阅读说明：本技术 一种同时表达FAdV-4纤突蛋白1与纤突蛋白2基因的DNA疫苗及其构建方法和应用 (DNA vaccine for simultaneously expressing FAdV-4 spike protein 1 and spike protein 2 genes and construction method and application thereof ) 是由 刘青涛 李银 杨婧 黄欣梅 赵冬敏 韩凯凯 刘宇卓 章丽娇 于 2021-07-01 设计创作，主要内容包括：本发明属于生物医药技术领域,具体涉及一种同时表达FAdV-4纤突蛋白1与纤突蛋白2基因的DNA疫苗及其构建方法和应用。本发明设计特异性引物,利用重叠PCR扩增技术,将FAdV-4的纤突蛋白1与纤突蛋白2基因通过2A肽基因连接形成融合基因fibers2A,然后将融合基因fibers2A插入pCDNA3.1真核表达载体,构建重组质粒pC-fibers2A,将重组质粒pC-fibers2A转化至大肠杆菌DH5α,扩大培养后提取重组质粒即为DNA疫苗。将该重组质粒用于免疫试验,结果表明该DNA疫苗可以免疫SPF鸡产生抗体,减小发病率和死亡率,提高免疫预防效果。(The invention belongs to the technical field of biological medicines, and particularly relates to a DNA vaccine for simultaneously expressing FAdV-4 spike protein 1 and spike protein 2 genes, and a construction method and application thereof. The invention designs a specific primer, utilizes an overlap PCR amplification technology to connect the fiber protein 1 of FAdV-4 and the fiber protein 2 gene through a 2A peptide gene to form a fusion gene fibers2A, then inserts the fusion gene fibers2A into a pCDNA3.1 eukaryotic expression vector to construct a recombinant plasmid pC-fibers2A, converts the recombinant plasmid pC-fibers2A into Escherichia coli DH5 alpha, and extracts the recombinant plasmid after amplification culture, namely the DNA vaccine. The recombinant plasmid is used for an immunity test, and the result shows that the DNA vaccine can immunize SPF chickens to generate antibodies, reduce morbidity and mortality and improve the immunity prevention effect.)

1. A DNA vaccine for simultaneously expressing FAdV-4 spike protein 1 and spike protein 2 genes is characterized in that the DNA vaccine contains a nucleotide sequence shown in SEQ ID No. 1.

2. The DNA vaccine of claim 1, wherein the DNA vaccine is obtained by cloning the nucleotide sequence shown in SEQ ID No.1 into an expression vector.

3. A construction method of a DNA vaccine for simultaneously expressing FAdV-4 spike protein 1 and spike protein 2 genes is characterized by comprising the following steps:

(1) designing specific primers P1 and P2 of FAdV-4 spike protein 1 and specific primers P3 and P4 of spike protein 2;

(2) extracting DNA of FAdV-4 as template DNA, and performing PCR amplification on the spike protein 1 by using specific primers P1 and P2; carrying out PCR amplification on the spike protein 2 by using specific primers P3 and P4;

(3) taking the PCR amplification product of the spike protein 1 and the PCR amplification product of the spike protein 2 in the step 2 as templates for overlapping PCR amplification, performing overlapping PCR amplification by using specific primers P1 and P4, and recovering the overlapping PCR amplification product by using glue to obtain a fusion gene fiber 2A;

(4) performing HindIII and NotI double enzyme digestion on the fusion gene fiber2A obtained in the step (3) and plasmid pCDNA3.1, and then connecting the fusion gene fibers2A and plasmid pCDNA3.1 by using T4 ligase to construct a recombinant plasmid pC-fibers2A for expressing FAdV-4 spike protein 1 and spike protein 2 genes;

(5) the recombinant plasmid pC-fibers2A is transformed into Escherichia coli DH5 alpha, and the recombinant expression plasmid is extracted after amplification culture, namely the DNA vaccine.

4. The method for constructing the DNA vaccine of claim 3, wherein the sequence of the specific primer P1 is SEQ ID No.2, the sequence of the specific primer P2 is SEQ ID No.3, the sequence of the specific primer P3 is SEQ ID No.4, and the sequence of the specific primer P4 is SEQ ID No. 5.

5. The method for constructing the DNA vaccine of claim 3, wherein the PCR amplification procedure for the spike protein 1 in the step (2) is as follows: 5min at 95 ℃; 30s at 95 ℃, 45s at 57 ℃, 1.5min at 72 ℃ and 35 cycles; 7min at 72 ℃.

6. The method for constructing the DNA vaccine of claim 3, wherein the PCR amplification procedure for the spike protein 2 in the step (2) is as follows: 5min at 95 ℃; 30s at 95 ℃, 45s at 59 ℃, 2min at 72 ℃ and 35 cycles; 7min at 72 ℃.

7. The method for constructing the DNA vaccine of claim 3, wherein the procedure of overlapping PCR amplification in the step (3) is as follows: 5min at 95 ℃; at 95 ℃ for 30s, at 65 ℃ for 45s, at 72 ℃ for 3min, for 35 cycles; 7min at 72 ℃.

8. The use of the DNA vaccine of claim 1 or 2 expressing both FAdV-4 spike protein 1 and spike protein 2 genes in the preparation of a medicament for preventing chicken pericardial effusion-inclusion body hepatitis syndrome.

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a DNA vaccine for simultaneously expressing FAdV-4 spike protein 1 and spike protein 2 genes, and a construction method and application thereof.

Background

At present, the group I avian adenovirus has 12 serotypes, wherein the avian adenovirus type 4 (FAdV-4) is the avian adenovirus which has the most serious harm to the chicken industry in China at present. In 2014, the pericardial effusion-inclusion body hepatitis syndrome caused by FAdV-4 is developed in chicken flocks in China, and is rapidly spread to the whole country, so that huge economic loss is caused to the chicken industry in China, but no vaccine is available at present. The literature reports show that the vaccine prepared by inactivating formaldehyde from the liver of infected chicken has good immune protection effect, the virus inactivated vaccine prepared by culturing chicken liver cancer cell line LMH can also provide effective protection, but the 2 vaccines relate to virus amplification culture in the preparation and use processes, and the risk of virus dispersion exists. Research shows that baculovirus expression products of the spike protein have good immunogenicity and can provide immune protection for chickens, but the baculovirus expression has the problems of low expression level, high cost and the like.

DNA vaccines, also known as genetic or nucleic acid vaccines, have advantages over other traditional vaccines and are therefore also referred to as third generation vaccines following inactivated vaccines, attenuated live vaccines and subunit vaccines. It can express vaccine antigens in vivo, thereby inducing specific humoral and cellular immune responses against the vaccine. The DNA vaccine is simple to prepare and low in cost, the production process does not involve amplification culture of virus, and the problem of virus dispersion does not exist; in addition, the DNA vaccine can simultaneously express different vaccine antigens, and is very stable and easy to store and transport. However, there is no development of a DNA vaccine in which both of the fiber protein 1 and the fiber protein 2 of FAdV-4 are used.

Disclosure of Invention

Aiming at overcoming the defects of virus dispersion, low expression level and the like of the vaccine against FAdV-4 in the prior art, the invention provides a DNA vaccine for simultaneously expressing FAdV-4 spike protein 1(fiber1) and spike protein 2(fiber2) genes and a construction method and application thereof. The invention firstly fuses the fiber1 and fiber2 genes together, then connects the fused gene to the eukaryotic expression vector, compared with the expression of a single protein gene, the invention not only improves the immunogenicity of the expressed antigen, but also ensures the synchronous expression of the fiber1 and the fiber2 genes, and the DNA vaccine has simple preparation, low cost, safety and effectiveness.

The invention is realized by the following technical scheme:

the invention provides a DNA vaccine for simultaneously expressing FAdV-4 spike protein 1 and spike protein 2 genes, which contains a nucleotide sequence shown in SEQ ID No. 1.

Preferably, the DNA vaccine is obtained by cloning the nucleotide sequence shown in SEQ ID No.1 into an expression vector.

The invention also provides a construction method of the DNA vaccine for simultaneously expressing the FAdV-4 spike protein 1 and spike protein 2 genes, which comprises the following steps:

(1) designing specific primers P1 and P2 of FAdV-4 spike protein 1 and specific primers P3 and P4 of spike protein 2;

(2) extracting DNA of FAdV-4 as template DNA, and performing PCR amplification on the spike protein 1 by using specific primers P1 and P2; carrying out PCR amplification on the spike protein 2 by using specific primers P3 and P4;

(3) taking the PCR amplification product of the spike protein 1 and the PCR amplification product of the spike protein 2 in the step 2 as templates for overlapping PCR amplification, performing overlapping PCR amplification by using specific primers P1 and P4, and recovering the overlapping PCR amplification product by using glue to obtain a fusion gene fiber 2A;

(4) performing HindIII and NotI double enzyme digestion on the fusion gene fiber2A obtained in the step (3) and plasmid pCDNA3.1, and then connecting the fusion gene fibers2A and plasmid pCDNA3.1 by using T4 ligase to construct a recombinant plasmid pC-fibers2A for expressing FAdV-4 spike protein 1 and spike protein 2 genes;

(5) the recombinant plasmid pC-fibers2A is transformed into Escherichia coli DH5 alpha, and the recombinant expression plasmid is extracted after amplification culture, namely the DNA vaccine.

Preferably, the sequence of the specific primer P1 is: 5'AATTAAGCTTATGTCGGCCCTAATCGCCTCCGCAGCCG 3' (shown as SEQ ID No. 2), the sequence of the specific primer P2 is: 5'CCCGCCTGCTTAAGCAGGCTAAAGTTGGTCGCGCCGCTGCCGGGGCCCGGAGCATT3' (shown as SEQ ID No. 3), the sequence of the specific primer P3 is: 5'GCTTAAGCAGGCGGGCGATGTGGAAGAAAACCCGGGCCCGATGCTCCGGGCCCCT 3' (shown as SEQ ID No. 4), the sequence of the specific primer P4 is: 5'TAAAGCGGCCGCTTACGGGAGGGAGGCCGCTGGACAGCTG 3' (shown in SEQ ID No. 5).

Preferably, the PCR amplification procedure for spike protein 1 in step (2) is: 5min at 95 ℃; 30s at 95 ℃, 45s at 57 ℃, 1.5min at 72 ℃ and 35 cycles; 7min at 72 ℃.

Preferably, the PCR amplification procedure for spike protein 2 in step (2) is: 5min at 95 ℃; 30s at 95 ℃, 45s at 59 ℃, 2min at 72 ℃ and 35 cycles; 7min at 72 ℃.

Preferably, the procedure of the overlapping PCR amplification in step (3) is: 5min at 95 ℃; at 95 ℃ for 30s, at 65 ℃ for 45s, at 72 ℃ for 3min, for 35 cycles; 7min at 72 ℃.

The invention also provides application of the DNA vaccine for simultaneously expressing the FAdV-4 spike protein 1 and the spike protein 2 genes in preparing a medicament for preventing the chicken pericardial effusion-inclusion body hepatitis syndrome.

Compared with the prior art, the invention has the beneficial effects that:

(1) the DNA vaccine prepared by the invention can simultaneously express the spike protein 1 and the spike protein 2 of the FAdV-4, so that the immunoprophylaxis effect on the FAdV-4 can be better improved, and the preparation and production process of the DNA vaccine does not involve amplification culture of the FAdV-4 virus, so that the virus scattering risk does not exist.

(2) Since the FAdV-4 genome uses different coding frames for the two antigens, i.e., fiber1 and fiber2, and the bases adjacent to the start codon of the fiber2 coding frame overlap with the bases adjacent to the stop codon of the fiber1 coding frame, the coding frames for the fiber1 and fiber2 antigens cannot be amplified simultaneously when FAdV-4 is amplified by PCR. The sequences of the fiber1 and fiber2 genes are respectively amplified, then the coding frames of the fiber1 and the fiber2 are amplified by an overlapping PCR amplification technology, and the two coding frames are connected together by a 2A peptide sequence, so that the two antigens can be simultaneously expressed, and the immunoprophylaxis effect on FAdV-4 is improved.

(3) The invention has important significance for promoting the immune control of the avian adenovirus serotype 4 (FAdV-4).

Drawings

FIG. 1 is a diagram showing the results of an electrophoresis nucleic acid test of PCR amplification products of the spike protein 1(fiber1) and spike protein 2(fiber2) genes;

wherein lane 1 is the amplification product of fiber1 gene, and lane 2 is the amplification product of fiber2 gene.

FIG. 2 is a diagram showing the results of agarose gel electrophoresis testing of the overlapping PCR amplification products of the fusion gene fibers 2A.

FIG. 3 is a diagram showing the results of restriction enzyme identification of the recombinant plasmid pC-fibers 2A.

FIG. 4 shows the results of indirect immunofluorescence assay of recombinant plasmid pC-fibers2A after transfection of DF1 cells, wherein (A) is fiber1, and (B) is fiber 2.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

A construction method of a DNA vaccine for simultaneously expressing FAdV-4 spike protein 1 and spike protein 2 genes comprises the following steps:

1. specific primers P1 and P2 of FAdV-4 spike protein 1 and specific primers P3 and P4 of spike protein 2 were designed, and the sequences were as follows:

p1: 5'AATTAAGCTTATGTCGGCCCTAATCGCCTCCGCAGCCG 3' (shown in SEQ ID No. 2);

p2: 5'CCCGCCTGCTTAAGCAGGCTAAAGTTGGTCGCGCCGCTGCCGGGGCCCGGAGCATT3' (shown in SEQ ID No. 3);

p3: 5'GCTTAAGCAGGCGGGCGATGTGGAAGAAAACCCGGGCCCGATGCTCCGGGCCCCT 3' (shown in SEQ ID No. 4);

p4: 5'TAAAGCGGCCGCTTACGGGAGGGAGGCCGCTGGACAGCTG 3' (shown in SEQ ID No. 5).

2. PCR amplification of the FAdV-4 spike protein 1(fiber1) Gene

(1) Extracting DNA of FAdV-4 as template DNA, and performing PCR amplification of fiber1 gene by using specific primers P1 and P2;

(2) PCR amplification System: specific primer P11 μ L, primer P21 μ L, reaction solution containing DNA polymerase 25 μ L, template DNA3 μ L, and sterilized deionized water to make up to 50 μ L;

(3) PCR amplification procedure: 5min at 95 ℃, 30s at 95 ℃, 45s at 57 ℃, 1.5min at 72 ℃ and 35 cycles; 7min at 72 ℃;

(4) the PCR-amplified product of fiber1 was subjected to nucleic acid electrophoresis, and the results are shown in FIG. 1: the desired fiber1 gene fragment was obtained and used in the overlap PCR amplification procedure after recovering the PCR amplification product.

3. PCR amplification of the FAdV-4 spike protein 2(fiber2) Gene

(1) Extracting DNA of FAdV-4 as template DNA, and performing PCR amplification of fiber2 gene by using specific primers P3 and P4;

(2) PCR amplification System: specific primer P31 μ L, primer P41 μ L, reaction solution containing DNA polymerase 25 μ L, template DNA3 μ L, and sterilized deionized water to make up to 50 μ L;

(3) PCR amplification procedure: 5min at 95 ℃, 30s at 95 ℃, 45s at 59 ℃, 2min at 72 ℃ and 35 cycles; 7min at 72 ℃;

(4) the PCR-amplified product of fiber1 was subjected to nucleic acid electrophoresis, and the results are shown in FIG. 1: the desired fiber2 gene fragment was obtained and used in the overlap PCR amplification procedure after recovering the PCR amplification product.

4. Overlapping PCR amplification of fusion gene fibers2A of FAdV-4 spike protein 1 and spike protein 2

(1) Preparing a template: performing gel recovery on the PCR amplification products of the FAdV-4 spike protein 1 gene and the spike protein 2 gene to serve as templates for overlapping PCR amplification;

(2) overlapping PCR amplification system: specific primer P11 μ L, specific primer P41 μ L, reaction solution containing DNA polymerase 25 μ L, FAdV-4 spike protein 1 gene PCR amplified glue recovery product 1.5uL, FAdV-4 spike protein 2 gene PCR amplified glue recovery product 1.5uL, sterilized deionized water to 50 μ L;

(3) PCR amplification procedure: 5min at 95 ℃; at 95 ℃ for 30s, at 65 ℃ for 45s, at 72 ℃ for 3min, for 35 cycles; 7min at 72 ℃;

(4) the products of the overlapping PCR amplification are subjected to agarose gel electrophoresis, as shown in FIG. 2, to obtain the desired fusion gene fibers2A gene fragment, and the products of the overlapping PCR amplification are recovered by gel, namely fusion gene fibers2A, which will be used for the construction of recombinant plasmids.

5. Construction of recombinant plasmid pC-fibers2A

(1) Performing Hind III and Not I double enzyme digestion on the fusion gene fibers2A and the plasmid pCDNA3.1 which are recovered by glue, then respectively recovering the glue, and connecting the fusion gene fibers2A and the plasmid pCDNA3.1 by using T4 ligase to construct a recombinant plasmid pC-fibers 2A;

(1) enzyme digestion of fusion gene fibers 2A: adding fusion gene fibers2A 15 μ L, Hind III endonuclease 1.5 μ L, Not I endonuclease 1.5 μ L, reaction solution 3 μ L and deionized water 9 μ L into a 1.5mL centrifuge tube, mixing uniformly, reacting in a 37 ℃ water bath kettle for 15min, and then recovering the enzyme digestion product by glue for ligation reaction;

(2) digestion of plasmid pcdna3.1: adding plasmid pCDNA3.110 μ L, Hind III endonuclease 1.5 μ L, Not I endonuclease 1.5 μ L, reaction liquid 3 μ L and deionized water 14 μ L into a 1.5ml centrifuge tube, mixing uniformly, reacting in a 37 ℃ water bath kettle for 15min, and then recovering the enzyme digestion product by glue for ligation reaction;

(3) and (3) connection reaction: 3 mu L of enzyme digestion recovery product of fusion gene fibers2A, 2 mu L of enzyme digestion recovery product of plasmid pCDNA3.1 and 5 mu L of reaction solution containing T4 ligase are added into a 1.5mL centrifuge tube, uniformly mixed and reacted for 30min in a metal bath at 16 ℃, then the product of the ligation reaction is transformed into escherichia coli DH5 alpha competent cells, a transformant is coated on an ampicillin (LB) agar plate, a clone is selected for sequence determination and enzyme digestion identification, and the enzyme digestion identification result is shown in figure 3, so that the recombinant plasmid pC-fibers2A which is the DNA vaccine is obtained.

Example 2: expression identification of recombinant plasmid pC-fibers2A

The recombinant plasmid pC-fibers2A constructed in example 1 was transfected and grown to DF1 cells, and the expression of spike protein 1 and spike protein 2 was detected by immunofluorescence 48h after transfection, the specific steps are as follows:

(1) 600ng of recombinant plasmid pC-fibers2A is dissolved in 50 mu L of DMEM, mixed evenly and then kept stand for 5min at room temperature; dissolving 2 μ L of transfection reagent lipofectamine 2000 in 50 μ L of DMEM, mixing uniformly, and standing at room temperature for 5 min; then mixing DMEM solution containing recombinant plasmid pC-fibers2A and transfection reagent lipofectamine 2000 into transfection solution, standing at room temperature for 20min, dripping the transfection solution into DF1 cells, culturing for 8h, replacing the cells with serum-containing DMEM, and continuously culturing for 48h for immunofluorescence test;

(2) gently washing DF1 cells with PBS for 2 times, adding pre-cooled 4% paraformaldehyde, and fixing at room temperature for 25 min; washing DF1 cells with PBS for 2 times, adding 0.25% TritonX-100, and reacting at room temperature for 5 min; washing DF1 cells with PBS for 2 times, adding the antibody of mouse anti-spike protein 1 and the antibody of rabbit anti-spike protein 2, and incubating for 1.5h at 37 ℃; DF1 cells were washed 2 times with PBS, added with FITC (green fluorescence) labeled secondary goat anti-mouse antibody and Cy3 (red fluorescence) labeled secondary goat anti-rabbit antibody, and incubated at 37 ℃ for 1 h; DF1 cells were washed 5 times with PBS and fluorescence was observed under an inverted fluorescence microscope. As shown in FIG. 4, both green and red fluorescence can be seen, indicating that both spike protein 1 and spike protein 2 are expressed in the cell.

Example 3: DNA vaccine immunoassay

(1) 10 SPF (specific pathogen free) chickens of 21 days old are inoculated with the recombinant plasmid pC-fibers2A, the inoculation amount of each SPF chicken is 200 mug, leg muscles are injected by 2 points, and the boosting immunization is carried out 14 days after the inoculation, wherein the inoculation amount and the method are the same as the first immunization and are used as an immunization group; meanwhile, the pCDNA3.1 empty vector injection is used as a control group, and 10 chickens in total are used;

(2) performing virus challenge 21 days after the boosting (HN15 strain is FAdV-4 and is originated from sick chicken infected with FAdV-4), wherein the virus challenge dose of each chicken is 100CLD50 (half lethal dose of chicken), the virus challenge mode is intramuscular injection, and the morbidity and mortality of the chicken are observed and recorded;

(3) the results show that part of the chickens in the control group start to get ill after 2 days after challenge, all the chickens get ill after 3 days, and the chickens mainly appear to be fluffy feather, depressed spirit and yellow-green sticky dilute manure and die after 14 days; the immunized group had 8 protected chicks and 2 had the disease without death.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> agricultural science and academy of Jiangsu province

<120> DNA vaccine for simultaneously expressing FAdV-4 spike protein 1 and spike protein 2 genes and construction method and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2799

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgtcggccc taatcgcctc cgcagccgat accgtctccg ccagcggaaa aaaacgaccc 60

cgcagggccc tatccgaacc tagccggtac ctttcggagg gcgacgagcg tcgaaaaccc 120

aaacgcgcgc gaccggccac ccgcgcgaat ggtcccctcc tcgatctggt gtatccattt 180

gacttcaatg cggggggagg aggtggcagc ggtggcggcg gtgggggagg tggaggtcag 240

cagatcgcgg tcgaccccga tgggccgctc gaactcactg gtgacctact gaccctcaac 300

accaaaacgc ccatttacgt cagcgatcga gcggtcagtc tgctcatcga tgacaatact 360

ttggccacta agcaagccaa cggggcgctc atggtcaaaa ccgcggcccc tctgaactcg 420

ggcactggtg gaggcgtcac gctaggcttc gaccctcgca ccatggcgct agattccgtc 480

accggggtgc tcaaagtgct cgtcgactca cagggacctc tacaagccga cacgggaggc 540

atcactctcc agttcgacac tcaagacttc gttgtcaaca atggcgtctt agcgctagcc 600

tcctcggtcg gtccgaccta tctgagcccc tttgcgacct acgaagtcac gcccgtcttg 660

ggaatatcgc agaggaacgg caacgtaaaa agcaagggct tgcaaaactg gtccataggc 720

tattacatct acatggtgag ctcagccggg ctagtcaacg gactcatcac tctggagcta 780

gcccatgacc tcacaggcgc gagcggagaa aacagcctga ctagcggtct caactttacc 840

tttgtgctca gccccatgta cccgatagaa acagaggtga atttgtccct catcgtgccg 900

cccacggtct cgccgaccaa tcaaaaccac gtgtttgtgc ccaatagcaa ccagagcgac 960

gtgggctatc tcgggctgcc gcctcatacc agggacaatt ggtacgtgcc catcgactcg 1020

cccggcctgc ggctcgtctc tttcatgccc accgccaccg gaaacgagaa attcggacag 1080

ggcacgttgg gatactgcgc cgccaccatc cagaacacgt ccagcggaac cacgccgtcg 1140

gatgcgatag ccttcactgt ctcgctgccg cagacctccg gctccaactg gtttgaccag 1200

aacgcgcccg acactgtggt gacgaccggt cctatccctt tttcctatca gggttacgtc 1260

tactccccca acgggaacaa tgctccgggc cccggcagcg gcgcgaccaa ctttagcctg 1320

cttaagcagg cgggcgatgt ggaagaaaac ccgggcccga tgctccgggc ccctaaaaga 1380

agacattccg aaaacgggaa gcccgagacc gaagcgggac cttccccggc tccaatcaag 1440

cgcgccaaac gcatggtgag agcatcccag cttgacctgg tttatccttt cgattacgtg 1500

gccgaccccg tcggagggct caacccgcct tttttgggag gctcaggacc cctagtggac 1560

cagggcggac agcttacgct caacgtcacc gatcccatca tcatcaagaa cagatcggtg 1620

gacttggccc acgaccccag tctcgatgtc aacgcccaag gtcaactggc ggtggccgtt 1680

gaccccgaag gggccctgga catcaccccc gatggactgg acgtcaaggt cgacggagtg 1740

accgtaatgg tcaacgatga ctgggaactg gccgtaaaag tcgacccgtc cggcggattg 1800

gattccaccg cgggtggact gggggtcagc gtggacgaca ccttgctcgt ggatcaggga 1860

gaactgggcg tacacctcaa ccaacaagga cccatcactg ccgatagcag tggtatcgac 1920

ctcgagatca atcctaacat gttcacggtc aacacctcga ccggaagcgg agtgctggaa 1980

ctcaacctaa aagcgcaggg aggcatccaa gccgacagtt cgggagtggg cgtttccgtg 2040

gatgaaagcc tacagattgt caacaacact ctggaagtga aaccggatcc cagcggaccg 2100

cttacggtct ccgccaatgg cctagggctg aagtacgaca ctaataccct agcggtgacc 2160

gcgggcgctt taaccgtggt cggagggggg agcgtctcca cacccatcgc tacttttgtc 2220

tcgggaagtc ccagcctcaa cacctacaat gccacgaccg tcaattccag cgcgaacgcc 2280

ttctcttgcg cctactacct tcaacagtgg aacatacagg ggctccttgt tacctccctc 2340

tacttgaaat tggacagcgc caccatgggg aatcgccctg gggacctcaa ctccgccaat 2400

gccaaatggt tcaccttttg ggtgtccgcc tatctccagc aatgcaaccc ctccgggatt 2460

caagcgggaa cggtcagccc ctccaccgcc accctcacgg actttgaacc catggccaat 2520

aggagcgtga ccagcccatg gacgtactcg gccaatggat actatgaacc atccatcggg 2580

gaattccaag tgttcagccc ggtggtaaca ggtgcctgga acccgggaaa catagggatc 2640

cgcgtcctcc ccgtgccggt ttcggcctcc ggagagcgat acacccttct atgctatagt 2700

ctgcagtgca cgaacgcgag catttttaat ccaaacaaca gcggaaccat gatcgtggga 2760

cccgtgctct acagctgtcc agcggcctcc ctcccgtaa 2799

<210> 2

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

aattaagctt atgtcggccc taatcgcctc cgcagccg 38

<210> 3

<211> 56

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cccgcctgct taagcaggct aaagttggtc gcgccgctgc cggggcccgg agcatt 56

<210> 4

<211> 55

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcttaagcag gcgggcgatg tggaagaaaa cccgggcccg atgctccggg cccct 55

<210> 5

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

taaagcggcc gcttacggga gggaggccgc tggacagctg 40