KR100474114B1 - Fusion protein having the enhanced immunogenecity to bovine theilerosis, and preparation process and use thereof - Google Patents

Abstract

The present invention relates to a fusion protein of p33 surface antigen and heat stimulating protein (HSP) of a domestic isolate, Theileria sergenti (Sungwhan stock), and a method and use thereof.

Description

Fusion protein having the enhanced immunogenecity to bovine theilerosis, and preparation process and use according to bovine tyreria disease

The present invention relates to a fusion protein with enhanced immunogenicity against bovine theilerosis. Fusion proteins of the More particularly, the present invention provides domestic strains (Korean isolate), tile LES Ria Advanced Performance tie seonghwan main highly immunogenic specific surface antigens with specific molecular adjuvant (molecular adjuvant) of (Theileria sergen t i, Sungwhan stock) , And methods for their preparation and use in connection with the prevention of bovine Tyreria disease.

Typhoid 's disease in cows is caused by infection of cattle through the tick ( Hamaphisalis longicornis ) of the genus Piroplasmida and the family Theileriidae . It is a disease that causes swelling, dyspnea, anemia and jaundice of the lymph nodes. In Korea, mites, the mediators of this disease, are distributed throughout the country, with varying degrees. In particular, since mites are actively active in spring, there are many cases of grazing in the grazing cows after about one month from the start of grazing. In addition, even if you are judging, you can get Tyreria disease if you eat the outdoor neat. The main symptoms include jaundice and anemia, usually a fever around 41 ℃ after one week, but most of the fever is not found in a few days. After 10-14 days of infection, the protozoa begins to appear in red blood cells and peaks in about 1 month. As the protozoa proliferate, appetite declines, anemia progresses rapidly, and the thorn mucosa becomes pale. In the end, the body temperature falls below 38 ℃, collapsed and can not stand up. These symptoms can be recovered after a few days with proper treatment early, but delayed treatment or sustained long-term anemia and jaundice even after recovery, and development in the rearing cows is markedly deteriorated. Prone to disease.

Currently in Korea, measures against this disease are limited to the prevention of infection through the treatment of ticks on pasture or the treatment of infected cows. In foreign countries, a method of immunizing immunity by separating the protozoan from the salivary glands of a disease-infected tick, inoculating the cow and inoculating it again and treating it is used. On the other hand, as a test for the development of domestic vaccines, for example, using protozoa isolated from blood and their metabolites. However, inoculation of protozoa and metabolites into cows may alleviate clinical symptoms somewhat, but not provide complete protection. In addition, these methods have the disadvantage that it is difficult to provide a sufficient amount of antigen when inoculated to many cattle.

As an alternative, there is a method of using a recombinant protein cloned and expressed by a surface protein having excellent immunogenicity, but recombinant proteins prepared using Escherichia coli are difficult to have sufficient protection by themselves and various immunoadjuvant should be used. . However, it is also difficult to obtain a suitable adjuvant that satisfies both the immune effect and economic aspects.

As described above, the cattle ileary disease is a chronic wasting disease, causing enormous economic damage to the farm, and the demand for a method that can effectively respond to this situation is increasing.

Accordingly, the present inventors conducted a continuous study to develop a new immunogen with more enhanced immunogenicity against bovine Tyreria disease. As a result, the fusion protein obtained by co-expression of the p33 surface antigen gene of the Tyrrhenian protozoan strain and a heat shock protein (HSP) gene having immunogenicity-promoting activity in a single vector is bovine Tyreria disease It was confirmed that it exhibits more enhanced immunogenicity for, and the present invention was completed.

Accordingly, an object of the present invention is to simultaneously bind the p33 surface antigen gene of the Tyreria protozoan and the Heat Shock Protein gene having immunogenicity-enhancing activity to the carboxyl terminus of the heat stimulating protein in one vector. To provide a fusion protein fused by expression, and a method for producing the same and use in the prevention of bovine Tyreria disease.

First, the present invention relates to a fusion protein in which p33 surface antigen of the Tyreria protozoan is fused to the carboxyl terminus of a thermostimulant protein and co-expressed through one expression vector.

In the present invention, it is preferable that the Tyreria protozoan is a domestic isolate, the Tyreria Seojeon Thai Sunghwan ( Theileria sergen t i , Sungwhan stock).

In addition, the p33 surface antigen may have a nucleotide sequence of SEQ ID NO: 1, the thermal stimulating protein may have a nucleotide sequence of SEQ ID NO: 2

Second, the present invention relates to a DNA encoding the fusion protein.

Third, the present invention relates to a recombinant vector comprising the DNA.

Fourth, the present invention relates to E. coli transformed with the recombinant vector.

Fifth, the present invention relates to a method for producing the fusion protein by culturing the E. coli.

Sixth, the present invention relates to a bovine Tyreria disease immunogen containing the fusion protein as an active ingredient.

Seventh, the present invention relates to a method of enhancing the immune antibody against bovine Tyreria disease by administering the fusion protein to the cow.

Hereinafter, the present invention will be described in detail.

The present invention relates to a prophylactic drug candidate for Tyreria disease, which is prevalent in Korea, and this disease is a chronic wasting disease, causing many economic damages to farmers, and the need for a vaccine against it is increasing. The present invention is to enhance the immunogenicity of p33 protein by expressing the domestic isolate of Tyreria p33 protein produced by genetic recombination with the heat stimulating protein. Tyreria surface protein p33 is known to exhibit a variety of forms, it is important to use genes extracted from domestic isolates for domestic diseases.

Among several surface antigens of the Theileria sergenti Sungwhan stock, p33 is known to have excellent immunological antigenicity, resulting in the production of recombinant proteins using E. coli . However, the challenge of inoculation after inoculation of the produced recombinant protein to the target animal did not show sufficient protective ability. Therefore, there is a need for a material that can reinforce it. The heat stimulating protein (HSP) present in the cell has the function of enhancing the immunogenicity of the vaccine antigens. An attempt was made to prepare a protein.

p33 cloned into pUC19 vector was treated with BamHI, Klenow fragment and KpnI, and hsp pRSET vector was treated with SphI, Klenow fragment and KpnI. Both treated DNAs were ligation and expressed using Novablue cells.

The antibody titers of surface antigen p33 and fusion protein (p33-hsp) in domestic isolates were compared. As a result, the antibody titers of p33 and fusion protein (p33-hsp) antigens were 1,800 and 25,600 fold, respectively, and significant differences were obtained between the two groups.

Therefore, the fusion protein according to the present invention is considered to be effectively used for the prevention of bovine tyreria disease. For this purpose, the fusion protein of the present invention may be formulated in conventional formulations, preferably injectables, in the pharmaceutical field together with pharmaceutically acceptable carriers. A typical dosage is 2 mg per 100 kg of cow's body weight based on the active ingredient, but this dose may be increased or decreased depending on the age, weight, health condition, etc. of the cow to be administered.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited by them in any way.

Example 1 Tyreria DNA Extraction, Cloning and Sequencing

Tyreria protozoa were isolated from infected cows in the breeder's improvement department of Chungnam. The protozoa were passaged using cattle in the laboratory, and the genes of the protozoa were extracted from the blood of the subfamily.

PMALc2 (BioLabs, USA), a maltose biding protein (MBP) fusion vector, was used as an expression vector of p33 in E. coli, and a pRSET vector as a fusion protein expression vector with heat stimulatory protein (manufacturer: Stanford university) was used.

Passage blood was extracted from the blood when the infection rate was more than 10%. After erythrocytes were separated from blood treated with EDTA, 700 μl of distilled water was added to 300 μl of red blood cells, mixed, and centrifuged at 15000 rpm for 10 minutes. 0.5% saponin was added to the precipitate, mixed well, and then washed twice with distilled water and the supernatant was removed. 500 μl of extraction buffer [10 mM Tris-HCl (pH 8.0), 100 mM EDTA (pH 8.0), 0.5% SDS, 20 μg / ml ribonuclease] was added to the precipitate, followed by reaction for 1 hour at 37 ° C. I was. 2.5 μl of proteinase K was added thereto, reacted at 50 ° C. for 30 minutes, and then cooled at room temperature for 10 minutes. Then, 500 μl of phenol was added, mixed well, and centrifuged at 15000 rpm for 10 minutes to separate only the supernatant. The same amount of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added to the supernatant, followed by centrifugation, and the same amount of phenol: chloroform: isoamyl alcohol (25: 24: 1) was added to the supernatant. Mixed. After centrifugation, the supernatant was separated, 50 μl of 10M ammonium acetate and 1000 μl of ethanol were added thereto, and the mixture was left at −70 ° C. for 30 minutes, followed by centrifugation at 15000 rpm for 10 minutes. The precipitated DNA was washed with 70% cold ethanol and resuspended with 30 μl of TE buffer and stored at -20 ° C. Prepare primer based on nucleotide sequence searched by Gene bank (Forward primer: 5'-TATGTTGTCCAAGAGATCGT-3'.Reverse primer: 5'-TGAGACTCAGTGCGCCTAGA-3 ') PCR was performed. PCR products were cloned into the pUC19 vector (FIG. 1) and the sequences of the cloned genes were analyzed. DNA sequencing was performed using a DNA sequencing kit (USB) and didedeoxy chain termination. In addition, sequencing was performed using a DNASIS program (Hitachi Software Engineering Co.) (FIG. 2: 849 bp).

Example 2: Expression of p33 and Fusion Protein Expression with Heat Stimulating Proteins

To express p33, p33 cloned into pUC19 was cut out by treatment with EcoRI, Klenow fragment and BamHI. PMAL-c2, an expression vector, was treated with HindIII, Klenow fragment and BamHI. Restriction enzyme-treated p33 and pMAL-c2 were treated with ligase and reacted at 16 ° C. to connect to p33 isolated from pUC19. The p33pMAL-c2 vector was expressed using E. coli DH5α (FIG. 3).

In order to prepare a fusion protein of p33 with a heat stimulating protein, p33 in the pUC19 vector was cut out by treatment with BamHI, Klenow fragment and KpnI. The hsp pRSET vector (isolated from bovine) was treated with Sph I, Klenow fragment and Kpn I. Two DNAs isolated were combined and expressed using E. coli Novablue (FIG. 4).

 The transformed Escherichia coli Novablue / pRSET / p33-HSP was deposited on Korean Collection for Type Cultures (KCTC) as Accession No. 10140 BP as of December 14, 2001.

Example 3 Expression of Recombinant p33 and Fusion Proteins by Western Blotting

Cell extracts were subjected to SDS-PAGE to confirm that proteins were expressed from Escherichia coli transformed with recombinant DNA using untransfected Escherichia coli as a negative control. That is, cells were lysed with lysis buffer (10 mM Tris, 300 mM NaCl, 0.5% NP-40, 1 mM PMSF, pH 7.5-8.0) at 1/50 times and treated for 1 minute in an ultrasonic crusher (Brinkmann). Then, centrifugation was performed at 12,000 rpm for 5 minutes to collect only the supernatant, and then subjected to electrophoresis after heat treatment with the SDS sample buffer solution. The transferred gel was transferred to a nitrocellulose filter and then recombinant p33 (FIG. 5; A: Western blotting using Tyreria positive serum) using monoclonal antibodies against maltose and heat stimulating protein. Western blotting with MBP antibody, 1: before expression induction, 2: after expression induction) and fusion proteins with heat stimulating protein (FIG. 6; A: SDS-PAGE pattern, B: Western blotting pattern, lanes 1 and 5) : p33-hsp fusion protein, lanes 2, 3, 4 and 6: non-fusion thermo stimulatory protein) were confirmed.

Example 4 Comparison of Immunogenicity of Recombinant p33 and Fusion Proteins

To investigate the immunogenicity of recombinant p33, 10 mg of recombinant p33 was inoculated in two cows at 10 day intervals and blood was collected 10 days after inoculation. Serum was separated from blood and western blotting was performed to determine antibody titer. Serum was diluted 100-fold, 200-fold, 400-fold, 600-fold, 800-fold, 1,000-fold, 1,200-fold, 1,400-fold, 1,600-fold and 1,800-fold with PBS buffer, and the secondary antibody was used diluted at 1,000-fold (Figure 7). ; 1: 100 times, 2: 200 times, 3: 400 times, 4: 600 times, 5: 800 times, 6: 1000 times, 7: 1200 times, 8: 1400 times, 9: 1600 times, 10: 1800 times ).

To investigate the immunogenicity of the p33-thermal stimulating protein (HSP) fusion antigen, rabbits were inoculated twice at 10 days intervals subcutaneously at 200 μg, and blood was collected 10 days after the final inoculation. The antibody titer was measured by performing ELISA by coating p33 protein with 2 µg / well. Serum was diluted 100-fold, 200-fold, 400-fold, 800-fold, 1,600-fold, 3,200-fold, 6,400-fold, 12,800-fold, 256,000-fold, 51,200-fold, and 102,400-fold in PBS buffer. Was used (FIG. 8).

As a result of investigating the antibody titers of p33 and the fusion protein, the antibody titer was confirmed at the dilution factor of 1,800-fold when inoculated with only p33 recombinant protein. In contrast, when inoculated in a fused form with heat stimulating protein, antibody titer was confirmed even at a dilution rate of 256,000 times. From this, it was confirmed that the heat stimulating protein significantly increased the immunogenicity of the main protein p33 as an auxiliary protein.

Tyreria disease, which is present in Korea, is a disease that prevailed in the past 7 and 80 years and is a chronic wasting disease that has been reemerged in recent years. However, up to now, by increasing the antibody value by inoculating the fusion protein according to the present invention in the absence of such a vaccine, it is expected to increase the growth rate and flow rate of domestic broilers by preventing infection of Tyreria disease.

1 is a schematic of cloning p33 into a pUC19 vector;

Figure 2 is a diagram showing the p33 gene sequence of the domestic isolates of Tyreria;

3 is a schematic diagram of cloning p33 into a pMALc-2 expression vector;

4 is a schematic of cloning of the p33-HSP fusion protein;

5 is a photograph confirming the presence of p33-HSP fusion protein using Western blotting;

Figure 6 is a photograph confirming the expression of p33-HSP fusion protein using SDS-PAGE and Western blotting;

7 is a photograph of the antibody titer of p33 using western blotting;

8 is a graph measuring the antibody titer of p33-HSP fusion protein using ELISA.

9 is a diagram showing the nucleotide sequence of the heat stimulating protein (HSP) of the Tyreria domestic isolate.

<110> National Veterinary Research and Quarantine Service <120> Fusion protein having the enhanced immunogenecity to bovine theilerosis, and preparation process and use <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 849 <212> DNA <213> Theileria sergenti <220> <221> gene (222) (1) .. (849) <400> 1 atgttgtcca agagatcgtt caacgtactt tgcctaggat acttccttat cgtctctgct 60 accgccgcag aggaaaaaaa agatgcaaag gctgaagaga agaaggactt aactctcgaa 120 gttaacgcca ccgcagccga acattttaaa gtcgacgcct caaacgccaa cgacgtcgtt 180 tttactgccg aagagggata ccgcatcaag acactcaagg tcggagataa gaacctgtat 240 accgtagata cttccaagtt caccccaact gtcgcccaca gactgaagca tgctgacgac 300 ctgttcttca agctcaacct gtcccacgca aagccattgc tgttcaagaa gaagactgac 360 aaggattggg ttcaattcag cttcgcccag tacctcgatg aagtggtatg gaaggagaag 420 aaggaagtaa aagacctcga cgcatccaag ttcgcagacg caggtctttt cgccgctgag 480 gctttcggta ccggaaagct gtacaacttc attggaaact tcaaggtcaa gaaggtcatg 540 ttcgaggaga aggacgttgg agattcaaac aaggccaaat acaccgctgt caaagtttac 600 gtcggtttcg atgagaagaa agtcgtaaga ctcgactact tctacactgg tgatgagaga 660 ttcaaggagg tttacttcaa attggtagac ggaaaatgga agaaggttga gcagagcgaa 720 gcaaacaagg atttgcacgc catgaacagt gcttggcttt cggactacaa gcctcttgtc 780 gacaagttct caccacttgc cgttctcagc gcggttctca tcgcctccct cgcagtattc 840 tattatctc 849 <210> 2 <211> 2100 <212> DNA <213> Theileria sergenti <220> <221> gene (222) (1) .. (2100) <400> 2 atctcgatcc cgcgaaatta atacgactca ctatagggag accacaacgg tttccctcta 60 gaataatttt gtttaacttt aagaaggaga tatacatatg tctaaaggac ctgcagttgg 120 cattgatctt ggcaccacct attcttgtgt gggtgtcttc cagcacggaa aggtggaaat 180 aattgccaat gatcagggga accgaaccac cccaagctat gtcgccttta ctgataccga 240 acggttaatc ggtgatgcag caaagaacca agtcgcaatg aatcccacca acacggtttt 300 tgatgccaaa cgacttattg gacgaagatt tgatgatgct gttgtccagt ctgatatgaa 360 acattggccc ttcatggtgg tgaatgatgc tggcaggcct aaggttcaag tagaatacaa 420 gggagagaca aagagttttt acccagagga ggtgtcatcc atggtcctga caaagatgaa 480 ggaaatcgca gaagcctacc ttgggaagac ggttaccaac gctgtcgtca cagtacctgc 540 ctattttaat gactctcagc gtcaggctac caaagatgct ggaactattg ctggtctcaa 600 cgtacttcga atcatcaatg agccaactgc tgctgctatt gcctatggct tagacaaaaa 660 ggttggagca gaaagaaacg tgctgatctt tgatttaggg ggtggcactt ttgatgtgtc 720 aatcctcact attgaggatg gaatctttga ggtcaaatct acagctggag atactcactt 780 gggtggagaa gactttgaca accgcatggt taaccatttt attgcggagt tcaagcgtaa 840 acacaagaag gatatcagtg aaaacaagag ggctgtccgt cgcctccgta ctgcttgtga 900 gcgtgctaag cgcactctct cttccagcac ccaggccagt attgagattg attccctcta 960 tgaaggaatc gacttctata cctctattac ccgtgcccga tttgaagaat tgaatgctga 1020 cttgttccgt ggcaccctgg accctgtgga gaaagccctt agggatgcca aactggacaa 1080 gtctcagatt catgatattg tcttggttgg tggctcaacc cgtatcccca agattcagaa 1140 acttctccag gacttcttca acgggaaaga actgaataag agcatcaacc ctgatgaggc 1200 tgttgcgtat ggcgccgctg tccaggcagc cattttgtct ggagacaaat ctgaaaatgt 1260 tcaagacttg ctgctgttgg atgtcactcc tctttccctt ggaattgaaa ctgctggtgg 1320 agtcatgact gtcctcatca agcgcaatac taccattcct accaagcaga cgcagacttt 1380 caccacctac tctgacaacc agcctggtgt gctcattcag gtttatgaag gtgagcgtgc 1440 catgaccaag gataacaacc tgcttggcaa gtttgaactc acgggcatac ctcctgcccc 1500 ccgtggtgtt cctcagattg aagtcacttt tgatattgat gccaatggca tcctcaatgt 1560 ttctgctgtg gataagagca caggaaaaga gaacaagatt accatcacta atgacaaggg 1620 ccgcttgagc aaggaagaca ttgaacgcat ggttcaagaa gcagagaagt acaaagctga 1680 agatgagaag cagcgggaca aggtgtcttc aaagaattcg cttgaatcct acgcgttcaa 1740 catgaaagct actgttgagg atgagaaact tcagggcagg attaatgatg aagacaaaca 1800 gaagattctt gacaagtgta atgaaataat caactggctg gataagaacc agactgcaga 1860 gaaggaagaa tttgaacatc agcagaagga gctggaaaaa gtctgcaacc ccatcattac 1920 caagctgtac cagagtgcag gaggcatgcc tgggggaatg ccaggaggaa tgcctggggg 1980 cttccctggt ggtggagctc ctccatcagg tggtgcctcc tctgggccca ccattgaaga 2040 ggttgattaa tctagagtcg agatctgcag ctggtaccat ggaattcgaa gcttgatccg 2100 2100

Claims (10)

Fusion of a heat shock protein (HSP) encoded by the gene sequence of SEQ ID NO: 2 to the amino terminus of the p33 surface antigen encoded by the gene sequence of SEQ ID NO: 1 of the Tyreria protozoan protein. 2. The fusion protein of claim 1, wherein the tiereria protozoan is a Korean isolate, Theileria sergenti , Sungwhan stock. delete delete DNA having a nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2 encoding the fusion protein according to any one of claims 1 to 2. Recombinant vector comprising a DNA according to claim 5. Escherichia coli transformed with the recombinant vector according to claim 6. A method for preparing a fusion protein according to claim 1 by culturing E. coli according to claim 7. An agent for preventing bovine tyreria disease containing the fusion protein according to any one of claims 1 to 2. A method for preventing bovine tyreria disease by administering to a cow a fusion protein according to any one of claims 1 to 2.