CN111850003A - A recombinantly expressed Pasteurella multocida thiamine periplasmic binding protein and its application - Google Patents

Abstract

The invention belongs to the technical field of animal genetic engineering, in particular to a recombinantly expressed Pasteurella multocida thiamine periplasmic binding protein and application thereof, and the nucleotide sequence of the Pasteurella multocida thiamine periplasmic binding protein gene As shown in SEQ ID NO: 1, the protein sequence encoded by the protein gene is shown in SEQ ID NO: 2. Using the genome of Pasteurella multocida capsule F strain HN07 as a template, the cloned Pasteurella multocida thiamine periplasmic binding protein gene was transformed into an E. High-purity protein was obtained by nickel-column affinity chromatography. The results of animal challenge protection test showed that thiamine periplasmic binding protein stimulated Th1/Th2 immune response, which could be used to challenge capsule type A strains HB03 and capsules respectively. Mice with the D-type strain HN06 provide good protection and can be used as an immunogenic additive and as a candidate molecule for subunit vaccines.

Description

A recombinantly expressed Pasteurella multocida thiamine periplasmic binding protein and its application

technical field

The invention belongs to the technical field of animal molecular biology, and particularly relates to a recombinantly expressed Pasteurella multocida thiamine periplasmic binding protein and its application.

Background technique

Pasteurella multocida is a Gram-negative bacterium that infects humans and a variety of animals, mainly causing respiratory disease and sepsis. Pigs of any age can be infected, with piglets and finishing pigs having a higher incidence. There are many serotypes of Pasteurella multocida, which can be divided into 5 serotypes A, B, D, E, and F according to the differences of capsular antigens. Clinical studies have shown that different serotypes of Pasteurella multocida show a certain "host preference" in epidemic and pathogenicity, and the resulting clinical symptoms are also different. This increases the difficulty of clinical prevention and control of Pasteurella multocida. The current clinical prevention against Pasteurella multocida is mainly inactivated vaccines, but inactivated vaccines have a single antigenic component and cannot provide better protection against the infection of different serotype strains. The highly protective immunogenic proteins of Pasteurella multocida provide limited protection against other serotype strains, and in pig herds, the serotypes are mainly A and D, so it is necessary to target pigs with multiple kills Subunit vaccine studies were conducted on common serotypes of Pasteurella spp.

The research group of the State Key Laboratory of Agricultural Microbiology of Huazhong Agricultural University and the Collaborative Innovation Center for Healthy Pig Breeding where the applicant is located obtained a thiamine-binding periplasmic protein gene of Pasteurella multocida through bioinformatics screening. The gene was amplified from Pasteurella multocida and recombinantly expressed. The recombinant protein expressed by the gene was verified by the animal challenge protection test to be the challenge capsule type A strain HB03 (GenBank accession number CP003328). , Capsular D-type strain HN06 (GenBank Accession No. CP003313) in mice all provided 100% protection, and has the potential as an immunogenic additive and a subunit vaccine candidate molecule.

SUMMARY OF THE INVENTION

The first object of the present invention is to amplify a Pasteurella multocida thiamine-binding periplasmic protein gene ThiB in the genome of Pasteurella multocida.

The second objective of the present invention is to express the thiamine-binding periplasmic protein gene of Pasteurella multocida efficiently in an E. coli expression system, thereby obtaining a recombinant protein rThiB.

The third object of the present invention is to provide the relevant application of the above recombinant protein rThiB in the preparation of Pasteurella multocida immunogenic additives and subunit vaccines.

The technical scheme of the present invention is as follows:

(1) Obtaining the thiamine-binding periplasmic protein gene of Pasteurella multocida: using the polymerase chain reaction (PCR) method, using the genome of Pasteurella multocida D-type strain HN06 as the template, PCR amplification The thiamine-binding periplasmic protein gene ThiB of Pasteurella multocida was increased, and the open reading frame (ORF) of the nucleotide sequence of the protein gene is shown in SEQ ID NO: 1 of the sequence table.

(2) The amino acid sequence encoded by the thiamine-binding periplasmic protein gene of Pasteurella multocida is shown in SEQ ID NO: 2 in the sequence listing.

(3) The present invention provides a method for preparing recombinant protein rThiB: the full-length part of the open reading frame (ORF) of the periplasmic protein gene of Pasteurella multocida thiamine shown in SEQ ID NO: 1 of the sequence table is combined The sequence corresponding to the signal peptide at the front was cut off, and the truncated 939bp (67bp-1005bp) was combined with the E. coli expression vector to construct a recombinant expression plasmid pET-30-ThiB, which was transformed into BL21 (DE3) by the competent method, and the Transformants with high resistance were selected by namycin and induced with 0.8mmol/L IPTG at 25°C for 14h. The above-mentioned bacterial liquid induced by IPTG expression is collected, separated and purified, and the final purified protein is the recombinant protein encoding the thiamine-binding periplasmic protein gene of Pasteurella multocida.

(4) Purification of the recombinant protein, inducing expression of 1 L of bacterial liquid according to the above method, and centrifuging the induced bacterial liquid at 4°C for 15 min at 7000 r/min to collect bacteria. Take 20 mL of PBS to resuspend the bacterial cell pellet and crush it four times with a pressure crusher; centrifuge at 4°C, 12000 r/min for 30 min, discard the pellet, collect the supernatant, filter, and purify with a HIS affinity purification column.

(5) The reactogenicity of rThiB was detected by western blot. After rThiB was separated by SDS-PAGE and transferred to PVDF membrane, the primary antibody was incubated with the positive serum of mice infected with the corresponding Pasteurella multocida for 2h, and the secondary antibody was incubated with HRP-labeled goat anti-mouse serum for 1h. The luminescence method was used for detection, and the results showed that a specific band was produced after the positive serum was incubated, and the size of the band was the same as that of the recombinant protein, indicating that the recombinant protein had good reactogenicity.

(6) The mice were immunized with the recombinant protein rThiB of the present invention, once every 14 days, for a total of 2 times. 14 days after the second immunization, mice were challenged with 5LD ₅₀ doses of HB03 and HN06, respectively, and the results showed that the recombinant protein could provide 100% protection to the mice, respectively.

The recombinant protein rThiB encoding the thiamine-binding periplasmic protein of Pasteurella multocida involved in the present invention can be respectively the challenge capsule type A strain HB03 (GenBank accession number CP003328) and the capsule D type strain HN06 (GenBank accession number). No. CP003313) mice all provide 100% protection and have good immunogenicity, which can be used as immunogenic additives and can also be used as candidate molecules for subunit vaccines.

Description of drawings

Figure 1: Electropherogram of the amplification of the thiamine-binding periplasmic protein gene of Pasteurella multocida. Description of reference numerals: Lane 1 in Figure 1: target gene amplified by cloning primers from the genome of Pasteurella multocida; Lane M in Figure 1: DNA molecular weight standard (purchased from Bao Bioengineering Dalian Co., Ltd.).

Figure 2: Amplification of the target fragment and vector electrophoresis using the enzyme digestion and ligation method. Explanation of reference numerals: Lane 1 in Figure 1: double-digested recombinant plasmid pET-30a-ThiB; Lane 2 in Figure 1: single-digested recombinant plasmid pET-30a-ThiB; Lane M in Figure 1: DNA molecular weight standard.

Figure 3: Map of the recombinant plasmid pET-30a-ThiB constructed by the present invention.

Figure 4: SDS-PAGE electrophoresis results for qualitative analysis of thiamine-bound periplasmic proteins of Pasteurella multocida. Description of reference numerals: Lane 1 in Figure 4: Unpurified rThiB; Description of reference numerals: Lane 2 in Figure 4: Purified rThiB; Description of reference numerals: Lane M in Figure 4: Protein molecular mass standard .

Figure 5: Results of the reactivity of P. multocida thiamine binding periplasmic protein recombinant protein rThiB with P. multocida HB03 positive sero. Description of reference numerals: Lane 1 in Figure 5: the recombinant protein of the present invention reacts with the positive serum of mice infected with Pasteurella multocida; Lane M in Figure 5: Protein molecular mass standard.

Figure 6: Results of the reactivity of P. multocida thiamine-binding periplasmic protein recombinant protein rThiB with P. multocida HN06 positive sera. Description of reference numerals: Lane 1 in Figure 6: the recombinant protein of the present invention reacts with the positive serum of mice infected with Pasteurella multocida; Lane M in Figure 6: Protein molecular mass standard.

Figure 7, Figure 8: Survival curves of animal challenge protection assay. Description of the reference numerals: Figures 7 and 8 are marked with the corresponding protection of the recombinant protein rThiB against D and F types of Pasteurella multocida and the survival status of the control group.

Figure 9: Antibody level status of mice in animal challenge protection assay.

Detailed ways

Explanation to the Sequence Listing:

Sequence Listing SEQ ID NO: 1 is the nucleotide sequence of a Pasteurella multocida thiamine-binding periplasmic protein gene ThiB cloned by the present invention, the sequence length is 1055bp, and 1-1055bp of the sequence is the protein The open reading frame (ORF, or CDS) sequence of a gene. Encoding 334 corresponding amino acid sequences.

Sequence Listing SEQ ID NO: 2 is the amino acid sequence encoded by the thiamine-binding periplasmic protein gene of Pasteurella multocida. Encodes 334 protein sequences.

The present invention will be described in detail below with reference to the embodiments.

Example 1: Cultivation of Pasteurella multocida

The lyophilized powder of Pasteurella multocida clinical strains (HB03 and HN06) isolated and preserved in our laboratory was inoculated on tryptic soy agar (TSA) medium containing 5% newborn bovine serum (purchased from American BD Company) , streaked with an inoculation loop and then placed in a 37°C incubator for 12h, picking a single colony in tryptic soy broth (TSB) (purchased from BD, USA) containing 5% newborn bovine serum at 37°C, Shake culture in a constant temperature shaker at 180rpm for 12-24h.

Example 2: Extraction of Pasteurella multocida genome

The cultured above-mentioned bacterial liquid was used for the extraction of genome, and the TIANGEN bacterial genomic DNA extraction kit was used to extract Pasteurella multocida HN06 genomic DNA, and the operation steps were carried out according to the kit instructions, specifically as follows:

(1) Take 5 mL of Pasteurella multocida HN06 bacterial solution, centrifuge at 10,000 r/min for 1 min, and aspirate the supernatant as much as possible.

(2) Add 200 μL of the buffer GA included in the kit to the cell pellet, and shake until the cells are completely suspended.

(3) Add 20 μL of the Proteinase K solution included in the kit to the tube, and mix well.

(4) Add 220 μL of buffer GB included in the kit, shake for 15 s, and place at 70° C. for 10 min. The solution should become clear, and centrifuge briefly to remove water beads on the inner wall of the tube cover.

(5) Add 220 μL of absolute ethanol, shake and mix well for 15 s. At this time, flocculent precipitation may appear. Briefly centrifuge to remove water droplets on the inner wall of the tube cover.

(6) Add the solution obtained in the previous step and the flocculent precipitate into the adsorption column CB3 that comes with the kit (put the adsorption column into the collection tube), centrifuge at 12000 r/min for 30s, pour off the waste liquid, and put the adsorption column CB3 into the collection tube.

(7) Add 500 μL of the buffer GD that comes with the kit to the adsorption column CB3, centrifuge at 12000 r/min for 30 s, discard the waste liquid, and put the adsorption column CB3 into the collection tube.

(8) Add 600 μL of the rinsing solution PW included in the kit to the adsorption column CB3 (please check whether absolute ethanol has been added before use), centrifuge at 12000r/min for 30s, pour out the waste liquid, and put the adsorption column CB3 into the collection in the tube.

(9) Repeat the operation of step (8).

(10) Put the adsorption column CB3 back into the collection tube, centrifuge at 12000 r/min for 2 min, and pour out the waste liquid. The adsorption column CB3 was placed at room temperature for 5 min to completely dry the residual rinse solution in the adsorption material.

(11) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50 μL of the elution buffer TE that comes with the kit to the middle part of the adsorption membrane, and place it at room temperature for 5 min. Centrifuge at 12000 r/min for 2 min. Collect into centrifuge tubes and store at -20°C until use.

Example 3: Construction of prokaryotic expression vector and inducible expression in Escherichia coli

Since Pasteurella multocida thiamine binds to periplasmic protein using Signal P4.1 software to predict that the first 22 amino acids in the protein amino acid sequence are signal peptide sequences, in order to facilitate prokaryotic expression, Pasteurella multocida thiamine was combined with The periplasmic protein gene was truncated and expressed, and its expression region was 67-1005bp.

The truncated fragment of the thiamine-binding periplasmic protein gene of Pasteurella multocida was ligated with the HIS tag vector pET-30a (purchased from Bao Bioengineering Dalian Co., Ltd.) by the method of enzymatic ligation.

(1) Primer design

Use Primer 5.0 software to design upstream and downstream primers:

Upstream primer F1: 5'-CGCGGATCCCAAACGCAAGCGGTCA-3';

Downstream primer R1: 5'-CCGCTCGAGTTATTGGGTTAGGGTCGTTT-3'.

(2) PCR amplification: The genome of Pasteurella multocida was used as the template, and the F1 and R1 primers were used for PCR amplification. The reaction system was 50 μL. The PCR reaction system was as follows:

PCR reaction conditions: 98°C for 5 min; 94°C for 45s, 56°C for 45s, 72°C for 1 min; 72°C for 10 min; a total of 30 cycles.

(3) Identification of PCR products: After the amplification is completed, take 5 μL of PCR products and mix them with 10× loading buffer for spotting, and observe the results after electrophoresis on 1.0% agarose gel, 1× TAE buffer, 120V for 30 minutes, and get target fragment (see the gel map shown in Figure 1).

(4) Connection and sequencing of target gene and T vector

Mix 1 μL (50ng/uL) of pMD19-T vector (purchased from Bao Bioengineering Dalian Co., Ltd.) with 4 μL of the target fragment recovered from the gel, add 5 μL of solution I (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and mix. Homogenize at 4°C overnight for ligation. Take 10 μL of the ligation product, add it to Escherichia coli DH5α competent cells under sterile conditions, slowly and repeatedly blow and mix with a pipette, and place in an ice bath for 30 min. Then use a 42°C water bath, heat shock for 90s, and then immediately cool in an ice bath for 3min (do not shake). Transfer the bacterial solution to an EP tube containing 600 μL of LB culture solution preheated to 37° C., and shake at 180 rpm at 37° C. for 45 minutes, so that the drug resistance gene on the vector can be expressed normally. Take 100 μL of bacterial solution and spread it evenly on a TSA plate containing ampicillin (AMP) (100 μg/mL). A single colony was inoculated into the LB medium containing 100 μg/mL AMP, shaken at 180 rpm and 37 °C for 12 to 16 hours, and then identified, that is, the positive clones were amplified by PCR, and the bacterial solution was sent to Shanghai Qingke Biotechnology Science and Technology Co., Ltd. performed sequencing analysis to obtain a 939bp genome sequence. This gene is the sequence of the signal peptide at the front of the open reading frame (ORF) full-length part. The full length of the sequence is a total of 939bp (67bp-1005bp). The (ORF) full-length sequence is shown in SEQ ID NO: 1 of the sequence listing.

Serial number: SEQ ID NO: 1

Sequence length: 1005bp

Source: Pasteurella multocida

Sequence characteristics: there is a correct open reading frame (ORF): 1005bp; decision position: the position where the start and stop codons exist: ATG, position 1; TAA, position 1005.

(5) The target gene was cut from the T vector, the restriction sites were BamHI and XhoI, the restriction system was 50 μL, and the reaction time was 2.5 h. At the same time, the pET-30a vector was digested with the same steps.

Enzyme cleavage reaction system:

(6) The ligation and sequencing of the target gene and the vector pET-30a

The gel recovery of the vector and the target fragment was carried out respectively, and the recovery concentration was determined. Take 1μL (7ng/μL) of pET-30a recovered product and mix with 7μL (40ng/μL) of ThiB recovered from gel, add 1μL T4 DNA Ligase (350ng/μL), 1μL 10×T4 DNA Ligase Buffer, and connect at 16°C after mixing. 45min. Take 10 μL of the ligation product, add it to Escherichia coli DH5α competent cells under sterile conditions, gently and repeatedly blow and mix with a pipette, and place in an ice bath for 30 min. 42°C water bath, heat shock for 90s, and then immediately ice bath for 2min to cool (be careful not to shake). Transfer the bacterial solution to 600 μL of LB medium preheated to 37° C., and gently shake at 180 rpm and 37° C. for 45 min to make the drug resistance gene on the vector express normally. Take 100uL of bacterial solution and spread it evenly on a TSA plate containing kanamycin (100μg/mL). Colonies were inoculated in LB medium containing 100 μg/mL kanamycin, and identified after 12-16 hours of constant temperature oscillation at 180 rpm and 37 °C. The positive clones were amplified by PCR, and the bacterial solution was sent to Shanghai Qingke Biotechnology Co., Ltd. for sequencing. Analysis yielded a genome sequence of 939 bp.

(7) Construction of pET-30a-ThiB expressing bacteria

The pET-30a-ThiB plasmid (1 μL) was added to Escherichia coli BL21 (DE3) competent cells (50 μL) under aseptic conditions, mixed with a pipette gently and repeatedly, and placed in an ice bath for 30 min. Then, in a water bath at 42° C., heat shock for 90 s, and then immediately cool in an ice bath for 2 min (be careful not to shake). Transfer the bacterial solution to 500 μL of LB culture solution preheated to 37°C, and shake at 150 rpm and 37 °C for 45 minutes to restore the resistance of bacteria; then take 150 μL of bacterial solution and spread it on kanamycin (KAN) (100 μg/mL) on LB agar plates. Invert the plate and incubate for 12-16h in a constant temperature incubator at 37°C, pick a single colony and inoculate it in LB medium containing 150μg/mL KAN, shake vigorously for 12-16h and identify it. At the same time, the pET-30a-ThiB empty vector without any foreign gene was transformed as a negative control.

Take 1 μL of bacterial solution for PCR, and the reaction system is 20 μL, as follows:

PCR reaction conditions: The reaction conditions were 94°C for 5 min; 94°C for 45s, 56°C for 45s, 72°C for 1 min; 72°C for 10 min; a total of 35 cycles.

Amplified positive clones can be used for prokaryotic expression of pET-30a-ThiB recombinant protein.

(8) Prokaryotic expression of recombinant protein rThiB

The positive pET-30a-ThiB/BL21(DE3) bacterial solution obtained above was added to the LB medium containing kanamycin at a volume ratio of 1:100, and cultured at 37°C and 200 rpm until the OD ₆₀₀ was about 0.6, adding 0.8 mmol/L IPTG, induced at 25°C for 14h. Collect a large amount of inducible bacterial liquid, centrifuge at 7000rpm for 15min at 4°C, discard the supernatant, add PBS (phosphate buffered saline, the formula is: 8.0g NaCl, 0.2g KCl, 1.56g Na2HPO4, 0.2g KH2PO4, add in order 800mL of double-distilled water, after completely dissolving, dilute to 1000.0mL, add HCl to adjust the pH to 7.8) After resuspending the bacterial cell precipitation, pressure crush 4 times; then centrifuge at 4°C, 12000rpm for 30min, discard the precipitate, and collect the supernatant , after filtration, the recombinantly expressed crude protein.

(9) Purification of recombinant crude protein

The obtained recombinant crude protein was purified by a nickel affinity chromatography column. The specific steps were as follows: take 5 mL of ddH ₂ O to pass through the column, wash away the absolute ethanol in the sealed column, then pass through the column with 5 mL of blinding buffer, take the broken liquid and pass it through the column. Column, after sample loading, equilibrate the column with 15mL lysis bufers, set the elution buffer according to 0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% Each of the 10 dilutions was prepared with 5 mL, passed through the column respectively, and finally passed through the column with 5 mL of blinding buffer and ddH ₂ O in sequence, and finally sealed the column with 5 mL of anhydrous ethanol. Take 40uL of 10 dilutions of the eluate to prepare protein electrophoresis samples, conduct SDS-PAGE electrophoresis, dialyze the eluate containing the target protein, concentrate, and store the purified recombinant protein at -80 °C for electrophoresis. The results are shown in Figure 4.

Example 4: Identification of Recombinant Protein Reactogenicity of Pasteurella multocida Thiamine-Binding Periplasmic Proteins

1) Identification of reactogenicity

The purified Pasteurella multocida thiamine-binding periplasmic protein recombinant protein (referred to as the recombinant protein of the present invention) was first subjected to SDS-PAGE electrophoresis, and then transferred to a PVDF membrane at a voltage of 100V, and the PVDF was transferred to the PVDF membrane after 40 minutes. The membrane was blocked with TBST-5% nonfat milk powder for 1 h, washed three times with TBST, 5 min each time; the PVDF membrane was placed in the 1:100 volume ratio of the mouse positive serum of Pasteurella multocida, incubated at room temperature for 1 h, and then washed with TBST. Wash 3 times with TBST for 5 min each; add secondary antibody (goat anti-mouse HRP-IgG diluted 1:5000 by volume, purchased from Abclone), incubate for 1 h at room temperature, and wash 3 times with TBST for 5 min each time , adding BCL (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.) to develop color. The results of Western blot assay showed that the purified recombinant protein of Pasteurella multocida thiamine-binding periplasmic protein could specifically react with the positive mouse serum of the capsular type A Pasteurella multocida strain HB03. (see Fig. 5), and also had a specific reaction with the positive mouse serum of the capsule D-type Pasteurella multocida strain HN06 (see Fig. 6).

Example 5: Mouse immune protection test of recombinant protein of Pasteurella multocida thiamine-binding periplasmic protein

Twenty clean 6-week Kunming rats were taken and divided into 4 groups with 5 rats in each group, and the groups were numbered as group A, group B, group C, and group D. At 0 d, blood was collected from the orbital vein of the mouse, and the purified recombinant protein of Pasteurella multocida thiamine-binding periplasmic protein (1 μg/mL) of the present invention was mixed and emulsified with an equal amount of Freund's complete adjuvant. Among them, each mouse in group A and group B was subcutaneously injected with 200uL protein adjuvant emulsion, and each mouse in group C and group D was subcutaneously injected with 200uL PBS (phosphate buffered saline). Orbital vein blood was collected, and the purified recombinant protein (1 μg/mL) of the present invention was mixed and emulsified with an equal amount of incomplete Freund’s adjuvant. Each mouse in group A and group B was subcutaneously injected with 200 μL protein adjuvant emulsion at multiple points. , each mouse in groups C and D were subcutaneously injected with _{200 μL} PBS (phosphate buffered saline) at multiple points. After 14 days, the mice underwent orbital vein blood collection. HN06 strain was challenged in groups B and D, and injected into mice by intraperitoneal injection, and the survival of mice was recorded. The results showed that all mice in groups A and B in this example survived, one in group C survived, and all mice in group D died. It is shown that the recombinant protein of the present invention can provide 100% protection to the challenged mice and has good immunogenicity (see Figure 7 and Figure 8 for the results).

Example 6: Detecting the antibody of the recombinant protein of the present invention by ELISA

In this example, the conventional square array titration method is used to determine the optimal coating concentration of the antigen. The specific steps are as follows: the purified recombinant protein of the present invention is sequentially diluted by 2 times starting from 8ug/mL with carbonate buffer (8ug/mL). /mL～0.25ug/mL), 3 wells for each dilution point, each well volume is 100μL, put it in a refrigerator at 4°C overnight, take it out the next day and wash it with PBST buffer 3 times, 3 min each time, each well Add 150 μL of 5% nonfat dry milk, block at 37 ° C for 1 h, discard the blocking solution in the well and wash 3 times, and use PBST buffer to dilute the recombinant protein immunized mouse serum and negative serum of the present invention in doubling (volume) respectively. The ratio is 1:200～1:12800), incubate at 37°C for 1h, wash 3 times, add 100uL of goat anti-mouse HRP-IgG enzyme-labeled secondary antibody (purchased from Abclone) diluted 5000 times to each well, and incubate at 37°C for 1h , wash 3 times, add 50uL of developer A and B in turn, incubate in the dark for 10min, add 50uL of H ₂ SO ₄ to develop color, measure the OD ₆₃₀ value with a microplate reader, and then divide the value of the positive hole by the negative hole Numerical value, select the dilution where the positive well with the largest quotient value is located as the optimal dilution concentration of the recombinant protein coating of the present invention, after determining the optimal coating concentration, detect the serum antibodies of the mice at 0d, 14d, and 28d after collecting the immune protein, The results are shown in Figure 9, indicating that the recombinantly expressed Pasteurella multocida thiamine-binding periplasmic protein can stimulate the body to produce higher levels of antibodies after immunizing mice.

sequence listing

<110> Huazhong Agricultural University

<120> A recombinantly expressed Pasteurella multocida thiamine periplasmic binding protein and its application

<141> 2020-07-09

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1005

<212> DNA

<213> Pasteurella multocida

<220>

<221> gene

<222> (1)..(1005)

<220>

<221> CDS

<222> (1)..(1005)

<400> 1

atg tct aga tta aaa act tcc ttt ttt ttc acc gca ctt tcc aca ctg 48

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

20 25 30

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

35 40 45

ttt gaa acc cat ttc cca caa tgc caa gtg aat ttt act gcg ttt ggt 192

Phe Glu Thr His Phe Pro Gln Cys Gln Val Asn Phe Thr Ala Phe Gly

50 55 60

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

65 70 75 80

aaa gcg gat gtg gtt gta ggg tta gat aac tac aac ctt gag gag gca 288

Lys Ala Asp Val Val Val Gly Leu Asp Asn Tyr Asn Leu Glu Glu Ala

85 90 95

gaa aaa agc ggc tta ttt gtt caa cat aaa gtc gat tta aca ccg ctg 336

Glu Lys Ser Gly Leu Phe Val Gln His Lys Val Asp Leu Thr Pro Leu

100 105 110

tca cta ccg gtt gag tgg aaa aat caa act ttt tta cct tat gat ttt 384

Ser Leu Pro Val Glu Trp Lys Asn Gln Thr Phe Leu Pro Tyr Asp Phe

115 120 125

ggg cag ttt gcc ttt att tat gac aaa acc aaa gtg caa cag cca cca 432

Gly Gln Phe Ala Phe Ile Tyr Asp Lys Thr Lys Val Gln Gln Pro Pro

130 135 140

aaa agc tta aaa gaa ttg gta gaa cgt gat gat tta aaa gtc att tat 480

Lys Ser Leu Lys Glu Leu Val Glu Arg Asp Asp Leu Lys Val Ile Tyr

145 150 155 160

caa gat cca cgt act agc agt gta gga cgt ggc tta ctc att tgg atg 528

Gln Asp Pro Arg Thr Ser Ser Val Gly Arg Gly Leu Leu Ile Trp Met

165 170 175

aac cat gtt tat acc gag aat gaa gtg gca caa gct tgg caa aaa tta 576

Asn His Val Tyr Thr Glu Asn Glu Val Ala Gln Ala Trp Gln Lys Leu

180 185 190

gcc aaa cat acg gtg act gtg ggg aaa ggt tgg tct gat act tat ggc 624

Ala Lys His Thr Val Thr Val Gly Lys Gly Trp Ser Asp Thr Tyr Gly

195 200 205

gcg ttt ttg aaa ggc gaa gct gat gtt gtc ttg agc tat aac acc tct 672

Ala Phe Leu Lys Gly Glu Ala Asp Val Val Leu Ser Tyr Asn Thr Ser

210 215 220

ccg tta tat cac atg gtg ttt gaa caa aaa gat cag tat ctc gcc acc 720

Pro Leu Tyr His Met Val Phe Glu Gln Lys Asp Gln Tyr Leu Ala Thr

225 230 235 240

gaa ttc gag gaa ggg ggc gta tta caa att gaa acg gca gca cgt gta 768

Glu Phe Glu Glu Gly Gly Val Leu Gln Ile Glu Thr Ala Ala Arg Val

245 250 255

gca caa cat gat aat cac tgt gcg gat cat ttc ttg gca ttt tta att 816

Ala Gln His Asp Asn His Cys Ala Asp His Phe Leu Ala Phe Leu Ile

260 265 270

cat cca gaa gca caa ggg cat tta gtc aag aat aat gtg atg tta ccg 864

His Pro Glu Ala Gln Gly His Leu Val Lys Asn Asn Val Met Leu Pro

275 280 285

gtg att aat acc aat att gaa ccg cac ttt gat gcc ctt aaa gcc acc 912

Val Ile Asn Thr Asn Ile Glu Pro His Phe Asp Ala Leu Lys Ala Thr

290 295 300

caa atg aac acg aaa gtg ctc gat acc tca aaa gta aat gcc gaa caa 960

Gln Met Asn Thr Lys Val Leu Asp Thr Ser Lys Val Asn Ala Glu Gln

305 310 315 320

gtc aaa aaa tgg att gct gtt tgg caa acg acc cta acc caa taa 1005

Val Lys Lys Trp Ile Ala Val Trp Gln Thr Thr Leu Thr Gln

325 330

<210> 2

<211> 334

<212> PRT

<213> Pasteurella multocida

<400> 2

Met Ser Arg Leu Lys Thr Ser Phe Phe Phe Thr Ala Leu Ser Thr Leu

1 5 10 15

Ser Leu Ser Val Phe Ala Gln Thr Gln Ala Val Asn Val Tyr Thr Tyr

20 25 30

Asp Ser Phe Thr Ser Glu Trp Gly Ala Gly Pro Lys Val Lys Lys Ala

35 40 45

Phe Glu Thr His Phe Pro Gln Cys Gln Val Asn Phe Thr Ala Phe Gly

50 55 60

Asp Ser Gly Thr Met Phe Asn Arg Leu Arg Leu Glu Gly Lys Lys Thr

65 70 75 80

Lys Ala Asp Val Val Val Gly Leu Asp Asn Tyr Asn Leu Glu Glu Ala

85 90 95

Glu Lys Ser Gly Leu Phe Val Gln His Lys Val Asp Leu Thr Pro Leu

100 105 110

Ser Leu Pro Val Glu Trp Lys Asn Gln Thr Phe Leu Pro Tyr Asp Phe

115 120 125

Gly Gln Phe Ala Phe Ile Tyr Asp Lys Thr Lys Val Gln Gln Pro Pro

130 135 140

Lys Ser Leu Lys Glu Leu Val Glu Arg Asp Asp Leu Lys Val Ile Tyr

145 150 155 160

Gln Asp Pro Arg Thr Ser Ser Val Gly Arg Gly Leu Leu Ile Trp Met

165 170 175

Asn His Val Tyr Thr Glu Asn Glu Val Ala Gln Ala Trp Gln Lys Leu

180 185 190

Ala Lys His Thr Val Thr Val Gly Lys Gly Trp Ser Asp Thr Tyr Gly

195 200 205

Ala Phe Leu Lys Gly Glu Ala Asp Val Val Leu Ser Tyr Asn Thr Ser

210 215 220

Pro Leu Tyr His Met Val Phe Glu Gln Lys Asp Gln Tyr Leu Ala Thr

225 230 235 240

Glu Phe Glu Glu Gly Gly Val Leu Gln Ile Glu Thr Ala Ala Arg Val

245 250 255

Ala Gln His Asp Asn His Cys Ala Asp His Phe Leu Ala Phe Leu Ile

260 265 270

His Pro Glu Ala Gln Gly His Leu Val Lys Asn Asn Val Met Leu Pro

275 280 285

Val Ile Asn Thr Asn Ile Glu Pro His Phe Asp Ala Leu Lys Ala Thr

290 295 300

Gln Met Asn Thr Lys Val Leu Asp Thr Ser Lys Val Asn Ala Glu Gln

305 310 315 320

Val Lys Lys Trp Ile Ala Val Trp Gln Thr Thr Leu Thr Gln

325 330

Claims (4)

1. A Pasteurella multocida thiamine-binding periplasmic protein gene, characterized in that the nucleotide sequence of the gene is shown in SEQ ID NO: 1. 2. A Pasteurella multocida thiamine-binding periplasmic protein gene, characterized in that the protein sequence encoded by the gene is shown in SEQ ID NO: 2. 3. The application of the Pasteurella multocida thiamine-binding periplasmic protein gene of claim 1 in the preparation of a Pasteurella multocida subunit vaccine. 4. Application of the Pasteurella multocida thiamine-binding periplasmic protein gene of claim 2 in the preparation of a Pasteurella multocida subunit vaccine.

Images