively, are amplified by PCR, where the oligonucleotides A, B, C and D are the sequences SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6
• Purify and clone the PCR products separately in pKOB vectors for amplification, to obtain the resulting pKOB::DwaaLR1 vector for its amplification, then enzymatically digest, purify and bind in the pDESTINATION vector.
• Transform the resulting vector pDEST::waaL into Escherichia coli and verify by PCR, miniprep and digestion;
• once constructed, the pDEST::waaL plasmid is electroporated in the S. enteritidis 3934vac and 3934vac sb13::clyA-fiberFAdV strains, where its integration is forced by growth at 42° C. and subsequent excision at 28° C.
• Verify the deletion of the waaL gene by double homologous recombination by PCR with oligos external to the construction (waaL E and F), where oligos E and F are SEQ ID NO:9 and SEQ ID NO:10.
• Recombinant avian vaccine of Salmonella enteritidis wherein it comprises a modified mutant strain of Salmonella enteritidis 3934 vac, a deletion of the waaL gene.

Method for generating a mutant strain of Salmonella enteritidis 3934vac wherein it comprises:

• Have a strain of Salmonella enteritidis 3934 vac.
• Insert a selection cassette by means of a first step of homologous recombination, wherein the cassette contains sequences complementary to the prophage ST64B in its flanking regions, in addition to the gene for resistance to antibiotic;
• where the bacterium is electroporated in the presence of the linear DNA carrying the selection cassette in the first step of homologous recombination.
• Select the transformant colonies in LB medium containing the antibiotic after the first step of homologous recombination.
• Confirm the presence of the cassette in the chromosome of Salmonella enteritidis 3934 by PCR with the oligos of sequences SEQ ID NO:23, SEQ ID NO:24 and SEQ ID NO:25.
• Insert a ClyA-fiberFAdV-His expression cassette by a second step of homologous recombination, where the expression cassette replaces the selection cassette, where the expression cassette comprises the sequence SEQ ID NO:21
• Select the strains of Salmonella enteritidis 3934vac sb13::clyA-fiberFAdV by verifying the product of the expression of the sequence SEQ ID NO:21 by Western Blot and SDS-PAGE.