CN117051021A - Salmonella typhimurium recombinant strain for non-inducible expression of salmonella pullorum Bcf pili and application thereof - Google Patents
Salmonella typhimurium recombinant strain for non-inducible expression of salmonella pullorum Bcf pili and application thereof Download PDFInfo
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Abstract
The invention discloses a salmonella pullorum Bcf pilus operon gene and a recombinant plasmid for non-inducible expression of salmonella pullorum Bcf pili. The invention also discloses a attenuated salmonella typhi SG01 recombinant strain for expressing salmonella pullorum Bcf pili, which is prepared from recombinant plasmid pBR322-SPbcfThe salmonella pullorum Bcf pilus can be expressed in chicken body in a non-inducible manner by introducing attenuated salmonella typhimurium SG01 strain. The invention expresses the coded salmonella pullorum Bcf pili in an in vitro non-induction mode through the vector pBR322bcfThe operon gene expresses and displays salmonella pullorum Bcf pili on the surface of the attenuated salmonella gallinarum. After chicken is immunized by the SG01 recombinant strain, an anti-salmonella pullorum Bcf pilus serum antibody can be generated in vivo in a non-inducible manner, the adhesion capability of salmonella pullorum to LMH can be obviously reduced, and the application potential of preventing and controlling salmonella pullorum infection is provided.
Description
Technical Field
The invention relates to the field of biotechnology application, in particular to salmonella typhi recombinant bacteria for non-inducible expression of salmonella pullorum Bcf pili and application thereof.
Background
Pili are a special filament-like structure on the bacterial surface, whose tip adhesin proteins bind to specific tissue cells of the host, thereby mediating bacterial adhesion and colonization of the host cells. Salmonella adhesion colonization of animals by expressing various pili is a key step in initiating pathogenic infection. It is reported in the literature that Bcf pili encoded by the bcf gene operon are expressed in most Salmonella except for the Salmonella enterica subspecies Dundon. Bcf pili are typical chaperone-advancing pili, comprising 7 subunits: bcfA, bcfB, bcfC, bcfD, bcfE, bcfF and bcfG. Wherein bcfA encodes the bcf major subunit, bcfB and bcfG encode the chaperone protein, bcfC encodes the propulsive protein, and bcfD encodes the top adhesion agent, bcfE and bcfF encode the two pilus-assisted subunits.
Under the feeding environment of prohibiting the growth-promoting antibacterial drug feed additive, the separation rate of the poultry source salmonella represented by the salmonella pullorum in the poultry farm in China is maintained at a higher level. In view of the recent impact of the above-mentioned serological salmonella as a dominant serological salmonella in the development of the poultry farming industry in China, there is an urgent need in the poultry farming industry to enhance control of infection with salmonella pullorum.
Disclosure of Invention
The invention aims to: the technical problem to be solved by the invention is to provide a novel salmonella pullorum Bcf pilus operon gene.
The invention also solves the technical problem of providing an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain containing the salmonella pullorum Bcf pilus operon gene.
The invention also solves the technical problem of providing the salmonella typhi recombinant strain for preventing and controlling salmonella pullorum infection in a poultry farm, which is non-inducible expression salmonella pullorum Bcf pilus.
The invention also solves the technical problem of providing the salmonella pullorum Bcf pilus operon gene, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain, and the application of the salmonella pullorum recombinant strain in preparing medicaments or vaccines for salmonella pullorum infection.
The invention also solves the technical problem of providing an anti-salmonella pullorum Bcf pilus serum antibody.
The invention also solves the technical problem of providing the application of the anti-salmonella pullorum Bcf pilus serum antibody in preparing medicaments for salmonella pullorum infection.
The invention finally solves the technical problem of providing a medicine or vaccine for preventing or treating salmonella pullorum infection.
The technical scheme is as follows: in order to solve the technical problems, the invention provides a salmonella pullorum Bcf pilus operon gene, and the Bcf pilus operon gene sequence is shown as SEQ ID NO.1. The gene is obtained from the genome of salmonella pullorum SP03 strain and contains 7 genes (bcfABCDEFG).
The invention also comprises an expression cassette, a recombinant vector, a recombinant cell or a recombinant strain containing the salmonella pullorum Bcf pilus operon gene.
The invention also comprises a recombinant plasmid, wherein the recombinant plasmid is obtained by inserting the chicken white diarrhea salmonella Bcf operon gene into an expression vector.
Wherein the expression vector is a non-inducible expression vector.
Wherein, the recombinant plasmid is recombinant plasmid pBR322-SPbcf, and the salmonella pullorum bcf operon gene encoding Bcf pili is inserted into a non-inducible expression vector pBR 322.
The invention also discloses a salmonella gallinarum recombinant strain which is used for non-inducible expression of salmonella pullorum Bcf pilus, and the salmonella gallinarum recombinant strain is obtained by introducing the recombinant plasmid into attenuated salmonella gallinarum.
Preferably, the recombinant strain is SG01 (pBR 322-SP bcf), and the recombinant plasmid pBR322-SPbcf is introduced into attenuated Salmonella typhimurium SG01 strain.
The invention also discloses a construction method of the recombinant strain, which comprises the following steps:
1) PCR (polymerase chain reaction) amplification of the complete operon gene of salmonella pullorum SP03 strain bcf, DNA sequencing and identification, and insertion of an expression non-inducible vector pBR322 (the nucleotide sequence of the complete operon gene of the salmonella SP03 strain bcf is shown in a sequence table), so as to construct a recombinant plasmid pBR322-SPbcf;
2) pBR322-SP bcf was introduced into Salmonella typhimurium SG01 strain.
Wherein the PCR amplification and the downstream primer sequences in the step 1) are respectively as follows
bcf-F：5’-GCTCTAGACCCTTATTTTTATATTTAAAAGGAGC-3’；
bcf-R：5’-CTAGCATGCTTAATGAATACGCGTCAGATCC-3’。
Wherein the underlined symbols represent Xba I and SphI cleavage sites, respectively.
Wherein, in step 2), pBR-SPbcf is introduced into Salmonella typhimurium SG01 strain electrotransformation competent cells by electrotransformation and suspected positive clones are screened by ampicillin LB plate. And identifying the correct positive clone as the salmonella typhi recombinant strain which is the non-inducible salmonella pullorum Bcf pilus expression, and naming the salmonella typhi recombinant strain as SG01 (pBR 322-SP bcf).
The invention also discloses application of the salmonella pullorum Bcf pilus operon gene, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain, and the salmonella typhimurium recombinant strain in preparation of medicaments or vaccines for salmonella pullorum infection.
The invention also discloses an anti-salmonella pullorum Bcf pilus serum antibody, which is obtained by separating the salmonella typhimurium recombinant bacteria inoculated with SPF chickens.
Wherein the inoculation method comprises oral administration or injection, and the inoculation dosage is 1×10 9 CFU～5×10 9 CFU。
The invention also discloses a method for preparing an anti-salmonella pullorum Bcf pilus serum antibody, which comprises the following steps:
1) SPF chicken of 2 weeks of age was orally immunized 5X 10 9 CFU doses of SG01 (pBR 322-SPbcf) strain.
2) After 2 weeks the wing vein was bled and serum was isolated.
The invention also discloses application of the anti-salmonella pullorum Bcf pilus serum antibody in preparing medicaments for salmonella pullorum infection.
Preferably, the invention also comprises the application potential of the anti-SG 01 (pBR 322-SP bcf) serum antibody for specifically blocking the adhesion capability of salmonella typhi and salmonella pullorum to hepatoma cells (LMH) of chickens in vitro and controlling the infection of salmonella typhimurium and salmonella pullorum.
The invention also comprises a medicine or vaccine for preventing or treating the infection of salmonella pullorum, which contains the salmonella pullorum Bcf pilus operon gene, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain and the salmonella typhimurium recombinant strain.
In summary, the invention comprises recombinant plasmid pBR322-SPbcf for expressing salmonella pullorum Bcf pilus and recombinant salmonella typhimurium SG01 (pBR 322-SP bcf) which is constructed based on the recombinant plasmid pBR322-SPbcf and is used for expressing salmonella pullorum Bcf pilus in vivo, and the recombinant salmonella pullorum Bcf pilus-expressing recombinant salmonella pullorum can generate antibody against salmonella pullorum Bcf pilus serum in chickens, thereby being expected to provide new thought and strategy for prevention and control of salmonella pullorum infection in poultry farms.
The beneficial effects are that: compared with the prior art, the invention has the advantages that: according to the invention, after chicken is immunized by SG01 (pBR 322-SP bcf) recombinant strain, a specific anti-salmonella pullorum Bcf pilus serum antibody can be generated in vivo in a non-induced manner, the adhesion capability of salmonella pullorum to hepatoma cells (LMH) of chicken can be obviously and specifically blocked, and the test result of the capability of the immune serum antibody of blocking bacteria from adhering to chicken-derived cells in vitro shows that the antibody has application potential for preventing and controlling salmonella pullorum infection.
Drawings
Fig. 1: and (5) carrying out PCR amplification on the complete operon gene of salmonella pullorum bcf. Lane M represents a Trans 15KDNA marker; lane 1 represents PCR amplification products of salmonella pullorum bcf operon gene.
Fig. 2: and (3) the digestion identification result of the recombinant plasmid pBR 322-SPbcf. Lane M represents a Trans 15K DNA marker; lane 1 represents the pBR322-SPbcf circular plasmid; lane 2 represents a linear fragment of pBR322-SPbcf after SphI single cleavage.
Fig. 3: identification of SG01. DELTA.bcf strain. Lane M represents Trans2K plusdnammarker; lane 1 represents SG01 wild strain (2251 bp); lane 2 represents the primary recombinant strain SG01 Δbcf: cat (1242 bp); lane 3 represents the secondary recombinant strain Sg01Δbcf (370 bp).
Fig. 4: functional verification results of the recombinant plasmid pBR 322-SPbcf. The left side is the agglutination result of SG01 delta bcf (pBR 322) bacterial suspension containing empty plasmid and salmonella pullorum positive serum (international standard substance), without specific agglutination particles; the right side shows the agglutination reaction result of SG01 delta bcf (pBR 322-SPbcf) bacterial suspension containing recombinant plasmid and salmonella pullorum positive serum (international standard substance), and specific agglutination particles are present (white arrow).
Fig. 5: and (5) verifying the function of the anti-pullorum salmonella Bcf pilus serum. The left side is the agglutination result of DH5 alpha (pBR 322-SPbcf) bacterial suspension and negative serum, negative, no specific agglutination particle; on the right side, there are specific agglutination particles (white arrows) positive as a result of agglutination reaction of DH5 alpha (pBR 322-SPbcf) bacterial suspension with anti-Salmonella pullorum serum.
Fig. 6: the anti-salmonella pullorum Bcf is used for specifically inhibiting the adhesion cell result of salmonella pullorum. Black bars represent adhesion rate of salmonella pullorum SP03 strain adhesion LMH cells; white bars represent the specific adhesion rate of LMH cells after incubation of Salmonella pullorum SP03 strain with anti-Salmonella pullorum Bcf strain crude serum for 30min; gray bars represent adhesion rate of adhesion LMH cells after the salmonella pullorum SP03 strain is incubated with negative serum for 30min; "×" represents that the difference was significant (p < 0.05).
Detailed Description
Before further describing the embodiments of the present invention, it should be understood that: the scope of the invention is not limited to the specific embodiments described below; it should also be appreciated that: the terminology used in the examples of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the scope of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.
The salmonella pullorum SP03 strain utilized by the invention is isolated from Jiangsu certain scale fowl farm, and is preserved in the laboratory of the applicant (publication document Wei Xing. Construction of recombinant salmonella gallinarum alpha roA gene deletion strain expressing APECI type pilus and safety efficacy evaluation [ D ]. University of Yangzhou, 2019.).
The invention discloses a chicken typhoid salmonella strain SG01 in an invention patent ZL2018114912006, wherein the preservation number is CGMCC No.16049, the preservation date is 2018, 7 months and 2 days, and the chicken typhoid salmonella strain is named as chicken typhoid salmonella (Salmonella gilnarum) and the strain code SG01.
The chicken liver cancer cells (LMH) utilized by the invention are derived from the university of Yangzhou She Jianjiang professor laboratory (open literature) of Jiangsu province.
Example 1: cloning and verification of the Salmonella pullorum bcf operon Gene encoding Bcf pili
A genome template of Salmonella pullorum SP03 strain was obtained by a boiling method, and a complete operon gene fragment SPbcf of Salmonella pullorum bcf was amplified using Expand Long Template PCR System (purchased from Haufmilo Biotechnology Co., ltd., switzerland). Based on the results of genome-wide sequencing of the SG01 strain of Salmonella typhi in the laboratory of the applicant of the present invention (publications: dai P, wu HC, ding HC, et al safety and protective effects of an avirulent SalmonellaGallinarum isolate as a vaccine candidate against SalmonellaGallinaruminfections in young chips.vet Immunol. Immunopathol.2022; 253:110501.) upstream and downstream primers were designed to amplify the sequence of the SPbcf fragment as shown below, wherein underlined symbols represent Xba I and SphI cleavage sites, respectively.
An upstream primer: 5' -GCTCTAGACCCTTATTTTTATATTTAAAAGGAGC-3’；
A downstream primer: 5' -CTAGCATGCTTAATGAATACGCGTCAGATCC-3’。
The PCR amplification system is shown in Table 1. The PCR amplification reaction procedure was: pre-denaturation at 94℃for 2min; denaturation at 94℃for 10s, annealing at 57℃for 30s, elongation at 68℃for 7min,10 cycles; denaturation at 94℃for 15s, annealing at 59℃for 30s, elongation at 68℃for 7min,20 cycles, an increase in elongation time of 20s after each cycle; the extension was complete at 68℃for 10min.
TABLE 1 PCR amplification System for complete operon gene fragment SPbcf of Salmonella pullorum bcf
The result of PCR amplified complete operon fragment of salmonella pullorum bcf is shown in figure 1, the length is about 6.6kb, and the sequencing result is shown in sequence table SEQ ID NO.1. Amplified fragments were digested with XbaI and SphI, digested with NheI and SphI, purified, ligated overnight with T4 DNA ligase, introduced into E.coli DH 5. Alpha. Competent cells, and screened for positive clones by ampicillin (Amp) resistant LB plates. The recombinant plasmid is identified by SphI single enzyme digestion, then agarose gel electrophoresis, and the target band is 10681bp (figure 2). The recombinant plasmid identified correctly by cleavage was designated pBR322-SPbcf, and the positive clone was designated DH 5. Alpha (pBR 322-SPbcf).
The Salmonella typhi SG01 strain is recovered, and a bcf gene deletion strain Sg01Δbcf of the SG01 strain is constructed by utilizing a lambda-Red homologous recombination technology and is used as a carrier for functional verification of recombinant plasmid pBR 322-SPbcf. The method comprises the following steps:
amplification and purification of cat Gene fragment: the nucleotide sequence of bcf (coding sequence see sequence table SEQ ID NO. 2) was obtained from SG01 strain genome-wide sequencing results (publication: dai P, wu HC, ding HC, et al safety and protective effects of an avirulent SalmonellaGallinarum isolate as a vaccine candidate against SalmonellaGallinarum infections in young chips, ve Immunol. 2022; 253:110501.) and primer design of SG01 bcf gene-deleted strain was performed using Oligo7 software (Table 2). PCR amplification of cat gene fragments was performed using plasmid pKD3 (publication: datsenko KA, wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products.PNAS.2000;97 (12): 6640-6645.) as template and P3 and P4 as primers. The amplification system is shown in Table 3. The PCR amplification reaction procedure was: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 30s, annealing at 52℃for 1min, extension at 72℃for 1min,10 cycles; denaturation at 95℃for 10s, annealing at 63℃for 1min, extension at 72℃for 1min,25 cycles; thoroughly extend at 72℃for 10min.
TABLE 2 construction of SG01 bcf Gene-deleted Strain and amplified fragment
Note that: the primer P1/P2 was used to identify the bcf gene. Homologous recombination primers P3/P4 are respectively designed on the inner sides of the P1/P2, wherein the underlined sequences are homologous to two wings of the bcf gene, and the underlined sequences are not homologous to two wings of the cat gene on the pKD 3.
Table 3 cat Gene fragment PCR amplification System
Preparation of competent cells of SG01 strain and transformation of the pKD46 plasmid (publication: datsenko KA, wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products.PNAS.2000;97 (12): 6640-6645.): SG01 strain electrotransformation competent cells were prepared by conventional methods. Single colony of SG01 strain is streaked and separated on LB solid plate, and placed in a constant temperature incubator at 37 ℃ for static culture for 24-36 h. Single colonies on LB solid plates were picked, inoculated into 4mL of LB liquid medium, and placed in shaking culture at 37℃overnight. Transferring the bacterial suspension cultured overnight into fresh LB liquid medium at a ratio of 1:100, shaking at 37deg.C to OD 600nm About 0.4 to 0.6, the supernatant was removed by centrifugation at 4000rpm for 10min at 4℃in an ice bath for 30min, the cells were resuspended in an equal volume of pre-chilled 10% glycerol and the washing was repeated 3 times. Finally, the supernatant is discarded, the sediment is resuspended by pre-cooled 10% glycerol, and the sediment is packaged in 40 mu L/tube and stored at 4 ℃ for standby. mu.L of pKD46 plasmid was taken40. Mu.L SG01 strain competent cells were mixed and added to a 0.1cm Bio-Rad electrode cup for shock transformation. After electric shock, 1mL of precooled SOC medium was added rapidly, shaking culture was performed at 30℃for 1.5h at 200rpm, centrifugation was performed at 4000rpm for 5min, the supernatant was discarded, and then resuspended in 200. Mu.L of SOC liquid medium and plated on LB solid plates containing 100. Mu.g/mL Amp resistance, and inverted overnight at 30 ℃. The next day, single colony on the solid plate is respectively picked up to be inoculated with an Amp-resistant LB liquid culture medium, plasmids are extracted after the amplification culture at 30 ℃ and the agarose gel electrophoresis identification is carried out.
Induction of Red homologous recombinase and preparation of SG01 competent cells harboring pKD46 plasmid: positive colonies identified as correct containing pKD46 plasmid were inoculated into 4mL of LB liquid medium (100. Mu.g/mL) containing Amp resistance, placed in a shaking table at 30℃for shaking culture overnight, inoculated into 40mL of LB medium containing Amp resistance at 1:100 the next day, placed in a shaking table at 30℃for 1.5h at 200rpm, and cultured to OD 600nm About 0.2 to 0.3, L-arabinose was added to a final concentration of 30mM, induced for 1 hour, then left on ice for pre-cooling for 30min, centrifuged at 4000rpm for 10min at 4℃and the supernatant discarded, the cells were resuspended in an equal volume of pre-cooled 10% glycerol and the washing was repeated three times. Finally, the supernatant is discarded, and finally, 100 times of the supernatant is concentrated to prepare 400 mu L of competent cells, and 40 mu L of competent cells are packaged in each tube and placed in a refrigerator at the temperature of minus 70 ℃ for standby.
Electrotransformation of Cm resistance gene PCR products containing homology arms: mu.L of the purified PCR product containing homologous sequences of cat gene and bcf gene was mixed with 40. Mu.L of SG01 strain competent cells containing pKD46 plasmid, and the mixture was added to a 0.1cm Bio-Rad electrode cup for electric shock. The mixture after electric shock was rapidly added to 1mL of pre-chilled SOC medium and incubated in a shaker at 200rpm,37℃for 2h. At normal temperature, centrifuging at 4000rpm for 5min, washing the thalli 3 times with 1mL of SOC culture medium, finally re-suspending the thalli with 200 mu L of SOC culture medium, coating the thalli on an LB solid plate containing chloramphenicol resistance (Cm), placing the solid plate in a constant temperature incubator at 37 ℃ for inversion culture for 24-36 h, and screening positive recombinants SG01 delta bcf: : cat.
Primary recombination identification: the above SG01 delta bcf was prepared by boiling: : the DNA template of cat is specifically selected positive recombinants Sg01Δbcf: : cat single colonies were cultured overnight at 37℃in LB liquid medium. Taking 1mL of bacterial liquid for 2min at 12000rpm in the next day, adding 1mL of ultrapure water after removing the supernatant, re-suspending the bacterial body after adding 12000rpm for 2min, adding 150 mu L of ultrapure water after removing the supernatant, and re-suspending to obtain SG01 delta bcf: : a DNA template of cat. And (3) performing PCR amplification reaction by using P1 and P2 as primers. Specific amplification systems and conditions are shown in Table 4. The PCR reaction procedure was: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 1min; annealing at 55 ℃ for 30s; extending at 72 ℃ for 30s and 30 cycles; thoroughly extend at 72℃for 10min. The results of the primary recombination assays are shown in FIG. 3.
Table 4 sg01Δbcf: : cat PCR amplification system
Elimination of pKD46 plasmid: the correct positive recombinants are identified by the previous PCR, inoculated in a Cm resistance LB liquid culture medium, cultured for 12 hours at 42 ℃, passaged for a plurality of times under the same condition, streaked LB solid plates are placed in a 37 ℃ culture, single colonies are randomly selected for Amp and Cm resistance detection, and clones sensitive to Amp and resistant to Cm are selected, namely the primary recombinant strains with eliminated pKD46 plasmids.
Construction and identification of the secondary recombinant strain: the primary recombinant strain Sg01Δbcf is prepared according to a conventional method: : electrotransformation competent cells of cat. Plasmid pCP20 (publication: datsenko KA, wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products.PNAS.2000;97 (12): 6640-6645.) was introduced into SG01. DELTA. Bcf by electrotransformation: : the cat electrotransformation competent cells were plated on LB solid plates containing Amp and Cm resistance, and suspected secondary recombination positive clones were selected at 30 ℃. And (3) carrying out PCR amplification identification on the suspected secondary recombination positive clone by taking the P1/P2 primer and the Sg01Δbcf as templates. Specific amplification systems and procedures were identical to the identification of the first recombinant strain (Table 4). The secondary recombination identification result is shown in figure 3, the successfully identified secondary recombinants are inoculated into an LB liquid culture medium without resistance, cultured for 12 hours at 42 ℃, passaged for a plurality of times under the same condition, an LB solid plate is streaked and placed into a 37 ℃ culture, single bacterial colony is randomly picked for Amp and Cm double resistance detection, and a mutant strain sensitive to both antibiotics, namely the secondary recombination strain, is obtained and named SG01 delta bcf.
SG01 delta bcf electrotransformation competent cells are prepared by a conventional method, recombinant plasmids pBR322-SPbcf and empty plasmid pBR322 are respectively introduced into SG01 delta bcf strain electrotransformation competent cells by an electrotransformation method, and positive clones, namely recombinant strains SG01 delta bcf (pBR 322-SPbcf) and empty plasmid control strains SG01 delta bcf (pBR 322) are screened by coating LB plates containing Amp after resuscitating for 1.5h at 37 ℃. SG01 delta bcf (pBR 322-SPbcf) and SG01 delta bcf (pBR 322) strains were cultured to logarithmic phase, 1mL of bacterial solutions were washed 1 time with 0.9% sterile physiological saline, and resuspended, and the bacterial solution concentration was adjusted to 5X 10 9 CFU/mL. And (3) dripping 10 mu L of bacteria on a clean glass slide, dripping 10 mu L of salmonella pullorum positive reference serum international standard Anti-S.pullorum serum (purchased from British national biological standard and assay institute), uniformly mixing for 2min, and observing an agglutination result by naked eyes. The results showed that the obvious aggregation particles appeared after the SG01. DELTA. Bcf (pBR 322-SP bcf) was mixed with the Salmonella pullorum positive reference serum international standard, as a positive reaction, whereas the empty plasmid control strain SG01. DELTA. Bcf (pBR 322) was mixed with the Salmonella pullorum positive reference serum international standard, as a negative reaction, without aggregation particles (FIG. 4). The above results demonstrate that the recombinant plasmid pBR322-SPbcf is functional.
Example 2: construction of recombinant salmonella typhi SG01 (pBR 322-SP bcf) and induction of specific anti-salmonella pullorum Bcf pilus serum antibody in chicken body
The method comprises the steps of preparing salmonella gallinarum SG01 strain electrotransformation competent cells by a conventional method, introducing recombinant plasmid pBR322-SPbcf which is successfully verified in functionality into the salmonella gallinarum SG01 strain electrotransformation competent cells by an electrotransformation method, resuscitating for 2 hours at 37 ℃, and then coating the cells on an LB plate containing ampicillin to screen positive clones, namely the recombinant salmonella gallinarum strain which is non-inducible and expresses salmonella gallinarum Bcf fimbriae, and the recombinant salmonella gallinarum strain is named SG01 (pBR 322-SPbcf).
Taking 2 SPF chickens of 2 weeks old, one of which is orally inoculated with 5×10 9 CFU doses of recombinant Salmonella typhi SG01 (pBR 322-SP bcf) strain and the other were inoculated orally with 1mL of sterile PBS (1M, pH 7.2). After 2 weeks of inoculation, immune chicken blood is collected and serum is separated, and anti-salmonella pullorum Bcf pilus serum and negative serum are obtained respectively. The serum was subjected to functional verification as follows:
culturing DH 5. Alpha (pBR 322-SPbcf) constructed in example 1 to logarithmic phase, collecting 1mL of bacterial liquid, washing with 0.9% sterile physiological saline for 1 time, and re-suspending to adjust bacterial liquid concentration to 5×10 9 CFU/mL. And (3) dripping 10 mu L of fungus on a clean glass slide, respectively dripping 10 mu L of negative serum and anti-pullorum salmonella Bcf pilus serum, uniformly mixing for 2min, and observing an agglutination result by naked eyes. As a result, as shown in FIG. 5, DH 5. Alpha. (pBR 322-SPbcf) did not produce specific agglutination particles after mixing with negative serum, and was a negative reaction (FIG. 5); and the mixture with the anti-pullorum salmonella Bcf pilus serum produces specific agglutination particles, which is a positive reaction (figure 5). The result proves that the recombinant plasmid has the function of resisting salmonella pullorum Bcf pilus serum.
Example 3: anti-SG 01 (pBR 322-SP bcf) serum antibody obviously and specifically inhibits adhesion level of salmonella pullorum to LMH (local tumor necrosis factor) cells
Culturing Salmonella pullorum SP03 strain to OD 600nm About 1.0, 3mL of the bacterial liquid was packed in 3 1.5mLEP tubes (1 mL per tube), centrifuged at 4000rpm for 5min, and the bacterial liquid was resuspended in DMEM medium, anti-Salmonella pullorum Bcf pilus serum antibody (1:50 dilution) and negative serum (1:50 dilution), respectively, and incubated for 30min. Resuscitating LMH cells, spreading in 96-well plate, respectively transferring the re-suspended bacterial liquid into each well (6 technical repeated wells are arranged in each group) according to MOI=1:100 ratio after the cells are fully adhered and spread and the incubation process is completed, and culturing at 37deg.C for 1 hr; the supernatant was discarded, and after 3 washes with PBS, 0.5% Triton X-100 lysate was added to each well and incubated for 30min; the liquid in the wells was transferred to a new centrifuge tube, washed 3 times with 150 μl of PBS, and the PBS was collected into the corresponding 1.5mL EP and repeated 2 times; in a ratio of 1:10The collected bacterial liquid was diluted stepwise with PBS and a suitable gradient dilution was selected (10 -1 、10 -2 、10 -3 And 10 -4 ) The bacterial liquid of (2) was pipetted into 10. Mu.L of the spot plate onto the LB solid plate, and the number of colonies growing on the next day plate was recorded. Two identical 96-well plates were additionally provided and the above steps were repeated as three biological replicates.
As shown in fig. 6, the adhesion amount of the salmonella pullorum SP03 strain to LMH cells was 100%, the cell adhesion amount of the SP03 strain after co-incubation with the salmonella pullorum Bcf pilus serum was found to be significantly reduced (p < 0.05), compared with the adhesion amount of the SP03 strain, by 80.7% (fig. 6), and the adhesion amount of the SP03 strain after incubation with negative serum was not significantly reduced (fig. 6), indicating that the salmonella pullorum Bcf pilus serum was able to significantly specifically inhibit the adhesion ability of the salmonella pullorum SP03 strain to LMH cells.
In conclusion, the anti-salmonella pullorum Bcf pilus serum antibody can remarkably and specifically inhibit the in-vitro adhesion capacity of salmonella pullorum to LMH cells, and has the application potential of controlling salmonella pullorum infection.
Claims (10)
1. The salmonella pullorum Bcf pilus operon gene is characterized in that the Bcf pilus operon gene sequence is shown in SEQ ID NO.1.
2. An expression cassette, recombinant vector, recombinant cell or recombinant strain comprising the salmonella pullorum Bcf pilus operon gene of claim 1.
3. A recombinant plasmid, which is obtained by inserting the gene of the salmonella pullorum Bcf operon in claim 1 into an expression vector.
4. The recombinant plasmid of claim 3, wherein the expression vector is a non-inducible expression vector.
5. A recombinant salmonella gallinarum expressing salmonella pullorum Bcf pili, wherein the recombinant salmonella gallinarum is obtained by introducing the recombinant plasmid of claim 3 or 4 into attenuated salmonella gallinarum.
6. The use of the salmonella gallinarum Bcf fimbriae operon gene of claim 1, the expression cassette, the recombinant vector, the recombinant cell or the recombinant strain of claim 2, and the salmonella gallinarum recombinant strain of claim 5 in the preparation of medicaments or vaccines for salmonella gallinarum infection.
7. An anti-salmonella pullorum Bcf pilus serum antibody, wherein the serum antibody is obtained by inoculating the salmonella typhimurium recombinant strain of claim 5 into an SPF chicken and then separating.
8. The use according to claim 7, wherein the inoculation method comprises oral administration or injection, and the inoculation dose is 1 x 10 9 CFU~5×10 9 CFU。
9. The use of an antibody against salmonella pullorum Bcf fimbriae serum according to claim 6, for the manufacture of a medicament for salmonella pullorum infection.
10. A medicament or vaccine for preventing or treating salmonella pullorum infection, comprising the salmonella pullorum Bcf pilus operon gene of claim 1, the expression cassette, recombinant vector, recombinant cell or recombinant strain of claim 2, and the salmonella typhimurium recombinant strain of claim 5.
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