Obtainment of a rough-type <i>Salmonella enteritidis </i>and its genetic modifications for use as an avian vaccine

Abstract

The present invention relates to a strain of Salmonella enteritidis 3934vac, which has been deleted the waaL gene to obtain a rough phenotype (3934vac DwaaL), the obtaining procedure and the oligos used with the objective of reducing toxicity and maintaining immunogenicity for its application as a vaccine. Another aspect of the present invention relates to a strain of Salmonella enteritidis 3934vac DwaaL, i.e. rough type, which has been modified to express the gene of the avian adenovirus type I fiber, in addition to the procedure for obtaining a Salmonella enteritidis 3034 vac DwaaL strain expressing an AvA-I fiber gene. The invention also comprises the development of a new, live, recombinant, effective and innocuous avian vaccine against the AvA-I virus developed via an insertion and integration process of AvA-I fiber genes in the chromosome of an attenuated and non-pathogenic strain of the bacterium Salmonella enteritidis.

Claims

1. A mutant strain of Salmonella enteritidis 3934vac wherein it comprises a deletion of the waaL gene.

2. 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::waaL 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 into the Salmonella 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; and Adding said modified mutant strain of Salmonella enteritidis 3934vac to a composition to make a vaccine comprising said modified mutant strain of Salmonella enteritidis 3934vac with a deletion of the waaL gene.

3. A mutant strain of Salmonella enteritidis 3934vac wherein it comprises the complete sequence of the AvA-I fiber gene and a deletion of the waaL gene.

4. Method for generating a mutant strain of Salmonella enteritidis 3934vac wherein it comprises: Have a strain of Salmonella enteritidis 3934vac; 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:257; 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; and 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.

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

6. Recombinant avian vaccine of Salmonella enteritidis wherein it comprises a modified mutant strain of Salmonella enteritidis 3934vac with a sequence of the AvA-I fiber gene and a deletion of the waaL gene.

7. A mutant strain of Salmonella enteritidis 3934vac wherein it comprises an expression cassette SEQ ID NO:21 and the deletion of waaL that is verified by the presence of the sequence SEQ ID NO:8.

8. A mutant strain of Salmonella enteritidis 3934vac wherein it comprises a sequence SEQ ID NO:26 and SEQ ID NO:8.

Description

LIST OF FIGURES

(1) FIG. 1: Allelic exchange method for the construction of 3934vacR.

(2) FIG. 2: Integrative plasmid pKO

(3) FIG. 3: Integrative plasmid pKO::waaL

(4) FIG. 4: Waa region (rfa) of Salmonella enteritidis 3934vac

(5) FIG. 5: Waa region (rfa) of Salmonella enteritidis 3934vacR

(6) FIG. 6: pKOB::sb13 Plasmid

(7) FIG. 7: pKOB::sb13 fiber Plasmid

(8) FIG. 8: Phenotypic differentiation of Salmonella strains on Congo red agar plates (left) and semisolid agar (right).

(9) FIG. 9: Flow chart for the detection and microbiological quantification of Salmonella. In addition, TSI: Triple sugar iron agar; LIA: Lysine iron agar.

(10) FIG. 10: Western blot results of the protein of interest clyA-fiberFAdV-His.

(11) FIG. 11: SDS-PAGE results of the protein of interest clyA-fiberFAdV-His, with positive controls A and B.

(12) FIG. 12: Coomassie staining of the SDS-PAGE and Western Blot of the protein extracts obtained after growth in LB medium at 37° C. or 28° C. In addition, MW: molecular weight marker, wt: Salmonella enteritidis 3934, A: Salmonella enteritidis 3934Vac; A-fiber: Salmonella enteritidis 3934Vac with ClyA-fiberFAdV-His fusion on the chromosome, A-pfiber: Salmonella enteritidis 3934Vac expressing the ClyA-fiberFAdV-His fusion from plasmid. The specific bands obtained in the western blot have been indicated (A, B and C).

(13) FIG. 13: Electrophoretic analysis of the PCR products reveals the presence of clones carrying the waaL deletion. For the selection of clones carrying the deletion, the bacterial chromosome was amplified using oligos external to the construct. In addition, M=molecular weight marker, wt=wild strain, c=colony. Expected sizes: Wt=2328 bp, ΔwaaL=1112 bp

(14) FIG. 14: Summary table of oligos used

PREFERRED EMBODIMENT OF THE INVENTION: EXAMPLES

(15) The following examples that are provided herein serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be interpreted as limitations of the invention claimed herein.

Example 1: Expression of the Fiber Antigen in the Salmonella enteritidis 3934 Vac Strain by Western Blot

(16) Protein Extract

(17) We plated 1 colony in 5 mL of LB culture medium for the ClyA fiber, which has the insertion in the chromosome. Incubate at 37° C. at 200 rpm overnight.

(18) We centrifuged at 11 0000 rpm for 5 minutes and added the lysis buffer 1 (Tris HCL 10 Mm, EDTA 5 mM, NaCl 50 mM)+protease inhibitor 40 μL (3 g/mL)+50 μL of lysozyme (10 mg/mL), plus the SDS loading buffer (50 μL simple buffer 2x+50 μL urea 8M+5 μL B-mercaptoethanol in 50 μL sample) per sample and all was homogenized and heated at 100° C. for 5 minutes and then placed on ice 5 minutes and from there 20 μL was taken for loading. 0.1 Amps were used for each gel by 1:30.

(19) The band of the protein of interest clyA-fiberFAdV-His was observed in the results of the Western blot. Markers were used; on the left the ladder P7709V (175 KDa), on the right ab 116029 (245 KDa) (FIG. 10).

(20) In addition, when two positive His+ controls are used, but the initials A and B are by weight, A is approximately 60 kDa and B is approximately 30 kDa. These results allow us concluding that the clyA-fiberFAdV-His fusion is expressed with a molecular weight of approx. of 85.4 kDa. (FIG. 11)

(21) The Western blot was performed for the wild strain: Salmonella enteritidis 3934, Δ: Salmonella enteritidis 3934Vac; Δ-fiber: Salmonella enteritidis 3934Vac with ClyA-fiberFAdV-His fusion on the chromosome, Δ-pfiber: Salmonella enteritidis 3934Vac expressing the cly-AfiberFAdV-His fusion from plasmid.

(22) Electrophoretic analysis of the PCR products reveals the presence of clones carrying the waaL deletion (FIG. 13)

(23) For the purposes of the present invention, the use of the Salmonella enteritidis 3934vac strain in experimental models suggested that it would work in murine and even avian model,

(24) In our trials, we have observed that when inoculated into birds, it does not work well because the infection develops anyway; in order to achieve immunity, a deletion was made in the waaL gene of Salmonella enteritidis 3934vac, with which immunity was achieved. Likewise, the challenge tests with the 3934vac sb13::clyA-fiberFAdV ΔwaaL strain demonstrated that it confers immunity against Salmonella and the avian type-I Adenovirus preventing the appearance of inclusion body hepatitis.

(25) Deposit of Microorganisms

(26) The strains of SG-9R sb:ClyA-FIBER 6His, 3934 vac-rough mutant and 3934 vac-Fiber rough mutant have been deposited in the Spanish Type Culture Collection (Paterna, Valencia, Spain), following the rules of the Budapest Treaty for the deposit of microorganisms for patent purposes on the following dates and they have been assigned the following deposit number:

(27) TABLE-US-00004 Material Deposit date Access Number SG-9Rsb:ClyA-FIBER 6His Apr. 5, 2017 CET 9331 3934 vac- rough mutant Apr. 5, 2017 CET 9332 3934 vac- Fiber rough Apr. 5, 2017 CET 9333 mutant

(28) The present invention is not limited to the scope of the microorganisms deposited in the patent, since they represent a specific illustration of an aspect of the invention. Any microorganism or plasmid that is functionally equivalent to those described in the invention are included within the invention.