CN111850003A - Recombinant expression pasteurella multocida thiamine periplasm binding protein and application thereof - Google Patents
Recombinant expression pasteurella multocida thiamine periplasm binding protein and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of animal genetic engineering, and particularly relates to a recombinant expression pasteurella multocida thiamine periplasmic binding protein and application thereof, wherein the nucleotide sequence of the pasteurella multocida thiamine periplasmic binding protein gene is shown as SEQ ID NO: 1, the protein sequence coded by the protein gene is shown as SEQ ID NO: 2, respectively. The cloned pasteurella multocida capsular F strain HN07 genome is used as a template, the cloned pasteurella multocida thiamine periplasmic binding protein gene is transformed into an escherichia coli expression strain, expression protein is induced by IPTG, and high-purity protein is obtained by a nickel column affinity chromatography method, and animal challenge protection test results show that the thiamine periplasmic binding protein stimulates Th1/Th2 immune response, can provide good protection for mice respectively attacking capsular A strain HB03 and capsular D strain HN06, can be used as an immunogenic additive, and can also be used as candidate molecules of subunit vaccines.
Description
Technical Field
The invention belongs to the technical field of animal molecular biology, and particularly relates to a recombinant expression pasteurella multocida thiamine periplasmic binding protein and application thereof.
Background
Pasteurella multocida is a gram-negative bacterium that infects humans and various animals, mainly causing respiratory diseases and septicemia. Pigs of any age group can be infected with the disease, with piglets and finishing pigs having a higher incidence. There are many serotypes of pasteurella multocida, which can be classified into 5 serotypes, A, B, D, E, F, etc., according to the difference of capsular antigens. Clinical studies have shown that pasteurella multocida of different serotypes shows a certain "host preference" both in prevalence and in pathogenesis, and the resulting clinical symptoms are also different. This increases the difficulty in preventing and controlling pasteurella multocida clinically. Currently, the clinical prevention of pasteurella multocida is mainly based on inactivated vaccines, but the inactivated vaccines have single antigen components and cannot provide better protection against infection of different serotype strains, and currently, many reported immunogenic proteins with better protection against homologous pasteurella multocida provide limited protection against other serotype strains, while in swine herds, serotypes mainly comprise a and D, so that it is necessary to perform research on subunit vaccines against common serotypes of pasteurella multocida.
The applicant selects a pasteurella multocida thiamine binding periplasmic protein gene by a bioinformatics method in the national center of agricultural microbiology of Huazhong agriculture university and the center of live pig healthy breeding synergy innovation, then the gene is obtained by amplification from pasteurella multocida, recombinant expression is carried out, and animal challenge protection tests prove that the recombinant protein expressed by the gene can respectively provide 100% of protection for the mice of challenge capsular type A strain HB03(GenBank accession number CP003328) and capsular type D strain HN06(GenBank accession number CP003313), so that the protein has the prospect of being used as an immunogenicity additive and subunit vaccine candidate molecules.
Disclosure of Invention
The first purpose of the invention is to amplify a pasteurella multocida thiamine-binding periplasmic protein gene ThiB in a pasteurella multocida genome.
The second purpose of the invention is to efficiently express the pasteurella multocida thiamine binding periplasmic protein gene in an escherichia coli expression system so as to obtain a recombinant protein rThiB.
The third purpose of the invention is to provide the related application of the recombinant protein rThiB in the preparation of pasteurella multocida immunogenic additive and subunit vaccine.
The technical scheme of the invention is as follows:
(1) acquisition of the pasteurella multocida thiamine binding periplasmic protein gene: by using a Polymerase Chain Reaction (PCR) method, taking a pasteurella multocida D type strain HN06 genome as a template, and carrying out PCR amplification to obtain a pasteurella multocida thiamine binding periplasmic protein gene ThiB, wherein an Open Reading Frame (ORF) of a nucleotide sequence of the protein gene is shown as a sequence table SEQID NO: 1 is shown.
(2) The coding amino acid sequence of the pasteurella multocida thiamine combined periplasmic protein gene is shown in a sequence table SEQ ID NO: 2, respectively.
(3) The invention provides a preparation method of recombinant protein rThiB, which comprises the following steps: the sequence table SEQ ID NO: 1, intercepting a sequence corresponding to a signal peptide at the front end of the whole Open Reading Frame (ORF) of the pasteurella multocida thiamine combined periplasmic protein gene, constructing a recombinant expression plasmid pET-30-ThiB by using the truncated 939bp (67bp-1005bp) and an escherichia coli expression vector, transforming the recombinant expression plasmid pET-30-ThiB into BL21(DE3) by using a competence method, screening a high-resistance transformant by using kanamycin, and inducing for 14 hours at 25 ℃ by using 0.8mmol/L IPTG. Collecting the IPTG induced expressed bacteria liquid, separating and purifying to obtain the final purified protein, i.e. the recombinant protein of the thiamine combined periplasmic protein gene of the pasteurella multocida.
(4) And (3) purifying the recombinant protein, performing induced expression on 1L of bacterial liquid according to the method, and centrifuging the induced bacterial liquid at 4 ℃ at 7000r/min for 15min to collect the bacterial liquid. After 20mL of PBS is taken for resuspending the thallus precipitate, crushing the thallus precipitate for 4 times by using a pressure crusher; centrifuging at 12000r/min for 30min at 4 deg.C, removing precipitate, collecting supernatant, filtering, and purifying with HIS affinity purification column.
(5) The reactogenicity of rThiB is detected by using a western blot method. After the rThiB is subjected to SDS-PAGE separation and transferred onto a PVDF membrane, primary antibody is incubated to infect the positive serum of a corresponding pasteurella multocida mouse for 2h, and secondary antibody is incubated to HRP-labeled goat anti-mouse serum for 1h, and then the detection is carried out by using an ECL chemiluminescence method, the result shows that a specific band is generated after the positive serum is incubated, the size of the band is the same as that of the recombinant protein, and the recombinant protein has good reactogenicity.
(6) The recombinant protein rThiB of the invention is used for immunizing mice once every 14 days for 2 times. Respectively using 5LD 14 days after the second immunization50The results of the challenge on mice by using HB03 and HN06 with the dosages show that the recombinant protein can respectively provide 100% of protection force for the mice.
The recombinant protein rThiB which encodes the thiamine-binding periplasmic protein of Pasteurella multocida can respectively provide 100% of protection power for mice of a challenge capsular type A strain HB03(GenBank accession number CP003328) and a capsular type D strain HN06(GenBank accession number CP003313), has good immunogenicity, can be used as an immunogenic additive, and can also be used as a candidate molecule of a subunit vaccine.
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FIG. 1: pasteurella multocida thiamine binding periplasmic protein gene amplification electrophoretogram. Description of reference numerals: lane 1 in fig. 1: cloning a target gene amplified by a primer from a pasteurella multocida genome; lane M in fig. 1: DNA molecular weight standards (available from Bao bioengineering, Dalian, Inc.).
FIG. 2: and amplifying the target fragment and the carrier electrophoresis picture by utilizing an enzyme digestion connection method. Description of reference numerals: lane 1 in fig. 1: double restriction enzyme digestion recombinant plasmid pET-30 a-ThiB; lane 2 in fig. 1: single restriction enzyme cutting recombinant plasmid pET-30 a-ThiB; lane M in fig. 1: and (5) DNA molecular weight standard.
FIG. 3: the map of the recombinant plasmid pET-30a-ThiB constructed by the invention.
FIG. 4: SDS-PAGE electrophoresis result of qualitative analysis of thiamine binding periplasmic protein of Pasteurella multocida. Description of reference numerals: lane 1 in fig. 4: unpurified rThiB; description of reference numerals: lane 2 in fig. 4: purified rThiB; description of reference numerals: lane M in fig. 4: and (3) protein molecular mass standard.
FIG. 5: the result of the reactivity of the pasteurella multocida thiamine binding periplasmic protein recombinant protein rThiB with the positive serum of pasteurella multocida HB 03. Description of reference numerals: lane 1 in fig. 5: the recombinant protein of the invention reacts with the positive serum of a mouse infected with Pasteurella multocida; lane M in fig. 5: and (3) protein molecular mass standard.
FIG. 6: the result of the reactivity of the pasteurella multocida thiamine binding periplasmic protein recombinant protein rThiB with the pasteurella multocida HN06 positive serum. Description of reference numerals: lane 1 in fig. 6: the recombinant protein of the invention reacts with the positive serum of a mouse infected with Pasteurella multocida; lane M in fig. 6: and (3) protein molecular mass standard.
Fig. 7 and 8: survival curves for animal challenge protection experiments. Description of reference numerals: FIGS. 7 and 8 are the graphs in which the protective power of rThiB recombinant protein against Pasteurella multocida D, F and the survival of the control group are plotted.
FIG. 9: status of antibody levels in mice in animal challenge protection experiments.
Detailed Description
Description of sequence listing:
sequence listing SEQ ID NO: 1 is the nucleotide sequence of the pasteurella multocida thiamine binding periplasmic protein gene ThiB cloned by the invention, the sequence length is 1055bp, wherein 1-1055bp of the sequence is the open reading frame (ORF, namely CDS) sequence of the protein gene. Encodes 334 corresponding amino acid sequences.
Sequence listing SEQ ID NO: 2 is the amino acid sequence encoded by the gene for the thiamine binding periplasmic protein of Pasteurella multocida. Coding 334 protein sequences.
The present invention will be described in detail with reference to examples.
Example 1: culture of Pasteurella multocida
The porcine pasteurella multocida clinical strains (HB03 and HN06) freeze-dried powder separated and stored in the laboratory are inoculated on a Tryptic Soy Agar (TSA) culture medium (purchased from American BD company) containing 5% newborn bovine serum, streaked by an inoculating loop and placed in a 37 ℃ incubator for culturing for 12h, and a single colony is picked up and placed in Tryptic Soy Broth (TSB) (purchased from American BD company) containing 5% newborn bovine serum to be shake-cultured for 12-24 h in a constant temperature shaker at 37 ℃ and 180 rpm.
Example 2: extraction of pasteurella multocida genome
The cultured bacterial liquid is used for extracting genome, the TIANGEN bacterial genome DNA extraction kit is used for extracting genome DNA of Pasteurella multocida HN06, and the operation steps are carried out according to the kit instruction, and specifically comprise the following steps:
(1) 5mL of bacterial liquid of pasteurella multocida HN06 is taken, centrifuged for 1min at 10000r/min, and the supernatant is sucked as clean as possible.
(2) 200. mu.L of buffer GA carried in the kit was added to the pellet, and the pellet was shaken until the pellet was completely suspended.
(3) Add 20. mu.L of the protease K solution carried in the kit to the tube and mix well.
(4) Adding buffer GB in a 220 mu L kit, shaking for 15s, standing at 70 ℃ for 10min, cleaning the solution, and centrifuging briefly to remove water beads on the inner wall of the tube cover.
(5) Add 220. mu.L of absolute ethanol, mix well with shaking for 15s, at which time a flocculent precipitate may appear, and centrifuge briefly to remove water droplets on the inner wall of the tube cover.
(6) Adding the solution and flocculent precipitate obtained in the previous step into adsorption column CB3 (placing the adsorption column into a collection tube) in the kit, centrifuging at 12000r/min for 30s, pouring off waste liquid, and placing adsorption column CB3 into the collection tube.
(7) Adding 500 μ L buffer GD in the kit, centrifuging at 12000r/min for 30s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube.
(8) Adding 600 μ L of rinsing liquid PW (added with anhydrous ethanol before use) in the kit into adsorption column CB3, centrifuging at 12000r/min for 30s, pouring off waste liquid, and placing adsorption column CB3 into a collection tube.
(9) And (5) repeating the operation of the step (8).
(10) The adsorption column CB3 is put back into the collecting tube, centrifuged at 12000r/min for 2min, and the waste liquid is poured off. The adsorption column CB3 was left at room temperature for 5min to thoroughly dry the residual rinse solution in the adsorption material.
(11) Transferring the adsorption column CB3 into a clean centrifuge tube, suspending and dripping elution buffer TE carried in a 50 mu L kit into the middle part of the adsorption membrane, standing for 5min at room temperature, centrifuging for 2min at 12000r/min, collecting the solution into the centrifuge tube, and storing at-20 ℃ for later use.
Example 3: construction of prokaryotic expression vector and induced expression in escherichia coli
As the pasteurella multocida thiamine combined periplasmic protein utilizes Signal P4.1 software to predict that the first 22 amino acids of the protein amino acid sequence are Signal peptide sequences, in order to facilitate prokaryotic expression, the pasteurella multocida thiamine combined periplasmic protein gene is truncated and expressed, and the expression region is 67-1005 bp.
The thionine-binding periplasmic protein gene truncated fragment of Pasteurella multocida is connected with an HIS tag vector pET-30a (purchased from Takara Bio-engineering Co., Ltd.) by using an enzyme digestion connection method.
(1) Primer design
Designing upstream and downstream primers by using Primer 5.0 software:
an upstream primer F1: 5'-CGCGGATCCCAAACGCAAGCGGTCA-3';
the downstream primer R1: 5'-CCGCTCGAGTTATTGGGTTAGGGTCGTTT-3'.
(2) And (3) PCR amplification: taking a Pasteurella multocida genome as a template, and carrying out PCR amplification by using F1 and R1 primers, wherein the reaction system is 50 mu L, and the PCR reaction system is as follows:
and (3) PCR reaction conditions: the reaction condition is 98 ℃ for 5 min; 45s at 94 ℃, 45s at 56 ℃ and 1min at 72 ℃; 10min at 72 ℃; for a total of 30 cycle periods.
(3) And (3) identifying a PCR product: after amplification, 5. mu.L of the PCR product was mixed with 10 × loading buffer, spotted, and subjected to electrophoresis for 30min in 1.0% agarose gel, 1 × TAE buffer, 120V, and the result was observed to obtain the desired fragment (see the gel chart shown in FIG. 1).
(4) Connection and sequencing of target gene and T vector
Mixing 1 μ L (50ng/uL) of pMD19-T vector (from Takara Bio-engineering, Inc.) with 4 μ L of the target fragment recovered from the gel, adding 5 μ L of solution I (from Tiangen Biotechnology, Inc. (Beijing) Co., Ltd.), mixing, and connecting at 4 deg.C overnight. 10 μ L of the ligation product was added to E.coli DH5 α competent cells under sterile conditions, gently and repeatedly pipetted and mixed, and placed in ice bath for 30 min. The reaction mixture was then heated in a 42 ℃ water bath for 90s, and immediately cooled in an ice bath for 3min (without shaking). Transferring the bacterial liquid to an EP tube filled with 600 microliter LB culture liquid preheated to 37 ℃, and oscillating at constant temperature of 37 ℃ for 45min at 180rpm to ensure that the drug-resistant gene on the vector is normally expressed. Uniformly coating 100 mu L of bacterial liquid on a TSA plate containing Ampicillin (AMP) (100 mu g/mL), placing the plate in a forward direction at 37 ℃ for 30min, inverting the plate, culturing the plate in a constant-temperature incubator at 37 ℃ for 12-16 h, selecting a single bacterial colony, inoculating the single bacterial colony in LB culture liquid containing 100 mu g/mL AMP, oscillating the plate at 180rpm and 37 ℃ for 12-16 h, and then identifying, namely, amplifying the PCR into positive clones, taking the bacterial liquid, sending the bacterial liquid to Hippocrate Biotech limited company for sequencing analysis to obtain a 939bp genome sequence, wherein the gene is a sequence of a signal peptide at the front end of an Open Reading Frame (ORF) full-length part, the sequence full-length is 939bp (67bp-1005bp) totally, and the Open Reading Frame (ORF) full-length sequence is as shown in a sequence table SEQ ID NO: 1 is shown.
Sequence number: SEQ ID NO: 1
Sequence length: 1005bp
The source is as follows: pasteurella multocida
Sequence characteristics: with correct Open Reading Frame (ORF): 1005 bp; and (4) determining the position: the positions of the initiation and termination codons: ATG, position 1; TAA, 1005 bit.
(5) The target gene is cut off from the T vector, the cutting sites are BamHI and XhoI, the cutting system is 50 mu L, the reaction time is 2.5h, and meanwhile, the pET-30a vector is cut off by the same steps.
An enzyme digestion reaction system:
(6) connection and sequencing of target gene and vector pET-30a
The gel recovery of the carrier and the target fragment was carried out separately, and the recovery concentration was measured. mu.L (7 ng/. mu.L) of the recovered pET-30a product was mixed with 7. mu.L (40 ng/. mu.L) of ThiB recovered from gel, and then 1. mu. L T4DNA Ligase (350 ng/. mu.L) and 1. mu.L of 10 XT 4DNA Ligase Buffer were added thereto, and they were mixed and ligated at 16 ℃ for 45 min. Taking 10 mu L of the ligation product, adding the ligation product into escherichia coli DH5 alpha competent cells under the aseptic condition, gently and repeatedly blowing and beating the cells evenly by a pipette, and placing the cells for 30min in ice bath. The mixture was heated in a water bath at 42 ℃ for 90s, and immediately cooled in an ice bath for 2min (taking care not to shake). Transferring the bacterial liquid into LB culture liquid containing 600 mul preheated to 37 ℃, and carrying out mild oscillation for 45min at 180rpm and 37 ℃ to ensure that the drug-resistant gene on the vector is normally expressed. Uniformly coating 100uL of bacterial liquid on a TSA plate containing kanamycin (100 mug/mL), placing the plate in the forward direction at 37 ℃ for 30min, inverting the plate, culturing the plate in a constant-temperature incubator at 37 ℃ for 12-16 h, selecting a single bacterial colony, inoculating the single bacterial colony in LB culture liquid containing 100 mug/mL kanamycin, oscillating the plate at 180rpm and the constant temperature of 37 ℃ for 12-16 h, identifying the bacterial colony, amplifying the PCR into positive clone, and taking the bacterial liquid to send the bacterial liquid to Hippocrate Biotech Limited company for sequencing analysis to obtain a 939bp genome sequence.
(7) Construction of pET-30 a-ThiB-expressing bacterium
pET-30a-ThiB plasmid (1. mu.L) was aseptically added to E.coli BL21(DE3) competent cells (50. mu.L), gently and repeatedly pipetted and mixed, and placed in an ice bath for 30 min. Then, the mixture was heated in a water bath at 42 ℃ for 90 seconds, and immediately cooled in an ice bath for 2min (taking care not to shake). Transferring the transfer bacteria liquid into LB culture liquid containing 500 μ L preheated to 37 deg.C, and gently oscillating at 150rpm and 37 deg.C for 45min to recover drug resistance of bacteria; further, 150. mu.L of the bacterial suspension was applied to LB agar plate containing Kanamycin (KAN) (100. mu.g/mL). And (3) inverting the plate, culturing for 12-16 h in a constant-temperature incubator at 37 ℃, selecting a single colony, inoculating the single colony in LB culture solution containing 150 mu g/mL KAN, and identifying after violently shaking for 12-16 h. Meanwhile, pET-30a-ThiB empty vector without any foreign gene added is transformed as a negative control.
Taking 1 mu L of bacterial liquid for PCR, wherein the reaction system is 20 mu L as follows:
and (3) PCR reaction conditions: the reaction condition is 94 ℃ for 5 min; 45s at 94 ℃, 45s at 56 ℃ and 1min at 72 ℃; 10min at 72 ℃; for a total of 35 cycle periods.
The clone which is amplified to be positive can be used for prokaryotic expression of pET-30a-ThiB recombinant protein.
(8) Prokaryotic expression of recombinant protein rThiB
According to the volume ratio of 1: 100 the positive pET-30a-ThiB/BL21(DE3) was cultured in LB medium containing kanamycin at 37 ℃ and 200rpm to OD 600About 0.6, 0.8mmol/L IPTG was added and induction was carried out at 25 ℃ for 14 h. Collecting a large amount of bacteria liquid for inducing expression, centrifuging at 4 ℃ at 7000rpm for 15min, discarding the supernatant, adding PBS (phosphate buffer solution, formula is: 8.0g NaCl, 0.2g KCl, 1.56g Na2HPO4, 0.2g KH2PO4, sequentially adding into 800mL double distilled water, after completely dissolving, fixing the volume to 1000.0mL, adding HCl to adjust pH to 7.8), resuspending the thallus precipitate, and crushing under pressure for 4 times; then centrifuging at 12000rpm for 30min at 4 ℃, discarding the precipitate, collecting the supernatant, and filtering to obtain the crude protein of recombinant expression.
(9) Purification of recombinant crude proteins
Purifying the obtained recombinant crude protein by using a nickel affinity chromatography column, which comprises the following specific steps: take 5mL ddH2Passing through a column, flushing absolute ethyl alcohol for sealing the column, passing through a column by using 5mL of binding buffer, passing the crushing liquid through the column, balancing the column by using 15mL of lysine buffers after the sample loading is finished, and passing through an elusion buffer according to the proportion of 0 to 10 percent,preparing 10 dilution of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% into 5mL, respectively passing through the column, and sequentially using 5mL of buffering, ddH2And (4) passing through the column, and finally sealing the column by using 5mL of absolute ethyl alcohol. Preparing protein electrophoresis samples by using 40uL of 10 diluted eluents, performing SDS-PAGE electrophoresis, dialyzing the eluents containing the target protein, concentrating, and storing the purified recombinant protein at-80 ℃, wherein the electrophoresis result is shown in figure 4.
Example 4: identification of the reactogenicity of recombinant proteins of the thiamine-binding periplasmic protein of Pasteurella multocida
1) Identification of reactogenicity
Carrying out SDS-PAGE electrophoresis on the purified recombinant protein (the recombinant protein of the invention for short) of the pasteurella multocida thiamine combined periplasmic protein, then transferring the protein to a PVDF membrane at 100V voltage, sealing the PVDF membrane for 40min by TBST-5% skimmed milk powder for 1h, washing for 3 times by TBST, and 5min each time; putting the PVDF membrane into the positive serum of the Pasteurella multocida mouse diluted by the volume ratio of 1:100, incubating for 1h at room temperature, washing for 3 times by TBST, and washing for 5min each time; adding secondary antibody (goat anti-mouse HRP-IgG diluted by 1:5000 volume ratio, purchased from Abclone company), incubating at room temperature for 1h, washing with TBST for 3 times, washing for 5min each time, adding BCL (purchased from Tiangen Biochemical technology (Beijing) Co., Ltd.) and developing. Western blot determination results show that the purified recombinant protein of the pasteurella multocida thiamine binding periplasmic protein can specifically react with the serum of a positive mouse of the capsular A type pasteurella multocida HB03 strain (see figure 5) and can also specifically react with the serum of a positive mouse of the capsular D type pasteurella multocida HN06 strain (see figure 6).
Example 5: mouse immunoprotection test of recombinant protein of Pasteurella multocida thiamine binding periplasmic protein
20 clean Kunming mice of 6 weeks are taken and divided into 4 groups of 5, and the groups are numbered as A group, B group, C group and D group. Collecting blood of orbital veins of mice at 0d, uniformly mixing and emulsifying purified recombinant protein (1 mu g/mL) of pasteurella multocida thiamine-binding periplasmic protein of the invention with equivalent Freund's complete adjuvant, wherein each of A group and B group is emulsifiedInjecting 200uL of protein adjuvant emulsion subcutaneously at multiple points for mice, injecting 200uL of PBS (phosphate buffer solution) subcutaneously for each mouse in groups C and D, collecting blood of orbital veins for 14D mice, mixing and emulsifying purified recombinant protein (1 mu g/mL) of the invention and an equivalent amount of Freund's incomplete adjuvant, injecting 200 mu L of protein adjuvant emulsion subcutaneously for each mouse in groups A and B, injecting 200 mu L of PBS (phosphate buffer solution) subcutaneously at multiple points for each mouse in groups C and D, collecting blood of orbital veins for 14D mice, and collecting 5LD of PBS (phosphate buffer solution) respectively50Dose HB03 strain attacking poison in A group, C group, 5LD50And (3) the HN06 strain is used for counteracting the toxin in the group B and the group D, and is injected into the mouse body through the abdominal cavity, and the survival condition of the mouse is recorded. The results showed that all mice in groups A and B survived, 1 survived in group C, and all mice in group D died. The recombinant protein of the invention is shown to provide 100% protection for challenge mice, and has good immunogenicity (the results are shown in fig. 7 and fig. 8).
Example 6: detection of antibodies to the recombinant proteins of the invention by ELISA
In this embodiment, the optimal coating concentration of the antigen is determined by a conventional matrix titration method, which comprises the following steps: diluting the purified recombinant protein of the invention by 2 times (8 ug/mL-0.25 ug/mL) from 8ug/mL in turn by using carbonate buffer solution, adding 3 holes at each dilution point, wherein the amount of each hole is 100 muL, placing the recombinant protein in a refrigerator at 4 ℃ overnight, taking the recombinant protein out the refrigerator the next day, washing the recombinant protein by using PBST buffer solution for 3 times, adding 150 muL and 5 percent of skimmed milk powder into each hole, sealing the holes at 37 ℃ for 1h, removing the sealing liquid in the holes, washing the holes for 3 times, incubating the recombinant protein immune mouse serum and the negative serum of the invention by using PBST buffer solution in a double ratio (the volume ratio is 1: 200-1: 12800), incubating for 1h at 37 ℃, washing for 3 times, adding 100uL of goat anti-mouse HRP-IgG enzyme-labeled secondary antibody (purchased from Abclone company) diluted by 5000 times into each hole, incubating for 1h at 37 ℃, washing for 3 times, adding 50uL of developing solution A, B in turn, and incubating for 10min in a dark place, addition of H2SO450uL, color development, OD determination with microplate reader630Dividing the value of the positive well by the value of the negative well, selecting the dilution of the positive well with the largest quotient as the optimal dilution concentration of the recombinant protein coating of the invention, determining the optimal coating concentration, The serum antibodies of 0d, 14d and 28d mice after immune protein collection are detected, and the result is shown in figure 9, which shows that the recombinant expression pasteurella multocida thiamine combined with periplasmic protein can stimulate the body to produce high-level antibodies after the mice are immunized.
Sequence listing
<110> university of agriculture in Huazhong
<120> recombinant expression pasteurella multocida thiamine periplasm binding protein and application thereof
<141>2020-07-09
<160>2
<170>SIPOSequenceListing 1.0
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<211>1005
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<213> Pasteurella multocida (Pasteurella multocida)
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<221>CDS
<222>(1)..(1005)
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Met Ser Arg Leu Lys Thr Ser Phe Phe Phe Thr Ala Leu Ser Thr Leu
1 5 10 15
tca cta tcc gtc ttt gct caa acg caa gcg gtc aat gtg tat act tat 96
Ser Leu Ser Val Phe Ala Gln Thr Gln Ala Val Asn Val Tyr Thr Tyr
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gat tcc ttc act tct gaa tgg ggg gca ggt ccg aaa gta aaa aaa gca 144
Asp Ser Phe Thr Ser Glu Trp Gly Ala Gly Pro Lys Val Lys Lys Ala
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Phe Glu Thr His Phe Pro Gln Cys Gln Val Asn Phe Thr Ala Phe Gly
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gat tct ggc act atg ttt aat cgc tta cgt tta gaa ggg aaa aaa aca 240
Asp Ser Gly Thr Met Phe Asn Arg Leu Arg Leu Glu Gly Lys Lys Thr
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Lys Ala Asp Val Val Val Gly Leu Asp Asn Tyr Asn Leu Glu Glu Ala
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<210>2
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<213> Pasteurella multocida (Pasteurella multocida)
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Asp Ser Gly Thr Met Phe Asn Arg Leu Arg Leu Glu Gly Lys Lys Thr
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Ala Phe Leu Lys Gly Glu Ala Asp Val Val Leu Ser Tyr Asn Thr Ser
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Claims (4)
1. A pasteurella multocida thiamine binding periplasmic protein gene is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown.
2. A pasteurella multocida thiamine binding periplasmic protein gene is characterized in that the protein sequence coded by the gene is shown as SEQ ID NO: 2, respectively.
3. Use of the pasteurella multocida thiamine binding periplasmic protein gene of claim 1 for the preparation of a pasteurella multocida subunit vaccine.
4. Use of the pasteurella multocida thiamine binding periplasmic protein gene of claim 2 for the preparation of a pasteurella multocida subunit vaccine.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113801812A (en) * | 2021-09-02 | 2021-12-17 | 华中农业大学 | Pasteurella multocida and application thereof |
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