Brucella melitensis polysaccharide conjugate vaccine and application thereof
1. The recombinant strain is obtained by introducing an expression vector into O9 serogroup enterocolitis yersinia; the expression vector contains an expression cassette A of Neisseria meningitidis glycosyltransferase PglL and an expression cassette B of cholera toxin B subunit fusion protein; the expression cassette B is located at the downstream of the expression cassette A; the cholera toxin B subunit fusion protein comprises a cholera toxin B subunit and a truncation of neisseria meningitidis pilin PilE; the truncation contains a glycosylation recognition site of Neisseria meningitidis glycosyltransferase PglL.
2. The recombinant bacterium according to claim 1, wherein: the O9 serogroup enterocolitis yersinia is enterocolitis yersinia 52212.
3. The recombinant bacterium according to claim 1 or 2, wherein:
the N-terminal amino acid residues are formed by the N-terminal polypeptide.
4. The recombinant bacterium according to claim 3, wherein: the cholera toxin B subunit fusion protein is a protein shown in SEQ ID No. 4.
5. A glycoprotein expressed by the recombinant bacterium of any one of claims 1 to 4.
6. The glycoprotein of claim 5 for use as a Brucella bovis vaccine.
7. The recombinant bacterium of any one of claims 1 to 4, or the use of the glycoprotein of claim 5, which is (a1) or (a2) or (a 3):
(a1) preparing a brucella vaccine of the cattle species;
(a2) preventing and/or treating brucella infection in cattle;
(a3) preparing a product for preventing and/or treating brucella abortus infection.
8. A Brucella vaccine comprising the glycoprotein according to claim 5 as an active ingredient.
9. The vaccine of claim 8, wherein: the vaccine also includes an immunological adjuvant.
10. A product for preventing and/or treating Brucella infection in cattle, which comprises the glycoprotein according to claim 5 as an active ingredient.
Background
Brucellosis is a common disease of people and animals which is popular in the world and frequently occurs in China, and causes huge economic loss every year. Currently, brucella vaccines are mainly attenuated live vaccines for animals, which cause abortion in female animals and infection of human beings, and no safe human vaccine exists. Brucella abortus, also known as Brucella abortus, belongs to a third-level biosafety-threatening organism, and mainly infects cattle and humans.
Disclosure of Invention
The invention aims to provide a brucellosis polysaccharide conjugate vaccine for cattle and application thereof.
In the first aspect, the recombinant strain is obtained by introducing an expression vector into Yersinia enterocolitica of O.9 serogroup; the expression vector contains an expression cassette A of Neisseria meningitidis glycosyltransferase PglL and an expression cassette B of cholera toxin B subunit fusion protein; the expression cassette B is located at the downstream of the expression cassette A; the cholera toxin B subunit fusion protein comprises a cholera toxin B subunit and a truncation of neisseria meningitidis pilin PilE; the truncation contains a glycosylation recognition site of Neisseria meningitidis glycosyltransferase PglL.
The yersinia enterocolitis of O9 serogroup may be yersinia enterocolitis 52212.
The truncation of the neisseria meningitidis pilin PilE can be specifically a polypeptide formed by the neisseria meningitidis pilin PilE from 45 th to 73 th amino acid residues at the N terminal.
The cholera toxin B subunit fusion protein is a protein shown in SEQ ID No. 4.
In the protein shown in SEQ ID No.4, the 1 st to 19 th positions from the N terminal are a DsbA signal peptide sequence, the 20 th to 122 th positions are an amino acid sequence of CTB (cholera toxin B subunit), the 123 th to 127 th positions are flexible linkers, and the 128 th to 156 th positions are amino acids 45 to 73 th positions of neisseria meningitidis pilin PilE. The serine at the 63 th site of the neisseria meningitidis pilin PilE (namely the 146 th site of SEQ ID No. 4) is a glycosylation site recognized by the neisseria meningitidis glycosyltransferase PglL.
The expression cassette B can be specifically shown as the 103-bit 921 th nucleotide from the 5' end of SEQ ID No. 3. Wherein, the 103-131 th nucleotide from the 5' end of SEQ ID No.3 is the sequence of the tac promoter, and the 178-645 th nucleotide is the coding sequence of the cholera toxin B subunit fusion protein.
The Neisseria meningitidis glycosyltransferase PglL can be specifically protein shown as SEQ ID No. 2.
The expression cassette A can be specifically shown as nucleotides 105 and 2240 from the 5' end of SEQ ID No. 1. Wherein, the 105 th and 133 th nucleotides from the 5' end of SEQ ID No.1 are the sequence of the tac promoter, and the 180 th and 1994 th nucleotides are the coding sequence of Neisseria meningitidis glycosyltransferase PglL.
The expression vector can be specifically an expression vector obtained by replacing a fragment between SacI and XhoI enzyme cutting sites of a recombinant vector pET28-tacpglL by a DNA molecule shown in the 7 th-1068 th position from the 5' end of SEQ ID No. 3. The recombinant vector pET28-tacpglL is a recombinant vector obtained by replacing a fragment between XbaI and XhoI cleavage sites of pET28a (+) vector with a DNA molecule shown in the 7 th to 2485 th positions from the 5' end of SEQ ID No. 1.
In a second aspect, the present invention protects the glycoprotein expressed by the recombinant bacteria described above.
The preparation method of the glycoprotein specifically comprises the following steps: (1) culturing the recombinant strain, inducing protein expression by IPTG, and centrifuging the culture system to collect the strain after induction; (2) resuspending the thalli, carrying out ultrasonic bacteria breaking, and centrifuging to collect supernatant (containing glycoprotein); (3) and (3) purifying the supernatant obtained in the step (2) by a nickel column and a molecular sieve, collecting a purified sample, and concentrating to obtain the glycoprotein.
In a third aspect, the present invention protects the use of the glycoprotein described hereinbefore as a vaccine against brucella bovis.
In a fourth aspect, the invention protects the application of the recombinant bacterium or glycoprotein described above, which is (a1) or (a2) or (a 3):
(a1) preparing a brucella vaccine of the cattle species;
(a2) preventing and/or treating brucella infection in cattle;
(a3) preparing a product for preventing and/or treating brucella abortus infection.
In a fifth aspect, the invention provides a vaccine for protecting brucella bovis, wherein the effective component is the glycoprotein described above.
The vaccine may also include an immunological adjuvant. The immunological adjuvant may specifically be an aluminium hydroxide adjuvant. The volume percentage content of the aluminum hydroxide adjuvant in the vaccine can be 10%.
In a sixth aspect, the present invention provides a product for preventing and/or treating brucella bovis infection, wherein the active ingredient is the glycoprotein described above.
The Brucella melitensis can be specifically Brucella melitensis vaccine strain A19.
The invention expresses an exogenous O-glycosylation system in O9 serogroup enterocolitis yersinia, leads O-antigen polysaccharide of the O9 serogroup enterocolitis yersinia to be covalently linked to a substrate protein cholera toxin B subunit, and takes host bacteria as glycosylation engineering bacteria to synthesize the polysaccharide conjugate vaccine. The glycoprotein has the same polysaccharide structure as the Brucella melitensis, and is expected to have the protection effect on the Brucella melitensis in immune animals. The O9 serogroup enterocolitis yersinia belongs to a secondary biosafety harm organism, and is easier to operate and culture than the bovine brucella, so that the O9 serogroup enterocolitis yersinia is selected as a host bacterium, the more dangerous bovine brucella in large-scale culture is avoided, and the production process is safer and more efficient.
Drawings
FIG. 1 shows protein expression verification.
FIG. 2 shows the result of Coomassie blue staining after passing glycoprotein through molecular sieves.
FIG. 3 shows the serum specificity and size validation of glycoproteins.
FIG. 4 shows the detection of serum IgG titer in immunized mice.
FIG. 5 is a graph showing the measurement of serum TNF-. alpha.expression levels after infection of immunized mice with a non-lethal dose of A19.
FIG. 6 is a spleen weight and bacterial load measurements after infection of immunized mice with a non-lethal dose A19.
FIG. 7 is a graph of the survival of immunized mice challenged with a lethal dose of A19.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
pET28a (+) vector: novagen Inc.
Yersinia enterocolitica 52212 is described in the literature: detection of yersinia enterocolitica, Zhang Xin, Zhang Hui Yuan, Wan Qi, et al [ J ] inspection and quarantine science, 2008,18(6): 23-26%; the public can be obtained from the military medical research institute of military science institute of the national people liberation military. Yersinia enterocolitis 52212 was detected as O:9 Yersinia enterocolitis serogroup.
The Brucella vaccine strain A19 is described in the following references: tanpofei [1], Nanwenlong [1], Pengchun [2], et al, research on SNP site of Brucella vaccine strain A19 of Chinese cattle [ J ]. J.Chinese veterinary J.2014, 48(7): 1-5.; the public can be obtained from the military medical research institute of military science institute of the national people liberation military.
Example 1 expression vector construction
First, construction of expression vector pET28-tacpglL-tacCTB4573
1. Artificially synthesizing a DNA molecule shown in SEQ ID No. 1. In SEQ ID No.1, the 1 st to 6 th nucleotides from the 5' end are XbaI recognition sites, the 105 th and 2240 th nucleotides are the sequence of a Neisseria meningitidis glycosyltransferase PglL expression cassette, wherein the 105 th and 133 th nucleotides are the sequence of a tac promoter, the 180 th and 1994 th nucleotides are the coding sequence (encoding the protein shown in SEQ ID No. 2) of the Neisseria meningitidis glycosyltransferase PglL, the 2475 th and 2480 th nucleotides are SacI recognition sequences, and the 2486 th and 2491 th nucleotides are XhoI recognition sites.
2. XbaI and XhoI are used for enzyme digestion of the DNA molecule shown in SEQ ID No.1 to obtain a gene fragment; using XbaI and XhoI to cut pET28a (+) vector to obtain vector large fragment; the gene fragment is connected with the large fragment of the vector to obtain a recombinant vector pET 28-tacpglL. Based on the sequencing results, the recombinant vector pET28-tacpglL was described as follows: the fragment between the XbaI and XhoI cleavage sites of the pET28a (+) vector was replaced by the DNA molecule shown in SEQ ID No.1 from positions 7-2485 of the 5' end.
3. Artificially synthesizing a DNA molecule shown in SEQ ID No. 3. In SEQ ID No.3, the 1 st to 6 th nucleotides from the 5' end are Sac I recognition site, the 103 nd-131 nd nucleotides are tac promoter sequence, the 178 nd-645 th nucleotides are recombinant protein rCTB4573 coding sequence (coding protein shown in SEQ ID No. 4), and the 1069 nd-1074 th nucleotides are XhoI recognition sequence.
In SEQ ID No.4, the 1 st to 19 th positions from the N-terminus are the DsbA signal peptide sequence, the 20 th to 122 th amino acid sequences of CTB (cholera toxin B subunit), the 123 th to 127 th positions are flexible linkers, and the 128 th to 156 th positions are the 45 th to 73 th amino acids of the pilin PilE of Neisseria meningitidis. The serine at the 63 th site of the neisseria meningitidis pilin PilE (namely the 146 th site of SEQ ID No. 4) is a glycosylation site recognized by the neisseria meningitidis glycosyltransferase PglL.
4. Amplifying a DNA molecule shown in SEQ ID No.3 by using a primer pair consisting of a primer SacI-UP and a primer XhoI-Down, and then performing double-enzyme digestion on the amplified product by using SacI and XhoI respectively to obtain a DNA fragment; carrying out double enzyme digestion on the recombinant vector pET28-tacpglL by SacI and XhoI to obtain a large vector fragment; the gene fragments were ligated with the vector fragments to obtain a recombinant plasmid, which was designated as pET28-tacpglL-tacCTB 4573.
SacI-UP：5’-gagctctgcataattcgtgtcgctcaaggcgcactc-3’；
XhoI-Down:5’-ctcgagcggccatcgatggaaaactgcagggcagtt-3’。
Based on the sequencing results, recombinant plasmid pET28-tacpglL-tacCTB4573 was described as follows: the fragment between the SacI and XhoI cleavage sites of the recombinant vector pET28-tacpglL was replaced by the DNA molecule shown in SEQ ID No.3 from position 7 to 1068 of the 5' end.
Second, construction of expression vector pET28-tacCTB4573
Amplifying a DNA molecule shown in SEQ ID No.3 by using a primer pair consisting of a primer XbaI-UP and a primer XhoI-Down, and then performing double-enzyme digestion on the amplified product by using XbaI and XhoI respectively to obtain a DNA fragment (SEQ ID No. 5); the only difference between this fragment and SEQ ID No.3 is that 5, the 1 st to 6 th nucleotides of the terminus are Xba I recognition sites, and the rest are identical and encode the recombinant protein rCTB4573 as a control. Using XbaI and XhoI to cut pET28a (+) vector to obtain vector large fragment; the gene fragment is connected with the large fragment of the vector to obtain a recombinant vector pET28-tacCTB 4573.
XbaI-UP：5’-tctagatgcataattcgtgtcgctcaaggcgcactc-3’；
XhoI-Down:5’-ctcgagcggccatcgatggaaaactgcagggcagtt-3’。
Based on the sequencing results, recombinant plasmid pET28-tacCTB4573 was described as follows: the fragment between the XbaI and XhoI cleavage sites of the pET28a (+) vector was replaced by the DNA molecule shown in positions 7-1068 from the 5' end in the order of SEQ ID No. 5.
Example 2 construction of recombinant bacterium
Preparation of competent cells of Yersinia enterocolitica 52212
Yersinia enterocolitica 52212 was streaked onto a BHI solid medium plate, cultured in an incubator at 25 ℃ for 24 hours, and single colonies were selected and inoculated into 5mL of BHI liquid medium, followed by overnight culture at 220rpm/min on a shaker at 25 ℃. Inoculating bacterial liquid 1:100 into 50mL of liquid BHI culture medium, culturing at 25 deg.C and 220rpm/min for 4-5h to make bacterial liquid OD600The bacterial liquid is placed on ice for ice bath for 20min when the bacterial liquid reaches about 0.5-0.6. The cells were collected by centrifugation at 5000 Xg/min for 10min at 4 ℃ and resuspended in 10% glycerol after pre-cooling and sterilization, centrifuged again, the supernatant discarded and the procedure repeated three times. After the last supernatant was discarded, the cells were resuspended in 500. mu.L of 10% glycerol and competent for 50. mu.L/tube, and could be stored at-80 ℃.
Second, construction of recombinant bacteria
The recombinant plasmid pET28-tacpglL-tacCTB4573 was introduced into the competent cells constructed in step one by the electrotransformation method, and 800. mu.L of BHI liquid medium was added, followed by culturing at 25 ℃ in a shaker at 220rpm/min for 4 hours, and all were plated with Kan (kanamycin) -resistant BHI solid culture plates to a final concentration of 50. mu.g/mL. Culturing at 25 deg.C for 24 hr. The single clone that grows out is 52212/pET28-tacpglL-tacCTB 4573.
The recombinant plasmid pET28-tacCTB4573 was introduced into the competent cells constructed in step one by the electrical transformation method, and 800. mu.L of BHI liquid medium was added thereto, followed by culturing at 25 ℃ in a shaker at 220rpm/min for 4 hours, and all were plated with Kan (kanamycin) -resistant BHI solid culture plates to a final concentration of 50. mu.g/mL. Culturing at 25 deg.C for 24 hr. The single clone that grows out is 52212/pET28-tacCTB 4573.
Example 3 expression, purification and detection of proteins
One, protein expression
The strains to be detected are as follows: 52212/pET28-tacpglL-tacCTB4573, 52212/pET28-tacCTB4573, Yersinia enterocolitica 52212.
1. Inoculating strains to be detected 52212/pET28-tacpglL-tacCTB4573 and 52212/pET28-tacCTB4573 into 5mL Kan liquid BHI culture medium with the final concentration of 50 mu g/mL, inoculating yersinia enterocolitica 52212 into non-resistant liquid BHI culture medium, carrying out overnight culture at 25 ℃ and 220rpm/min, respectively inoculating 5mL Kan liquid BHI culture medium with the final concentration of 50 mu g/mL and non-resistant liquid BHI culture medium according to the volume ratio of 1:100 the next day, and carrying out OD culture at 25 ℃ and 220rpm/min for 4-5h to ensure that the bacterial liquid OD600Reaching about 0.6-0.8.
2. After completing step 1, adding into the culture systemAdding 5 μ L IPTG, culturing overnight at 25 deg.C, centrifuging 1.5ml bacterial culture solution 5000 Xg/min for 10min, collecting thallus, and adding 100 μ L ddH2And O, resuspending the thallus.
3. To the resuspended bacterial solution in step 2, 100. mu.L of 2 XSDS Loading Buffer (32g SDS, 6.17g DTT, 0.4g bromophenol blue, 160mL glycerol, 100mL 1mol/LpH6.8 Tris-HCl, ddH2O to volume of 1L) was added, mixed well and subjected to boiling water bath for 10 min. Then, 8000 Xg/min, 10min centrifugation, and supernatant fluid is taken.
4. Taking 10 mu L of the supernatant obtained in the step 3 as a sample to be detected, carrying out electrophoresis by using 12% SDS-PAGE gel, after the bromophenol blue gel comes out, staining for 24h by using Coomassie brilliant blue staining solution (1.2g G-250, 510mL of 40% ammonium sulfate, 408mL of anhydrous methanol, 36mL of phosphoric acid and ddH2O for constant volume of 1L), and then decolorizing for 24h by using decolorizing solution (glacial acetic acid solution with volume concentration of 0.5%). The same sample was subjected to electrophoresis on 12% SDS-PAGE, transferred to PVDF membrane at 15V for 1 hour by a Bio-RadWB membrane transfer apparatus, and detected by using murine HRP-Anti-His tag monoclonal antibody (antibody is Abmart product, cat # M20020).
The results are shown in FIG. 1. The results show that, when the whole bacteria sample is subjected to WB detection after SDS-PAGE, when PglL and CTB4573 are co-expressed, the O-antigen polysaccharide of the bacteria is transferred to the carrier protein CTB4573 under the catalysis of glycosyltransferase PglL (polysaccharide and carrier protein CTB4573 are connected to glycosylation sites through covalent bonds), so that upward migration of molecular weight can be seen in a WB diagram, and in addition, because the bacterial polysaccharide is in a tandem repeat structure and O-antigen polysaccharide of Yersinia colitis-producing serovar O9 is a homopolymer, a 35-40kDa band is shown, which indicates that the protein is glycosylated and is combined with the bacterial polysaccharide, and when the carrier without PglL is used as a control, only the carrier protein is expressed, the size is about 15kDa and is slightly lower than the glycosylated protein, which indicates that the substrate protein is fully glycosylated when the PglL is expressed; yersinia enterocolitica 52212, which does not express any vector, does not have any bands.
II, glycoprotein purification
1. Inoculating 52212/pET28-tacpglL-tacCTB4573 with 7L liquid BHI medium, culturing at 25 deg.C and 220rpm/min to bacterial liquid OD600And reaches 0.About 6-0.8.
2. After the step 1 is completed, 1mL of 1M/L IPTG is added into each 1L culture system for induction, the culture is continued overnight at 25 ℃, and then the bacteria are collected by centrifugation at 5000 Xg/min for 10 min.
3. Resuspend the cells from step 2 with about 200mL of BufferA1(100mL of 5mol/LNaCl, 20mL of 1mol/LpH7.5 Tris-HCl, 10mL of 1mol/L imidazole, volume fixed to 1L with distilled water, pH adjusted to 7.0 with concentrated HCI), sonicate in an ice water bath (sonication time 3 hours for 5s at rest, 5s at rest), then centrifuge at 4 ℃ for 10min at 8,000 Xg/min to collect the supernatant and repeat once. The supernatant is the sample.
4. And (3) purifying the sample obtained in the step (3), wherein the specific method comprises the following steps:
(1) installing a nickel column (the filler of the nickel column is GE Healthcare product, the product number is 42068700), opening a liquid chromatograph, setting the flow rate to be 4mL/min, and firstly washing 4-5 column volumes by using 0.5M NaOH solution; then washing the column bed with distilled water until the pH value is neutral and stabilizing the column volume by about 5 columns; with 0.5M NiSO4Washing about 3 column volumes, repeatedly washing the column bed with distilled water again until the pH is neutral, and stabilizing about 5 column volumes; the bed was washed with Buffer B1(100mL of 5mol/L NaCl, 20mL of 1mol/L pH7.5 Tris-HCl, 500mL of 1mol/L imidazole, distilled water to 1L, pH adjusted to 7.0 with concentrated HCl) for about 1-2 column volumes, and finally equilibrated with Buffer A1 for about 5 column volumes. The sample was loaded to completely pass through the nickel column. The bed was further flushed with Buffer a1 until the uv absorbance and ionic strength values had decreased to a plateau and elution was continued for 5-6 column volumes. Then eluting with Buffer B1, collecting eluate, verifying, and centrifuging with ultrafiltration tube (the ultrafiltration tube is product of Merk company, and has a product number of UFC901096) with a cut-off molecular weight of 10kDa at 4000 Xg for concentrating to a final volume of 10 mL.
(2) Passing the eluent concentrated in the step (1) through a molecular sieve (the filler is Superdex)TM200 prep grade, GE Healthcare product, cat #: 10270287) was further separated, and the mobile phase was 1 XPBS (8g NaCl, 1.44g Na)2HPO4，0.2g KCI，0.24g KH2PO4And the volume is adjusted to 1L by using distilled water). Collection of the ultraviolet absorption valueThe effluent at the time of significant elevation was separated by SDS-PAGE and verified by Coomassie blue staining.
The results are shown in FIG. 2. In FIG. 2, the left panel shows the time of the molecular sieve peak, and the right panel shows the Coomassie blue staining results of the samples collected during the peak discharge. The results show that the glycoprotein peaks at about 100mL after loading, and the peak is about 120mL, so that relatively pure glycoprotein can be obtained after purification. Collecting the sample with higher purity, concentrating, and quantifying sugar, and naming the purified glycoprotein as C-OPSBa。
For purified glycoprotein C-OPSBQuantification was performed in the following manner: preparing a 100mg/mL glucose solution with distilled water, mixing the solution with distilled water 1: a1000 dilution of 100. mu.g/mL glucose solution was used as the mother liquor. And (3) sucking 50, 100, 200, 300, 400, 600 and 800 mu L of glucose mother liquor by using a pipette gun, adding the glucose mother liquor into a small test tube, sequentially adding 950, 900, 800, 700, 600, 400 and 200 mu L of distilled water, namely glucose standard solutions with the concentrations of 5, 10, 20, 30, 40, 60 and 80 mu g/mL, and sucking 1mL of distilled water by using the pipette gun, adding the distilled water into the small test tube, namely a standard product with the concentration of 0. A sample such as glycoprotein is diluted with distilled water to a final volume of 1mL and added to a small tube. 4mL of a 2mg/mL anthrone-sulfuric acid solution was added quickly, immediately followed by insertion into an ice bath until the liquid was completely cooled. The test tube cap was covered, the test tube was placed in a boiling water bath for 10min, and after removal, the tube was again ice-cooled until the temperature of the liquid was reduced to room temperature. Adding the standard substance and the sample into a 96-well plate at 200 muL/well, reading at 620nm by using a spectrophotometer (enzyme-linked immunosorbent assay), drawing a standard substance concentration-light absorption value curve according to the result, and calculating the sample concentration according to the standard curve, wherein the standard curve R2 > 0.99 can be used for calculating the sample concentration.
The quantitative determination of the sugar after purification of the glycoprotein was 44.74. mu.g/mL. The average yield was about 150. mu.g sugar/L medium.
Third, glycoprotein specificity and size characterization
And (3) mixing the glycoprotein prepared in the second step with 2 xSDS Loading Buffer 1:1, carrying out boiling water bath for 10min, carrying out SDS-PAGE, carrying out Coomassie brilliant blue staining and WB respectively, and detecting by using a murine HRP-Anti-His tag monoclonal antibody (the antibody is an Abmart product, the product number is M20020). Meanwhile, in order to prove that the polysaccharide in the glycoprotein has cross over on the serum of both the Brucella and the Yersinia enterocolitica of O:9 serogroup, WB is carried out by using a Brucella monoclonal antibody (antibody is Thermo Fisher product, cat # TD2548562) and a Yersinia enterocolitica monoclonal antibody of O:9 serogroup (antibody is Fitzgerald product, cat # 10R-Y100 a).
The results are shown in FIG. 3A and show that the purified glycoprotein clearly shows a large glycosylation band, most of the contaminating proteins are removed, and the glycoprotein can cross-react with the sera of Brucella bovis (Anti B.a) and Yersinia enterocolitica O:9 (Anti Y.e), showing a glycosylation band at 35-40kDa, and the lower glycoprotein band is not shown, probably because of the less polysaccharide binding in the low molecular weight glycoprotein.
Since SDS-PAGE gels disaggregate the dimeric protein, whereas CTB is in pentameric form in nature, to verify whether glycosylated CTB is also present in the multimeric form, it was verified using non-reducing electrophoresis and stained with coomassie brilliant blue.
The results are shown in FIG. 3B. The results showed that the size of the glycoprotein was around 242kDa and the monomer size was around 35-40kDa, indicating that the substrate protein CTB was still present as a polymer after glycosylation.
Four, LPS and OPS extraction
1. Yersinia enterocolitica 52212 was streaked on BHI plates, cultured at 25 ℃ for 24 hours, and a single clone was picked up and inoculated into 5mLBHI liquid medium, and cultured overnight at 25 ℃ and 220 rpm/min.
2. After the step 1 is finished, the bacterial liquid 1:100 is inoculated in 1L BHI liquid culture medium, cultured for 16-18h at 25 ℃ and 220rpm/min, and centrifuged at 6000 Xg/min at 4 ℃ for 10min to collect thalli.
3. After completion of step 2, the cells were resuspended in ice-precooled distilled water, centrifuged again at 6000 Xg/min at 4 ℃ for 10min, the supernatant discarded and the procedure repeated three times. And finally, discarding the supernatant for the last time, weighing the wet weight (g) of the thallus, re-suspending the thallus by using distilled water which is 3 times of the wet weight of the thallus, carrying out ice bath for 3min, immediately placing the thallus in a water bath at 68 ℃ for 3min, and repeating the step for three times.
4. After the step 3 is completed, 90% phenol with the same volume is added, the mixture is shaken for 30min at 220rpm/min in a water bath at 68 ℃, centrifuged for 15min at 10000 Xg/min and 4 ℃, and the upper aqueous phase is absorbed. Adding distilled water with the same volume as the absorbed water phase into the rest precipitate and phenol phase part, shaking vigorously and mixing, repeating the above step, mixing the water phases obtained twice, adding dialysis bag with molecular weight cutoff of 3500Da, dialyzing with distilled water for three days to remove phenol, and changing water every 8 h. The crude LPS is obtained. Adding DNase with the final concentration of 5 mug/mL and RNase with the final concentration of 1 mug/mL into LPS, incubating for 3h at 37 ℃, adding Proteinase K with the final concentration of 10 mug/mL, incubating for 1h at 68 ℃, and finally boiling in a water bath for 10min to obtain the relatively pure LPS.
5. Adding glacial acetic acid with the final concentration of 1% (volume percentage content) into the LPS obtained in the step 4, carrying out boiling water bath for 90min, adjusting the pH to 7.0 by using NaOH solution, then centrifuging for 5h at 40000 Xg/min, and collecting the supernatant, namely the OPS.
6. The LPS and OPS obtained in step 4 and step 5 were subjected to sugar quantification.
The LPS quantification of Yersinia enterocolitica 52212 was 447.30. mu.g/mL, and the OPS quantification was about 204.31. mu.g/mL, and was designated as OPSBa。
Example 4 animal immunization experiment
Experimental animals: SPF grade female 5-6 week old BALB/c mice, purchased from Witongli.
1. The C-OPS prepared in example 3BaAnd OPSBaDiluted with 1 XPBS to a concentration of 2.5. mu.g/100. mu.L, respectively, to give C-OPSBaSolutions and OPS solutions.
2. To the C-OPS obtained in step 1BaSolutions and OPSBaAluminum hydroxide adjuvant (product of Invivo Gen, cat # 5569) was added to the solution to obtain C-OPSBa+ Al solution and OPSBa+ Al solution. Aluminum hydroxide adjuvant to C-OPSBa+ Al solution and OPSBaThe volume percentage content of the + Al solution is 10 percent.
3. Experimental animals were housed in 10 cages, and mice were administered by intraperitoneal injection on day 1 (primary), day 15 (secondary), and day 29 (tertiary) in groups of 20 mice each.
PBS group: each mouse 100 u L1 x PBS.
OPSBaGroup (2): mu.L OPS per mouseBaAnd (3) solution.
OPSBa+ Al group: mu.L OPS per mouseBa+ Al solution.
C-OPSBaGroup (2): each mouse 100 u L C-OPSBaAnd (3) solution.
C-OPSBa+ Al group: each mouse 100 u L C-OPSBa+ Al solution.
4. Serum potency detection
On day 10 after the triple immunization in step 3, 10 mice tail blood were taken per group to isolate serum. A96-well plate is coated with LPS (the preparation method of the LPS is the same as that of the LPS in the example 3) of the bovine brucella vaccine strain A19, and the mouse serum titer is measured by ELISA (enzyme-linked immunosorbent assay) by using donkey anti-mouse IgG marked by HRP (horse radish peroxidase) as a secondary antibody (the antibody is an Abcam product, and the product has the code number of GR 160830-14).
The results are shown in FIG. 4. The results show that OPS is comparable to the PBS groupBaGroup and C-OPSBaIgG titer of the immune group serum against the A19 strain LPS is obviously improved, and C-OPS is also improvedBaThe group rise is more significant and is more pronounced than the OPSBaThere was some difference in the groups, but the presence or absence of adjuvant had essentially no effect on serum titers.
5. Determination of serum inflammatory factor after non-lethal A19 infection
Streaking a Brucella vaccine strain A19 of a cattle species on a TSA plate, culturing for 2-3 days at 37 ℃ in an incubator, selecting a single clone, inoculating the single clone on 5mL of TSB liquid culture medium, culturing for 24h at 37 ℃ and 220rpm/min, and then 1:100 transfer to 5ml LTSB liquid culture medium, 37 ℃, 220rpm/min culture for 9-10h, to OD600When the concentration is 2.0, the bacterial solution is diluted in a gradient manner, applied to a TSA solid culture medium, cultured in a 37 ℃ incubator for 3 days, counted, and the concentration of the bacterial solution is calculated. According to the concentration of bacterial liquid, female BALB/c mice of 7-8 weeks are prepared to carry out challenge experiments, the lethal dose and the maximum non-lethal dose of A19 to the mice are groped, and the specific method is as follows: 15 BALB/c mice were divided into 3 mice/cage (group), and based on the concentration of the bacterial solution determined,diluting the bacterial liquid to 105、106、107、108And 109CFU/200 uL, injecting bacterial liquid with different concentrations into the abdominal cavity of mice in groups, each 200 uL, and observing the survival condition of the mice within 7 days.
Results display 107CFU/mouse is the maximum dose that will not cause death to the mouse, 109CFU/mouse is the minimum dose lethal to the mouse. Further on average, within this range, the dose is subdivided into 5 concentrations (10)7、5×107、108、5×108、109CFU/200 μ L), 40 BALB/c mice 8 weeks old were divided into 8 mice/cage (group), 200 μ L per mouse was subjected to intraperitoneal challenge with diluted bacterial solution at a subdivided concentration, and survival of mice within 7 days was observed. Half Lethal Dose (LD) of the mice is obtained50) About 5X 107CFU/mouse, the lethal dose is about 1X 108CFU/mouse.
On day 14 after the triple immunization in step 3, 10 mice per group were injected intraperitoneally with a non-lethal dose (1.03X 10)7CFU/mouse) of brucella vaccine a19, 3 mice bled daily on days 0, 1, 3, 5 and 7 post-infection, and ELISA kits pre-coated with mouse TNF- α (dake, inc., cat #: 1217202) to determine the expression level of TNF-alpha in serum.
The results are shown in FIG. 5. The results showed that there was no significant change in the levels of TNF- α expression in each group on day 0 and day 1 post-infection, starting on day three, PBS, OPSBa(with and without adjuvant) and C-OPSBaThe level of TNF-alpha expression is increased in the adjuvanted group, and the level is increased with C-OPSBaThe non-adjuvanted group had significant differences, with the highest values reached by day five, which continued until day 7. In this process, C-OPSBaThe expression level of TNF-alpha in the non-adjuvant group is stable and basically not obviously increased all the time, which indicates that C-OPS is infected by A19BaThe mice without adjuvant group can rapidly eliminate bacteria and reduce the expression of inflammatory factors.
6. Spleen load determination after non-lethal dose A19 infection
On day 7 after infection with A19 in step 5, 5 mice were dissected out of each group, and the spleens of the mice were weighed and soaked in 1mL of physiological saline and homogenized by cell sieving. Centrifuging at 6000 Xg/min for 10min, removing supernatant, collecting spleen precipitate, adding 1mL of physiological saline to resuspend the precipitate, repeating the step once, finally resuspending the precipitate with sterile 0.1% deoxycholate sodium solution, coating TSA solid culture medium after gradient dilution, culturing for 3 days at 37 ℃ in an incubator, counting, and calculating the spleen bacterial load of each mouse.
The results are shown in FIG. 6. In FIG. 6, the weight of spleen in mice is shown on the left, the weight of spleen in Control mice is shown in normal mice, and the weight of spleen in mice in each group after infection with A19 is increased compared with the weight of spleen in normal mice, but C-OPS is presentBaThe weight gain of the groups (adjuvanted and unadjuvanted) was minimal, significantly different from the other groups, OPSBaThe group weight gain (with and without adjuvant) was the greatest. The right panel shows the results of spleen bacterial load of mice, C-OPSBaThe spleen load in the (adjuvanted and unadjuvanted) groups was 103.0-103.5Left and right of the spleen, all other groups were 104.0-104.5Around the spleen, there are significant differences. Thus, C-OPS can be usedBaAfter the mice are immunized, the mice can effectively eliminate the Brucella melitensis in vivo after infection, and the damage of bacteria to organisms is reduced.
7. Evaluation of mouse protection after lethal dose A19 challenge
On day 14 after the third immunization in step 3, 10 mice per group were injected intraperitoneally with a lethal dose (actual challenge dose is 1.54 × 10)8CFU/mouse, 3 × LD50) A19, observing the survival of mice in each group.
The results are shown in FIG. 7, where all PBS mice died within two days after challenge with a lethal dose of A19, OPSBaSurvival rates of the unadjuvanted and adjuvanted groups were 60% and 30%, respectively, and C-OPSBaSurvival rates of both (adjuvanted and unadjuvanted) groups were 100%, indicating C-OPSBaIndeed protection against Brucella melitensis could be generated in immunized mice without the need for adjuvants.
Sequence listing
<110> military medical research institute of military science institute of people's liberation force of China
<120> bovine brucellosis polysaccharide conjugate vaccine and application thereof
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2491
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
tctagaactg cataattcgt gtcgctcaag gcgcactccc gttctggata atgttttttg 60
cgccgacatc ataacggttc tggcaaatat tctgaaatga gctgttgaca attaatcatc 120
ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagaattca 180
tgcccgctga aacgaccgta tccggcgcgc accccgccgc caaactgccg atttacatcc 240
tgccctgctt cctttggata ggcatcgtcc cctttacctt cgcgctcaaa ctgaaaccgt 300
cgcccgactt ttaccacgat gccgccgccg cagccggcct gattgtcctg ttgttcctca 360
cggcaggaaa aaaactgttt gatgtcaaaa tccccgccat cagcttcctt ctgtttgcaa 420
tggcggcgtt ttggtatctt caggcacgcc tgatgaacct gatttacccc ggtatgaacg 480
acatcgtctc ttggattttc atcttgctcg ccgtcagcgc gtgggcctgc cggagcttgg 540
tcgcacactt cggacaagaa cgcatcgtga ccctgtttgc ctggtcgctg cttatcggct 600
ccctgcttca atcctgcatc gtcgtcatcc agtttgccgg ctgggaagac acccctctgt 660
ttcaaaacat catcgtttac agcgggcaag gcgtaatcgg acacatcggg cagcgcaaca 720
acctcggaca ctacctcatg tggggcatac tcgccgccgc ctacctcaac ggacaacgaa 780
aaatccccgc cgccctcggc gtaatctgcc tgattatgca gaccgccgtt ttaggtttgg 840
tcaactcgcg caccatcttg acctacatag ccgccatcgc cctcatcctt cccttctggt 900
atttccgttc ggacaaatcc aacaggcgga cgatgctcgg catagccgca gccgtattcc 960
ttaccgcgct gttccaattt tccatgaaca ccattctgga aacctttact ggcatccgct 1020
acgaaactgc cgtcgaacgc gtcgccaacg gcggtttcac agacttgccg cgccaaatcg 1080
aatggaataa agcccttgcc gccttccagt ccgccccgat attcgggcac ggctggaaca 1140
gttttgccca acaaaccttc ctcatcaatg ccgaacagca caacatatac gacaacctcc 1200
tcagcaactt gttcacccat tcccacaaca tcgtcctcca actccttgca gagatgggaa 1260
tcagcggcac gcttctggtt gccgcaaccc tgctgacggg cattgccggg ctgcttaaac 1320
gccccctgac ccccgcatcg cttttcctaa tctgcacgct tgccgtcagt atgtgccaca 1380
gtatgctcga atatcctttg tggtatgtct atttcctcat ccctttcgga ctgatgctct 1440
tcctgtcccc cgcagaggct tcagacggca tcgccttcaa aaaagccgcc aatctcggca 1500
tactgaccgc ctccgccgcc atattcgcag gattgctgca cttggactgg acatacaccc 1560
ggctggttaa cgccttttcc cccgccactg acgacagtgc caaaaccctc aaccggaaaa 1620
tcaacgagtt gcgctatatt tccgcaaaca gtccgatgct gtccttttat gccgacttct 1680
ccctcgtaaa cttcgccctg ccggaatacc ccgaaaccca gacttgggcg gaagaagcaa 1740
ccctcaaatc actaaaatac cgcccccact ccgccaccta ccgcatcgcc ctctacctga 1800
tgcggcaagg caaagttgca gaagcaaaac aatggatgcg ggcgacacag tcctattacc 1860
cctacctgat gccccgatac gccgacgaaa tccgcaaact gcccgtatgg gcgccgctgc 1920
tacccgaact gctcaaagac tgcaaagcct tcgccgccgc gcccggtcat ccggaagcaa 1980
aaccctgcaa atgaaagctt ggctgttttg gcggatgaga gaagattttc agcctgatac 2040
agattaaatc agaacgcaga agcggtctga taaaacagaa tttgcctggc ggcagtagcg 2100
cggtggtccc acctgacccc atgccgaact cagaagtgaa acgccgtagc gccgatggta 2160
gtgtggggtc tccccatgcg agagtaggga actgccaggc atcaaataaa acgaaaggct 2220
cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt 2280
aggacaaatc cgccgggagc ggatttgaac gttgcgaagc aacggcccgg agggtggcgg 2340
gcaggacgcc cgccataaac tgccaggcat caaattaagc agaaggccat cctgacggat 2400
ggcctttttg cgtttctaca aactcttttg tttatttttc taaatacatt caaatatgta 2460
tccgctcatg agacgagctc ggccgctcga g 2491
<210> 2
<211> 604
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Pro Ala Glu Thr Thr Val Ser Gly Ala His Pro Ala Ala Lys Leu
1 5 10 15
Pro Ile Tyr Ile Leu Pro Cys Phe Leu Trp Ile Gly Ile Val Pro Phe
20 25 30
Thr Phe Ala Leu Lys Leu Lys Pro Ser Pro Asp Phe Tyr His Asp Ala
35 40 45
Ala Ala Ala Ala Gly Leu Ile Val Leu Leu Phe Leu Thr Ala Gly Lys
50 55 60
Lys Leu Phe Asp Val Lys Ile Pro Ala Ile Ser Phe Leu Leu Phe Ala
65 70 75 80
Met Ala Ala Phe Trp Tyr Leu Gln Ala Arg Leu Met Asn Leu Ile Tyr
85 90 95
Pro Gly Met Asn Asp Ile Val Ser Trp Ile Phe Ile Leu Leu Ala Val
100 105 110
Ser Ala Trp Ala Cys Arg Ser Leu Val Ala His Phe Gly Gln Glu Arg
115 120 125
Ile Val Thr Leu Phe Ala Trp Ser Leu Leu Ile Gly Ser Leu Leu Gln
130 135 140
Ser Cys Ile Val Val Ile Gln Phe Ala Gly Trp Glu Asp Thr Pro Leu
145 150 155 160
Phe Gln Asn Ile Ile Val Tyr Ser Gly Gln Gly Val Ile Gly His Ile
165 170 175
Gly Gln Arg Asn Asn Leu Gly His Tyr Leu Met Trp Gly Ile Leu Ala
180 185 190
Ala Ala Tyr Leu Asn Gly Gln Arg Lys Ile Pro Ala Ala Leu Gly Val
195 200 205
Ile Cys Leu Ile Met Gln Thr Ala Val Leu Gly Leu Val Asn Ser Arg
210 215 220
Thr Ile Leu Thr Tyr Ile Ala Ala Ile Ala Leu Ile Leu Pro Phe Trp
225 230 235 240
Tyr Phe Arg Ser Asp Lys Ser Asn Arg Arg Thr Met Leu Gly Ile Ala
245 250 255
Ala Ala Val Phe Leu Thr Ala Leu Phe Gln Phe Ser Met Asn Thr Ile
260 265 270
Leu Glu Thr Phe Thr Gly Ile Arg Tyr Glu Thr Ala Val Glu Arg Val
275 280 285
Ala Asn Gly Gly Phe Thr Asp Leu Pro Arg Gln Ile Glu Trp Asn Lys
290 295 300
Ala Leu Ala Ala Phe Gln Ser Ala Pro Ile Phe Gly His Gly Trp Asn
305 310 315 320
Ser Phe Ala Gln Gln Thr Phe Leu Ile Asn Ala Glu Gln His Asn Ile
325 330 335
Tyr Asp Asn Leu Leu Ser Asn Leu Phe Thr His Ser His Asn Ile Val
340 345 350
Leu Gln Leu Leu Ala Glu Met Gly Ile Ser Gly Thr Leu Leu Val Ala
355 360 365
Ala Thr Leu Leu Thr Gly Ile Ala Gly Leu Leu Lys Arg Pro Leu Thr
370 375 380
Pro Ala Ser Leu Phe Leu Ile Cys Thr Leu Ala Val Ser Met Cys His
385 390 395 400
Ser Met Leu Glu Tyr Pro Leu Trp Tyr Val Tyr Phe Leu Ile Pro Phe
405 410 415
Gly Leu Met Leu Phe Leu Ser Pro Ala Glu Ala Ser Asp Gly Ile Ala
420 425 430
Phe Lys Lys Ala Ala Asn Leu Gly Ile Leu Thr Ala Ser Ala Ala Ile
435 440 445
Phe Ala Gly Leu Leu His Leu Asp Trp Thr Tyr Thr Arg Leu Val Asn
450 455 460
Ala Phe Ser Pro Ala Thr Asp Asp Ser Ala Lys Thr Leu Asn Arg Lys
465 470 475 480
Ile Asn Glu Leu Arg Tyr Ile Ser Ala Asn Ser Pro Met Leu Ser Phe
485 490 495
Tyr Ala Asp Phe Ser Leu Val Asn Phe Ala Leu Pro Glu Tyr Pro Glu
500 505 510
Thr Gln Thr Trp Ala Glu Glu Ala Thr Leu Lys Ser Leu Lys Tyr Arg
515 520 525
Pro His Ser Ala Thr Tyr Arg Ile Ala Leu Tyr Leu Met Arg Gln Gly
530 535 540
Lys Val Ala Glu Ala Lys Gln Trp Met Arg Ala Thr Gln Ser Tyr Tyr
545 550 555 560
Pro Tyr Leu Met Pro Arg Tyr Ala Asp Glu Ile Arg Lys Leu Pro Val
565 570 575
Trp Ala Pro Leu Leu Pro Glu Leu Leu Lys Asp Cys Lys Ala Phe Ala
580 585 590
Ala Ala Pro Gly His Pro Glu Ala Lys Pro Cys Lys
595 600
<210> 3
<211> 1074
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gagctctgca taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg 60
ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg 120
ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac agaattcatg 180
aagaaaattt ggctggcctt agccggcctg gttctggcat tcagcgccag cgcaaccccg 240
cagaacatca ccgacctgtg cgccgagtac cacaacaccc aaatttatac cctgaacgac 300
aaaattttta gctacaccga gagcctggca ggcaagcgcg agatggccat catcaccttc 360
aagaacggcg ccattttcca ggtggaggtg ccgggcagcc agcacatcga cagtcagaag 420
aaggccatcg agcgcatgaa ggacaccctg cgcatcgcct acctgaccga ggccaaggtg 480
gagaagctgt gcgtgtggaa caacaagacc ccgcacgcca tcgccgcaat cagcatggcc 540
aacgaccaga acgccaccag cgccgtgacc gagtactatc tgaaccatgg cgagtggccg 600
ggtaataaca ccagcgccgg cgtggccaca agcagtgaga tcaagggcgg cggccaccat 660
caccaccacc attaaaagct tggctgtttt ggcggatgag agaagatttt cagcctgata 720
cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc 780
gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt 840
agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc 900
tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag 960
taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg 1020
ggcaggacgc ccgccataaa ctgccctgca gttttccatc gatggccgct cgag 1074
<210> 4
<211> 156
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser
1 5 10 15
Ala Ser Ala Thr Pro Gln Asn Ile Thr Asp Leu Cys Ala Glu Tyr His
20 25 30
Asn Thr Gln Ile Tyr Thr Leu Asn Asp Lys Ile Phe Ser Tyr Thr Glu
35 40 45
Ser Leu Ala Gly Lys Arg Glu Met Ala Ile Ile Thr Phe Lys Asn Gly
50 55 60
Ala Ile Phe Gln Val Glu Val Pro Gly Ser Gln His Ile Asp Ser Gln
65 70 75 80
Lys Lys Ala Ile Glu Arg Met Lys Asp Thr Leu Arg Ile Ala Tyr Leu
85 90 95
Thr Glu Ala Lys Val Glu Lys Leu Cys Val Trp Asn Asn Lys Thr Pro
100 105 110
His Ala Ile Ala Ala Ile Ser Met Ala Asn Asp Gln Asn Ala Thr Ser
115 120 125
Ala Val Thr Glu Tyr Tyr Leu Asn His Gly Glu Trp Pro Gly Asn Asn
130 135 140
Thr Ser Ala Gly Val Ala Thr Ser Ser Glu Ile Lys
145 150 155
<210> 5
<211> 1074
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tctagatgca taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg 60
ccgacatcat aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg 120
ctcgtataat gtgtggaatt gtgagcggat aacaatttca cacaggaaac agaattcatg 180
aagaaaattt ggctggcctt agccggcctg gttctggcat tcagcgccag cgcaaccccg 240
cagaacatca ccgacctgtg cgccgagtac cacaacaccc aaatttatac cctgaacgac 300
aaaattttta gctacaccga gagcctggca ggcaagcgcg agatggccat catcaccttc 360
aagaacggcg ccattttcca ggtggaggtg ccgggcagcc agcacatcga cagtcagaag 420
aaggccatcg agcgcatgaa ggacaccctg cgcatcgcct acctgaccga ggccaaggtg 480
gagaagctgt gcgtgtggaa caacaagacc ccgcacgcca tcgccgcaat cagcatggcc 540
aacgaccaga acgccaccag cgccgtgacc gagtactatc tgaaccatgg cgagtggccg 600
ggtaataaca ccagcgccgg cgtggccaca agcagtgaga tcaagggcgg cggccaccat 660
caccaccacc attaaaagct tggctgtttt ggcggatgag agaagatttt cagcctgata 720
cagattaaat cagaacgcag aagcggtctg ataaaacaga atttgcctgg cggcagtagc 780
gcggtggtcc cacctgaccc catgccgaac tcagaagtga aacgccgtag cgccgatggt 840
agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa aacgaaaggc 900
tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag 960
taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg 1020
ggcaggacgc ccgccataaa ctgccctgca gttttccatc gatggccgct cgag 1074
