CN113583978A - 3 kinds of recombinant adenovirus, RBD of SARS-CoV-2 Spike protein and their application - Google Patents

3 kinds of recombinant adenovirus, RBD of SARS-CoV-2 Spike protein and their application Download PDF

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CN113583978A
CN113583978A CN202010369075.2A CN202010369075A CN113583978A CN 113583978 A CN113583978 A CN 113583978A CN 202010369075 A CN202010369075 A CN 202010369075A CN 113583978 A CN113583978 A CN 113583978A
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张林琦
周东明
李明茜
史宣玲
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Tsinghua University
Tianjin Medical University
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Tianjin Medical University
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Abstract

The invention discloses 3 kinds of recombinant adenovirus, RBD of SARS-CoV-2 Spike protein and their application. The present invention provides recombinant adenoviruses: expressing the recombinant adenovirus of the protein shown in the sequence 3 of the sequence table; expressing the recombinant adenovirus of the protein shown in the sequence 5 of the sequence table; a recombinant adenovirus expressing the protein shown in the sequence 1 of the sequence table. The protein shown in the sequence 1 of the sequence table is the full-length SARS-CoV-2 Spike protein. The protein shown in sequence 3 of the sequence table is the SARS-CoV-2 receptor binding domain RBD protein with membrane anchoring signal peptide. The protein shown in the sequence 5 of the sequence table is SARS-CoV-2 receptor binding domain RBD protein with secretory signal peptide. The vaccine developed by the invention aiming at the novel coronavirus SARS-CoV-2 has important theoretical guidance value and wide application prospect, and provides possibility for radical treatment of the novel coronavirus pneumonia.

Description

3 kinds of recombinant adenovirus, RBD of SARS-CoV-2 Spike protein and their application
Technical Field
The present invention relates to 3 kinds of recombinant adenovirus, RBD of SARS-CoV-2 Spike protein and their application.
Background
The majority of SARS-CoV-2 infected patients develop severe respiratory disease with clinical symptoms very similar to those developed in 2003 by SARS-CoV. This disease is highly worldwide because it can be transmitted to humans. To date, no specific drugs or vaccines have been available for the treatment or prevention of this disease.
The current outbreak of the new Coronavirus pneumonia epidemic and the SARS epidemic that has been outbreak in 2002 are caused by previously unknown Coronavirus (CoV). Coronavirus can be transmitted through a spray, respiratory secretion contact and other ways due to unpredictability, thereby bringing serious consequences and becoming one of the great threats affecting human health.
Disclosure of Invention
The invention aims to provide 3 kinds of recombinant adenovirus, RBD of SARS-CoV-2 Spike protein and application thereof.
The recombinant adenovirus provided by the invention is (a1), (a2), (a3) or (a 4):
(a1) expressing the recombinant adenovirus of the protein shown in the sequence 3 of the sequence table;
(a2) expressing the recombinant adenovirus of the protein shown in the sequence 5 of the sequence table;
(a3) expressing the recombinant adenovirus of the protein shown in the sequence 1 of the sequence table;
(a4) expressing the recombinant adenovirus of the protein consisting of the 14 th to 236 th amino acid residues in the sequence 3 of the sequence table.
The protein shown in the sequence 1 of the sequence table is the full-length SARS-CoV-2 Spike protein.
The protein shown in sequence 3 of the sequence table is the SARS-CoV-2 receptor binding domain RBD protein with membrane anchoring signal peptide.
The protein shown in the sequence 5 of the sequence table is SARS-CoV-2 receptor binding domain RBD protein with secretory signal peptide.
The protein composed of 14 th to 236 th amino acid residues in the sequence 3 of the sequence table is SARS-CoV-2 receptor binding domain RBD protein.
The invention also protects the protein as follows (b1), (b2) or (b 3):
(b1) protein shown in a sequence 3 in a sequence table;
(b2) protein shown in a sequence 5 in a sequence table;
(b3) and (b) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of (b1) or (b 2).
Illustratively, the labels are shown in table 1. The relationship between the labels in table 1 can be and or can be a relationship of or.
TABLE 1
Label (R) Residue of Sequence of
Poly-Arg 5-6 (typically 5) RRRRR
Poly-His 2-10 (typically 6)One) HHHHHH
FLAG 8 DYKDDDDK
Strep-tag II 8 WSHPQFEK
c-myc 10 EQKLISEEDL
The invention also protects nucleic acid molecules encoding said proteins.
The nucleic acid molecule may specifically be a DNA molecule.
The DNA molecule may specifically be (c1) or (c2) or (c3) as follows:
(c1) a DNA molecule shown in a sequence 4 of a sequence table;
(c2) a DNA molecule shown in a sequence 6 of a sequence table;
(c3) a DNA molecule shown in a sequence 2 of a sequence table.
The invention also protects the recombinant plasmid which is (d1), (d2), (d3) or (d 4):
(d1) inserting a DNA molecule of a protein shown in a sequence 3 of a coding sequence table into a chimpanzee adenovirus vector to obtain a recombinant plasmid;
(d2) inserting a DNA molecule of a protein shown in a sequence 5 of a coding sequence table into a chimpanzee adenovirus vector to obtain a recombinant plasmid;
(d3) inserting a DNA molecule of a protein shown in a sequence 1 of a coding sequence table into a chimpanzee adenovirus vector to obtain a recombinant plasmid;
(d4) the recombinant plasmid is obtained by inserting DNA molecules of protein consisting of 14 th to 236 th amino acid residues in a sequence 3 of a coding sequence table into a chimpanzee adenovirus vector.
The DNA molecule of the protein shown in the sequence 3 of the coding sequence table can be specifically the DNA molecule shown in the sequence 4 of the sequence table.
The DNA molecule of the protein shown in the sequence 5 of the coding sequence table can be specifically the DNA molecule shown in the sequence 6 of the sequence table.
The DNA molecule of the protein shown in the sequence 1 of the coding sequence table can be specifically the DNA molecule shown in the sequence 2 of the sequence table.
The DNA molecule of the protein consisting of 14 th to 236 th amino acid residues in the sequence 3 of the coding sequence table can be specifically a DNA molecule shown by 40 th to 708 th nucleotides in the sequence 4 of the sequence table.
The chimpanzee adenoviral vector can be an adenoviral vector derived from Simian adenoviruses 25(NCBI Reference Sequence: AC _ 000011.1).
In the chimpanzee adenovirus vector, Simian adenoviruses 25 is modified as follows: both E1 and E3 partial regions have been deleted, the E1 partial region is deleted so that the produced recombinant virus cannot replicate in normal cells to enhance the biosafety thereof, and the E3 partial region is deleted to increase the insertion capacity of foreign genes.
The chimpanzee adenovirus vector can be pAdC68XY4 pAdC 68-delta E1(GFP) -delta E3(SwaI) -E4(orf3-6Hu5) vector.
In the recombinant plasmid, a DNA molecule is inserted into a delta E1 region of a chimpanzee adenovirus vector.
In the recombinant plasmid, the DNA molecule was used to replace a small fragment between the two SrfI cleavage sites of the pAdC68XY4 pAdC 68-delta E1(GFP) -delta E3(SwaI) -E4(orf3-6Hu5) vector.
The invention also protects the recombinant adenovirus, which is obtained by transfecting any recombinant plasmid into an adenovirus packaging cell and then carrying out cell culture.
The cell culture may be a single cell culture.
The cell culture may be a multiple continuous cell culture.
When the cell culture is repeated continuous cell culture, the first cell culture is to transfect recombinant plasmid into adenovirus packaging cells, and then culture. When the cell culture is multiple continuous cell culture, starting from the second cell culture, the steps of each cell culture are as follows: after the last generation of cells are cultured, the cells are collected and crushed, and then the collected supernatant is infected with new adenovirus packaging cells and then cultured. When the cell culture is repeated continuous cell culture, after the last cell culture, the cells are collected and crushed, and then the collected supernatant is subjected to virus purification to obtain virus liquid.
The preparation method of the recombinant adenovirus sequentially comprises the following steps:
(1) transfecting the linearized recombinant plasmid into adenovirus packaging cells, and then culturing until about 80% of the cells can observe plaque formation;
(2) collecting cells, performing cell disruption by adopting a repeated freeze-thaw mode, and then centrifuging to collect supernatant.
The preparation method of the recombinant adenovirus sequentially comprises the following steps:
(1) transfecting the linearized recombinant plasmid into adenovirus packaging cells, and then culturing until about 80% of the cells can observe plaque formation;
(2) collecting cells, performing cell disruption by adopting a repeated freeze thawing mode, and then centrifuging to collect supernatant;
(3) infecting adenovirus packaging cells with the supernatant, and culturing until most cells are in a floating state;
(4) collecting cells, performing cell disruption by adopting a repeated freeze thawing mode, and then centrifuging to collect supernatant;
(5) infecting adenovirus packaging cells with the supernatant, and culturing until most cells are in a floating state;
(6) collecting cells, performing cell disruption by adopting a repeated freeze thawing mode, and then centrifuging to collect supernatant;
(7) infecting adenovirus packaging cells with the supernatant, and culturing until most cells are in a floating state;
(8) collecting cells, performing cell disruption by adopting a repeated freeze thawing mode, and then centrifuging to collect supernatant;
(9) and taking the supernatant, and performing cesium chloride density gradient centrifugation purification to obtain virus liquid.
The linearized recombinant plasmid is specifically: the large fragment (about 31kb) obtained by digesting the recombinant plasmid with the restriction enzyme PacI.
The invention also provides a kit for preparing the recombinant adenovirus, which comprises any one of the recombinant plasmid and the adenovirus packaging cell.
The adenovirus packaging cell has adenovirus E1 gene. The adenovirus packaging cell is specifically HEK293A cell.
The invention also protects the application of any recombinant adenovirus in the preparation of a novel coronavirus vaccine.
The invention also protects the application of any protein or any nucleic acid molecule or any recombinant plasmid in the preparation of novel coronavirus vaccines.
The invention also protects the application of any recombinant adenovirus in preparing a novel coronavirus resistant medicament. The recombinant adenovirus can cause organisms to produce antibodies, and the antibodies have a neutralizing effect on the novel coronavirus.
The invention also protects the application of any protein or any nucleic acid molecule or any recombinant plasmid in the preparation of the novel coronavirus resistant medicine.
The invention also protects a product, and the active ingredient of the product is any one of the recombinant adenovirus.
The invention also protects a product, the active component of which is any one of the proteins or any one of the nucleic acid molecules or any one of the recombinant plasmids.
The application of the product is (e1) or (e 2):
(e1) as a novel coronavirus vaccine;
(e2) can be used as an anti-novel coronavirus drug.
The invention also protects the application of the kit in the preparation of a novel coronavirus vaccine.
The invention also protects the application of the kit in preparing the novel coronavirus resistant medicine.
The invention comprises 3 SARS-CoV-2 antigen protein forms: the full-length S protein ensures the original S structure as much as possible, not only retains the membrane anchoring signal peptide, but also retains the transmembrane region and the intracellular region at the rear part of S2, and simulates the state that the spike protein is anchored on the cell surface as much as possible, thereby being used as a powerful immunogen; the SARS-CoV-2 receptor binding domain RBD protein with membrane anchoring signal peptide retained the original membrane anchoring signal peptide, mimicking the region of the membrane protein that binds to the target cell receptor ACE 2; SARS-CoV-2 receptor binding domain RBD protein having a secretory signal peptide A secretory signal peptide is used to secrete soluble RBD protein out of the cell membrane. The invention inserts full-length SARS-CoV-2 Spike protein or SARS-CoV-2 receptor binding domain RBD protein with membrane anchoring signal peptide or SARS-CoV-2 receptor binding domain RBD protein with secretory signal peptide into delta E1 region of chimpanzee adenovirus vector, and introduces genetic material into target cell to transcribe and express corresponding antigen protein by adenovirus infected cell. The full-length SARS-CoV-2 Spike protein has membrane anchoring signal peptide, transmembrane region and intracellular region, so that the expressed protein is not secreted to the outside of cell, but rather simulates the surface Spike of virus particle and is inserted into the membrane of target cell to produce the action of immunogen and stimulate the body to produce immune reaction. The RBD protein of SARS-CoV-2 receptor binding domain with membrane anchoring signal peptide is secreted out of cell, and can play the role of immunogen and stimulate organism to produce immune response. The SARS-CoV-2 receptor binding domain RBD protein with secretory signal peptide can enhance yield, secrete to the outside of cell, play the role of immunogen and stimulate organism to produce immune response.
The invention avoids the defect of pre-existing immunity of human type 5 adenovirus vectors, simultaneously retains the advantages of high titer and easy production and storage of adenovirus, provides an effective strategy of vaccine for radically treating novel coronavirus pneumonia, and has wide application prospect. The vaccine developed by the invention aiming at the novel coronavirus SARS-CoV-2 has important theoretical guidance value and wide application prospect, and provides possibility for radical treatment of the novel coronavirus pneumonia.
Drawings
FIG. 1 is a graph showing the results of antigen expression identification in example 1.
Fig. 2 shows the results of the vaccine-induced total antibody detection (logarithmic result with ED50 value base 10).
FIG. 3 shows the results of the antibody neutralization activity assay (logarithmic results with ID50 value at base 10) in the serum of animals immunized with the vaccine.
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. The HEK293A cell is an adenovirus packaging cell and has an E1 gene.
Example 1 preparation and characterization of recombinant viruses
Construction of recombinant plasmid
The vector pAdC68XY4 pAdC 68-delta E1(GFP) -delta E3(SwaI) -E4(orf3-6Hu5) is a circular plasmid, and is shown as a sequence 7 in a sequence table. pAdC68XY4 pAdC68- Δ E1(GFP) - Δ E3(SwaI) -E4(orf3-6Hu5) the vector was derived from Simian adonovirus 25(NCBI Reference Sequence: AC _000011.1) and was engineered as follows: the partial regions of E1 and E3 are deleted, the partial region of E1 prevents the produced recombinant virus from replicating in common cells to enhance the biosafety, and the partial deletion of E3 increases the insertion capacity of the exogenous gene. The vector pAdC68XY4 pAdC 68-delta E1(GFP) -delta E3(SwaI) -E4(orf3-6Hu5) has two SrfI cleavage recognition sequences, and a small fragment between the two cleavage recognition sequences has the EGFP gene. When a recombinant plasmid was constructed using the vector pAdC68XY4 pAdC68- Δ E1(GFP) - Δ E3(SwaI) -E4(orf3-6Hu5), a site between two SrfI cleavage sites was selected as an insertion site for a foreign DNA molecule, which is the Δ E1 region.
A small fragment between two SrfI enzyme cutting sites of the pAdC68XY4 pAdC 68-delta E1(GFP) -delta E3(SwaI) -E4(orf3-6Hu5) vector is replaced by a double-stranded DNA molecule shown in a sequence 2 of a sequence table to obtain a recombinant plasmid ChAdTS-COVID-19S. The DNA molecule shown in sequence 2 of the sequence table encodes the protein shown in sequence 1 of the sequence table, and the protein shown in sequence 1 of the sequence table is the full-length SARS-CoV-2 Spike protein.
A small fragment between two SrfI enzyme cutting sites of the pAdC68XY4 pAdC 68-delta E1(GFP) -delta E3(SwaI) -E4(orf3-6Hu5) vector is replaced by a double-stranded DNA molecule shown in a sequence 4 of a sequence table to obtain a recombinant plasmid ChAdTS-COVID-19 RBD. The DNA molecule shown in sequence 4 of the sequence table encodes the protein shown in sequence 3 of the sequence table, and the protein shown in sequence 3 of the sequence table is the SARS-CoV-2 receptor binding domain RBD protein with membrane anchoring signal peptide. The protein shown in sequence 3 of the sequence table has a membrane anchoring signal peptide "MFVFLVLLPLVSS".
A small fragment between two SrfI enzyme cutting sites of the pAdC68XY4 pAdC 68-delta E1(GFP) -delta E3(SwaI) -E4(orf3-6Hu5) vector is replaced by a double-stranded DNA molecule shown in a sequence 6 of a sequence table to obtain a recombinant plasmid ChAdTS-COVID-19 RBDs. The DNA molecule shown in sequence 6 of the sequence table encodes the protein shown in sequence 5 of the sequence table, and the protein shown in sequence 5 of the sequence table is SARS-CoV-2 receptor binding domain RBD protein with secretory signal peptide. In sequence 5 of the sequence table, there is a secretory signal peptide "MGWSCIILFLVATATCVHS".
Secondly, preparing virus liquid
Cell culture conditions: 37 ℃ and 5% CO2The constant temperature incubator.
1. HEK293A cells were cultured in DMEM medium containing 10% fetal bovine serum until the cell density reached 80%.
2. The recombinant plasmid is taken and digested by restriction enzyme PacI to release the recombinant virus genome, and a large fragment of about 31kb is recovered.
3. The large fragment obtained in step 2 was transfected into the cells completing step 1 by means of Lipofectamine2000 and cultured for 2 hours (with serum-free DMEM medium), and then the cells were transferred to DMEM medium containing 10% fetal bovine serum until plaque formation was observed in about 80% of the cells (about 10-14 days).
4. After completion of step 3, the cells were collected, resuspended in serum-free DMEM medium, then repeatedly frozen and thawed 3 times, and then centrifuged at 3000g for 10 minutes at 4 ℃ to collect the supernatant (P0 generation supernatant).
5. HEK293A cells were cultured in DMEM medium containing 10% fetal bovine serum until the cell density reached 80%.
6. The supernatant of P0 generation was infected with the cells that completed step 5, and cultured until the vast majority of the cells were in a floating state (about 24-48 hours).
7. After completion of step 6, the cells were collected, resuspended in serum-free DMEM medium, then freeze-thawed 3 times repeatedly, and then centrifuged at 3000g for 10 minutes at 4 ℃ to collect the supernatant (P1 generation supernatant).
8. HEK293A cells were cultured in DMEM medium containing 10% fetal bovine serum until the cell density reached 80%.
9. The supernatant of P1 generation was infected with the cells that completed step 8, and cultured until the vast majority of the cells were in a floating state (approximately 24-48 hours).
10. After completion of step 9, the cells were collected, resuspended in serum-free DMEM medium, then freeze-thawed 3 times repeatedly, and then centrifuged at 3000g for 10 minutes at 4 ℃ to collect the supernatant (P2 generation supernatant).
11. HEK293A cells were cultured in DMEM medium containing 10% fetal bovine serum until the cell density reached 80%.
12. The supernatant of P2 generation was infected with the cells that completed step 11, and cultured until the vast majority of the cells were in a floating state (about 24-48 hours).
13. After completion of step 12, the cells were collected, resuspended in serum-free DMEM medium, then freeze-thawed 3 times repeatedly, and then centrifuged at 3000g for 10 minutes at 4 ℃ to collect the supernatant (P3 generation supernatant).
14. And (4) taking the supernatant P3, and performing cesium chloride density gradient centrifugation purification to obtain virus liquid.
When the recombinant plasmid ChAdTS-COVID-19S is adopted, the obtained virus liquid is named as ChAdTS-COVID-19S virus liquid.
When the recombinant plasmid ChAdTS-COVID-19RBD is adopted, the obtained virus liquid is named as ChAdTS-COVID-19RBD virus liquid.
When the recombinant plasmid ChAdTS-COVID-19RBDs is adopted, the obtained virus liquid is named as ChAdTS-COVID-19RBDs virus liquid.
The above procedure was carried out using pAdC68XY4 pAdC68- Δ E1(GFP) - Δ E3(SwaI) -E4(orf3-6Hu5) vector in place of the recombinant plasmid, and the resulting virus solution was designated as ChAdTS virus solution.
Thirdly, enzyme digestion identification
Test virus solutions: ChAdTS-COVID-19S virus liquid, ChAdTS-COVID-19RBD virus liquid, ChAdTS-COVID-19RBDs virus liquid or ChAdTS virus liquid. And taking the virus liquid to be tested, and extracting genome DNA. Taking genome DNA, adopting restriction enzyme MfeI to perform enzyme digestion, and then performing electrophoresis detection. The ChAdTS-COVID-19S virus liquid shows 12 bands, the ChAdTS-COVID-19RBD virus liquid and the ChAdTS-COVID-19RBDs virus liquid show 11 bands, and the ChAdTS virus liquid shows 9 bands.
Positive control: recombinant plasmid ChAdTS-COVID-19S, recombinant plasmid ChAdTS-COVID-19RBD, recombinant plasmid ChAdTS-COVID-19RBDs or pAdC68XY4 pAdC 68-delta E1(GFP) -delta E3(SwaI) -E4(orf3-6Hu5) vectors. Taking a positive control, carrying out enzyme digestion by using restriction enzyme MfeI and restriction enzyme PacI, and carrying out electrophoresis detection. The recombinant plasmid ChAdTS-COVID-19S showed 12 bands, the recombinant plasmid ChAdTS-COVID-19RBD and the recombinant plasmid ChAdTS-COVID-19RBDs showed 11 bands, and the vector pAdC68XY4 pAdC68- Δ E1(GFP) - Δ E3(SwaI) -E4(orf3-6Hu5) showed 9 bands.
Fourth, identification of the virus
1. Titer identification
Test virus solutions: ChAdTS-COVID-19S virus liquid, ChAdTS-COVID-19RBD virus liquid, ChAdTS-COVID-19RBDs virus liquid or ChAdTS virus liquid.
The titer of the test virus fluid was measured by absorptiometry.
Viral titer OD260 × dilution fold × 1.1 × 1012。
The unit of the virus titer is the number of virus particles per milliliter (vp/mL)
The titer of the ChAdTS-COVID-19S virus solution is 7.5 multiplied by 1012vp/mL。
The titer of the ChAdTS-COVID-19RBD virus solution is 6 multiplied by 1012vp/mL。
The titer of the ChAdTS-COVID-19RBDs virus liquid is 1×1013vp/mL。
The titer of the ChAdTS virus solution is 1.5X 1013vp/mL。
2. Identification of antigen expression
Test virus solutions: ChAdTS-COVID-19S virus liquid, ChAdTS-COVID-19RBD virus liquid, ChAdTS-COVID-19RBDs virus liquid or ChAdTS virus liquid.
Cell culture conditions: 37 ℃ and 5% CO2The constant temperature incubator.
Firstly, 5X 10 HEK293A cells are cultured5Six-hole plates are inoculated at the density of each cell/hole, and the cells are cultured until the cell density is about 90 percent.
② infecting the cells of the step I with test virus liquid, the infecting dose is 109vp/well, set to 3 replicates. After 24 hours of culture, the cells were collected.
Thirdly, the cells collected in the second step are respectively subjected to cell lysis, then the supernatant is collected and subjected to polyacrylamide gel electrophoresis, and then Western Blot (primary antibody adopts rabbit polyclonal antibody against SARS-CoV-2 Spike). Beta-actin protein is used as an internal reference. The information for the rabbit polyclonal antibody against SARS-CoV-2 Spike is as follows: SARS-CoV Spike Antibody, Rabbit PAb, Antibody Affinity Purified: zhou Yi Qiao Shen, 40150-T62-COV 2.
The results are shown in FIG. 1.
Example 2 use of recombinant viruses
The virus solutions used for animal immunization were all the virus solutions prepared in example 1.
First, animal immunization
Female BALB/C mice, 6 weeks old, were divided into 24 groups of 5 mice each, and treated as follows:
first group (group G1): a single immunization is carried out by nasal drip, and the immune product is ChAdTS virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)8vp)；
Second group (group G2): a single immunization is carried out by nasal drip, and the immune product is ChAdTS virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)9vp)；
Third group (group G3): single immunization by nasal dropThe immune material is ChAdTS virus liquid (the virus amount given to a single mouse is 2X 10)10vp)；
Fourth group (group G4): a single immunization is carried out by intramuscular injection, and the immune product is ChAdTS virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)8vp)；
Fifth group (group G5): a single immunization is carried out by intramuscular injection, and the immune product is ChAdTS virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)9vp)；
Sixth group (group G6): a single immunization is carried out by intramuscular injection, and the immune product is ChAdTS virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)10vp)；
Seventh group (group G7): single immunization is carried out by nasal drip, and the immune product is ChAdTS-COVID-19S virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)8vp)；
Eighth group (group G8): single immunization is carried out by nasal drip, and the immune product is ChAdTS-COVID-19S virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)9vp)；
Ninth group (group G9): single immunization is carried out by nasal drip, and the immune product is ChAdTS-COVID-19S virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)10vp)；
Tenth group (group G10): the single immunization is carried out by intramuscular injection, and the immune product is ChAdTS-COVID-19S virus solution (the virus dose given to a single mouse is 2 multiplied by 10)8vp)；
Eleventh group (group G11): the single immunization is carried out by intramuscular injection, and the immune product is ChAdTS-COVID-19S virus solution (the virus dose given to a single mouse is 2 multiplied by 10)9vp)；
Tenth group (group G12): the single immunization is carried out by intramuscular injection, and the immune product is ChAdTS-COVID-19S virus solution (the virus dose given to a single mouse is 2 multiplied by 10)10vp)；
Third group (group G13): the single immunization is carried out by nasal drip, and the immune material is ChAdTS-COVID-19RBD virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)8vp)；
Fourteenth aspect of the inventionGroup (G14 group): the single immunization is carried out by nasal drip, and the immune material is ChAdTS-COVID-19RBD virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)9vp)；
Fifteenth group (group G15): the single immunization is carried out by nasal drip, and the immune material is ChAdTS-COVID-19RBD virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)10vp)；
Sixteenth group (group G16): the single immunization is carried out by intramuscular injection, and the immune product is ChAdTS-COVID-19RBD virus solution (the virus dose given to a single mouse is 2 x 10)8vp)；
Seventeenth group (group G17): the single immunization is carried out by intramuscular injection, and the immune product is ChAdTS-COVID-19RBD virus solution (the virus dose given to a single mouse is 2 x 10)9vp)；
Eighteenth group (group G18): the single immunization is carried out by intramuscular injection, and the immune product is ChAdTS-COVID-19RBD virus solution (the virus dose given to a single mouse is 2 x 10)10vp)；
Nineteenth group (group G19): single immunization is carried out by nasal drip, and the immune material is ChAdTS-COVID-19RBDs virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)8vp)；
Twentieth group (group G20): single immunization is carried out by nasal drip, and the immune material is ChAdTS-COVID-19RBDs virus liquid (the virus dose given to a single mouse is 2 multiplied by 10)9vp)；
Twentieth group (group G21): single immunization is carried out by nasal drip, and the immune product is ChAdTS-COVID-19RBDs virus solution (the virus dose given to a single mouse is 2 multiplied by 10)10vp)；
Twenty-second group (group G22): the single immunization is carried out by intramuscular injection, and the immune product is ChAdTS-COVID-19RBDs virus solution (the virus dose given to a single mouse is 2 multiplied by 10)8vp)；
Twenty-third group (group G23): the single immunization is carried out by intramuscular injection, and the immune product is ChAdTS-COVID-19RBDs virus solution (the virus dose given to a single mouse is 2 multiplied by 10)9vp)；
Twenty-fourth group (group G24): single injection by intramuscular injectionImmunization, the immune material is ChAdTS-COVID-19RBDs virus liquid (the virus amount given to a single mouse is 2 x 10)10vp)。
In each of the above groups, the immunization volume for the nasal drip immunization was 10. mu.l per mouse, and the immunization volume for the intramuscular injection immunization was 100. mu.l per mouse, and the virus concentration was adjusted using PBS buffer solution of pH7.2 as a solvent.
Blood was collected once before immunization (cheek blood).
Blood was collected every two weeks after immunization (cheek blood).
Preparation of SARS-CoV-2 pseudovirus
The plasmid expressing the full-length SARS-CoV-2 Spike protein (named as SARS-CoV-2 plasmid) and the skeleton plasmid pNL4-3R-E-luciferase transfect 293T cells together, after incubation, SARS-CoV-2 pseudotyped virus with infectivity but without replication capacity can be obtained, and the infectivity is similar to that of live virus. Backbone plasmid pNL4-3R-E-Luciferase, i.e.a backbone plasmid pNL4-3R-E containing Luciferase (i.e.vector with the Luciferase conjugation backbone pNL4-3R-E in the literature): wang Q, Liu L, Ren W, Gettie a, Wang H, Liang Q, Shi X, Montefiori DC, Zhou T, Zhang l.cell rep.2019.
Inserting the double-stranded DNA molecule shown in the sequence 2 of the sequence table between the enzyme cutting sites of BamHI and EcoRI of the pcDNA3.1(+) vector to obtain SARS-CoV-2 plasmid.
The SARS-CoV-2 plasmid and the skeleton plasmid pNL4-3R-E-luciferase are co-transfected into 293T cells, and are kept still at 37 ℃ for incubation (a DMEM culture medium containing 10 percent fetal calf serum is adopted), cell culture supernatant is collected after transfection for 48 hours, and virus liquid containing SARS-CoV-2 pseudovirus (SARS-CoV-2 virus liquid for short) is obtained.
Preparation of SARS-CoV-2RBD protein
1. Inserting the double-stranded DNA molecule shown in the sequence 8 of the sequence table into the NheI and HindIII enzyme cutting sites of the pcDNA3.1(+) vector to obtain the recombinant plasmid. The recombinant plasmid expresses the fusion protein, and the signal peptide is cut off to form mature protein. The mature protein sequentially consists of the following elements from N end to C end: SARS-CoV-2RBD (composed of 14 th to 236 th amino acid residues in sequence 3 of the sequence table), Strep-tag II tag and FLAG tag.
2. The recombinant plasmid obtained in step 1 is transfected into 293T cells growing to 90% density by PEI transfection reagent, and is firstly cultured for 6-8 hours in serum-free DMEM medium, and then is cultured for 72 hours in DMEM medium containing 10% fetal calf serum.
3. After step 2 is completed, the supernatant is collected, affinity purification is performed using streptavidin, and then the purified protein solution is collected.
4. And (3) concentrating the protein solution obtained in the step (3) and replacing the system, and replacing the protein system with PBS (phosphate buffer solution) with pH7.2 to obtain the SARS-CoV-2RBD protein solution.
Fourth, detection of vaccine-induced Total antibodies
And (4) taking the blood sample obtained in the step one, separating serum, and detecting the total IgG by adopting ELISA. In the total IgG detection, an ELISA plate (100ng protein/well) is coated with SARS-CoV-2RBD protein prepared in the third step, serum is firstly diluted to 200 times of volume and then subjected to gradient dilution by 3 times (8 dilutions are performed in PBS buffer solution with the solvent of pH7.2), and the secondary antibody is Anti-mouse IgG HRP.
ED50 value (half maximal effect dilution factor): dilution factor that caused 50% of the maximal effect.
The log base 10 ED50 values for sera from several groups of mice are shown in figure 2(W for weeks).
The ED50 values of the sera of the groups of mice at 4 weeks of immunization are shown in Table 2 (N.D. test result is negative).
TABLE 2
Figure BDA0002477363690000081
Figure BDA0002477363690000091
Fifth, detecting the neutralizing activity of the antibody in the animal serum after vaccine immunization
Solution to be tested: taking the blood sample obtained in the first step, and separating the obtained serum.
1. And (3) diluting the solution to be detected to 48 times by adopting a DMEM medium containing 10% FBS, and then diluting the solution to be detected in a gradient manner by 3 times to obtain diluents with different serum concentrations in sequence.
2. 100 microliters of the dilution obtained in step 1 was mixed with 50 microliters of SARS-CoV-2 virus solution (virus content: 100TCID50) prepared in step two, and incubated at 37 ℃ for 1 hour. A blank control was set up with 100 μ l DMEM medium containing 10% FBS instead of 100 μ l of diluent.
3. After completion of step 2, 50. mu.l of cell fluid of Huh7 cells (about 2X 10 cells) was added4Huh7 cells), and standing and incubating for 48 hours (in practical application, 48-72 hours can be used) at 37 ℃.
4. After completion of step 3, 100. mu.l of PBS buffer and 50. mu.l of cell lysate (Bright-Glo) were addedTMLuciferase Assay System, Promega, E2650), left for 2min, and then Luciferase activity was detected using a chemiluminescence apparatus.
Each treatment was set up with 3 replicates and the results averaged.
Neutralization activity ═ (fluorescence intensity of blank-fluorescence intensity of experimental group to which diluent was added)/fluorescence intensity of blank × 100%.
The serum dilution at 50% neutralization activity corresponds to position ID 50.
The log base 10 of serum ID50 values for several groups of mice is shown in FIG. 3(W for weeks).
The serum ID50 values of each group of mice at 4 weeks of immunization are shown in Table 3 (N.D. test result is negative).
TABLE 3
Group of ID50 value Group of ID50 value
Group G1 N.D. Group G13 106.710587
Group G2 N.D. Group G14 86.0079676
Group G3 N.D. Group G15 96.6392092
Group G4 N.D. Group G16 N.D.
Group G5 N.D. Group G17 N.D.
Group G6 N.D. Group G18 34.883539
Group G7 67.7670975 Group G19 165.407734
Group G8 101.175906 Group G20 82.252005
Group G9 285.446729 Group G21 104.185559
Group G10 N.D. Group G22 N.D.
Group G11 127.326478 Group G23 49.0249729
Group G12 119.746191 Group G24 36.1500306
The results show that immune sera are very good at neutralizing SARS-CoV-2 pseudovirus.
SEQUENCE LISTING
<110> Qinghua university
Tianjin medical university
<120> 3 kinds of recombinant adenovirus, RBD of SARS-CoV-2 Spike protein and their application
<130> CGGNQAYX206031
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 1273
<212> PRT
<213> SARS-CoV-2
<400> 1
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala
675 680 685
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
690 695 700
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
705 710 715 720
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
725 730 735
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
740 745 750
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
755 760 765
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
770 775 780
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
785 790 795 800
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
820 825 830
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
835 840 845
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
850 855 860
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
865 870 875 880
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
885 890 895
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
900 905 910
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
915 920 925
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
945 950 955 960
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
965 970 975
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
980 985 990
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
995 1000 1005
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn
1010 1015 1020
Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys
1025 1030 1035
Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
1040 1045 1050
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val
1055 1060 1065
Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His
1070 1075 1080
Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn
1085 1090 1095
Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln
1100 1105 1110
Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val
1115 1120 1125
Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro
1130 1135 1140
Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1145 1150 1155
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
1160 1165 1170
Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu
1175 1180 1185
Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu
1205 1210 1215
Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met
1220 1225 1230
Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys
1235 1240 1245
Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro
1250 1255 1260
Val Leu Lys Gly Val Lys Leu His Tyr Thr
1265 1270
<210> 2
<211> 3822
<212> DNA
<213> SARS-CoV-2
<400> 2
atgttcgtgt tcctggtgct gctgcctctg gtgagcagcc agtgcgtgaa tctgaccacc 60
agaacccagc tgcctcctgc ctacaccaat agcttcacca gaggagttta ttatcccgat 120
aaggtgttca gaagtagtgt attacatagt acccaggacc tgttcctacc tttcttcagt 180
aacgtgacct ggttccacgc catccacgtg agcggcacca atggcaccaa gagattcgac 240
aatcctgtgc tgcctttcaa tgacggcgtg tacttcgcca gcaccgagaa gagcaatatc 300
atcagaggct ggatcttcgg caccaccttg gattccaaga ctcagagcct gctgattgta 360
aacaacgcta caaatgtggt gatcaaggtg tgcgagttcc agttctgcaa tgaccctttc 420
ctgggtgttt attatcataa gaacaacaag agctggatgg agagcgagtt ccgcgtatat 480
tcgtcggcta ataattgcac cttcgagtac gtgagccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaatttcaa gaatctgaga gagttcgtgt tcaagaatat cgacggctac 600
ttcaagatct acagcaagca cacacccatt aatctggtga gagacctgcc tcagggcttc 660
agcgccctgg agcctctggt ggacctgcct atcggcatca atatcaccag attccagacc 720
ctgctggccc tgcacagatc atatcttaca ccaggcgatt cgtcaagcgg ttggaccgct 780
ggagctgcgg catattacgt gggctacctg cagcctagaa ccttcctgct gaagtacaat 840
gagaatggta cgataaccga cgcagttgat tgtgccctgg accctctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa tttcagagtg 960
cagcctaccg agagcatcgt gagattccct aatatcacca atctgtgccc tttcggcgag 1020
gtgttcaatg ccaccagatt cgccagcgtg tacgcatgga accgcaagcg gataagcaat 1080
tgcgtggccg actacagcgt gctgtacaat agcgccagct tcagcacctt caaatgttat 1140
ggtgtttcgc caacaaagct gaatgacctg tgcttcacca atgtgtacgc cgacagcttc 1200
gtgatcagag gcgacgaggt gagacagatc gcgccagggc agaccggcaa gatcgccgac 1260
tacaattaca agctgcctga cgacttcacc ggctgcgtga tcgcgtggaa ctctaacaat 1320
ctagattcga aagttggagg caattacaat tacctgtaca gactgttcag aaagagcaat 1380
ctgaagcctt tcgagagaga catcagcacc gagatctacc aggccggcag cacaccgtgt 1440
aatggcgtgg agggcttcaa ttgctacttc cctctgcaga gctacggctt ccagcctacc 1500
aatggcgtgg gctaccagcc ttacagagtg gtggtgctga gcttcgagct gctgcacgct 1560
cccgctaccg tgtgcggccc taagaagagc accaatctgg tgaagaataa gtgcgtgaat 1620
ttcaatttca atggtctaac tggaacgggc gtgctgaccg agagcaataa gaagtttctt 1680
ccctttcaac aattcggcag agacatcgcc gacaccacag atgctgtaag agaccctcag 1740
accctggaga tcctggacat cactccgtgt agcttcggcg gcgtgagcgt gatcacaccg 1800
ggtaccaata ccagcaatca ggtggccgtg ctgtaccagg acgtgaattg caccgaggtg 1860
cctgtggcca tccacgccga ccagctgact cccacttgga gggtatattc cacgggaagc 1920
aatgtgttcc agaccagagc cggctgcctg atcggcgccg agcacgtgaa taatagctac 1980
gagtgcgaca tccctatcgg cgccggcatc tgcgccagct accagaccca gaccaatagc 2040
cctagaagag ccagaagcgt ggccagccag agcatcatcg cctacaccat gagcctgggc 2100
gccgagaata gcgtggccta cagcaataat agcatcgcca tccctaccaa tttcaccatc 2160
agcgtgacca ccgaaatatt accagtctcc atgaccaaga ccagcgtgga ctgcaccatg 2220
tacatctgcg gcgacagcac cgagtgcagc aatctgctgc tgcagtacgg cagcttctgc 2280
acccagctga atagagccct gaccggcatc gccgtggagc aggacaagaa tacccaggag 2340
gtgttcgccc aggtgaagca gatctacaag actccgccga tcaaggactt cggcggcttc 2400
aatttcagcc aaatactccc agatccaagc aagcctagca agaggagctt catcgaggac 2460
ctgctgttca ataaggtgac cctggccgac gccggcttca tcaagcagta cggcgactgc 2520
ctaggtgata ttgcggcaag agacctgatc tgcgcccaga agtttaacgg tttgacagta 2580
ctacctcctc tgctgaccga cgagatgata gcacaatata cgtcggcatt gctcgctggc 2640
acgatcacat cgggctggac tttcggcgcc ggagcagcgt tgcaaatccc tttcgccatg 2700
cagatggcct acagattcaa tggcatcggc gtgacccaga atgtgctgta cgagaatcag 2760
aagctgatcg ccaatcagtt caatagcgcc atcggcaaga tccaggacag cctgagcagc 2820
accgccagcg ccctgggcaa gctgcaggac gtggtgaatc agaatgccca ggccctgaat 2880
accctggtga agcagctgag cagcaatttc ggcgccatca gtagtgtact caacgatatc 2940
ctgagcagac tggacaaggt ggaggccgag gtgcaaattg atcgtcttat tactggcaga 3000
ctgcagagcc tgcagaccta cgtgacccag cagctgatca gagccgccga gatcagagcc 3060
agcgccaatc tggccgccac caagatgagc gagtgcgtgc tgggccagag caagagagtg 3120
gacttctgcg gcaagggcta ccacctgatg agcttccctc agagcgctcc acatggcgtg 3180
gtgttcctgc acgtgaccta cgtgcctgcc caggagaaga atttcaccac cgcacccgca 3240
atctgccacg acggcaaggc ccacttccct agagagggcg tgttcgtgag caatggcacc 3300
cactggttcg tgacccagag aaatttctac gagcctcaga tcatcaccac cgacaatacc 3360
ttcgtgagcg gcaattgcga cgtggtgatc gggatagtca ataatactgt ctacgaccct 3420
ctgcagcctg agctggacag cttcaaggag gagctggaca agtacttcaa gaatcacacc 3480
agccctgacg tggacctcgg tgatatttcg ggaatcaatg ccagcgtggt gaatatccag 3540
aaggaaattg atcggctcaa cgaagtggcc aagaatctga atgagagcct gatcgacctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggcctt ggtacatctg gctgggcttc 3660
atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gacctcctgt 3720
tgttcctgtt tgaaagggtg ttgttcgtgt gggtcctgct gcaagttcga cgaggacgac 3780
agcgagcctg tgctgaaggg cgtgaagctg cactacacct ag 3822
<210> 3
<211> 236
<212> PRT
<213> Artificial sequence
<400> 3
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Arg Val Gln
1 5 10 15
Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro
20 25 30
Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp
35 40 45
Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr
50 55 60
Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr
65 70 75 80
Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val
85 90 95
Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys
100 105 110
Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val
115 120 125
Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr
130 135 140
Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu
145 150 155 160
Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn
165 170 175
Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe
180 185 190
Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu
195 200 205
Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys
210 215 220
Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
225 230 235
<210> 4
<211> 711
<212> DNA
<213> Artificial sequence
<400> 4
atgttcgtgt tcctggtgct gctgcctctg gtgagcagca gagtgcagcc taccgagagc 60
atcgtgagat tccctaatat caccaatctg tgccctttcg gcgaggtgtt caatgccacc 120
agattcgcca gcgtgtacgc atggaaccgc aagcggataa gcaattgcgt ggccgactac 180
agcgtgctgt acaatagcgc cagcttcagc accttcaaat gttatggtgt ttcgccaaca 240
aagctgaatg acctgtgctt caccaatgtg tacgccgaca gcttcgtgat cagaggcgac 300
gaggtgagac agatcgcgcc agggcagacc ggcaagatcg ccgactacaa ttacaagctg 360
cctgacgact tcaccggctg cgtgatcgcg tggaactcta acaatctaga ttcgaaagtt 420
ggaggcaatt acaattacct gtacagactg ttcagaaaga gcaatctgaa gcctttcgag 480
agagacatca gcaccgagat ctaccaggcc ggcagcacac cgtgtaatgg cgtggagggc 540
ttcaattgct acttccctct gcagagctac ggcttccagc ctaccaatgg cgtgggctac 600
cagccttaca gagtggtggt gctgagcttc gagctgctgc acgctcccgc taccgtgtgc 660
ggccctaaga agagcaccaa tctggtgaag aataagtgcg tgaatttcta g 711
<210> 5
<211> 242
<212> PRT
<213> Artificial sequence
<400> 5
Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Cys
1 5 10 15
Val His Ser Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn
20 25 30
Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
35 40 45
Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala
50 55 60
Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys
65 70 75 80
Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val
85 90 95
Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala
100 105 110
Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp
115 120 125
Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser
130 135 140
Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser
145 150 155 160
Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala
165 170 175
Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro
180 185 190
Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro
195 200 205
Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr
210 215 220
Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val
225 230 235 240
Asn Phe
<210> 6
<211> 729
<212> DNA
<213> Artificial sequence
<400> 6
atgggatggt catgtatcat cctttttcta gtagcaactg caacctgtgt acattcaaga 60
gtgcagccta ccgagagcat cgtgagattc cctaatatca ccaatctgtg ccctttcggc 120
gaggtgttca atgccaccag attcgccagc gtgtacgcat ggaaccgcaa gcggataagc 180
aattgcgtgg ccgactacag cgtgctgtac aatagcgcca gcttcagcac cttcaaatgt 240
tatggtgttt cgccaacaaa gctgaatgac ctgtgcttca ccaatgtgta cgccgacagc 300
ttcgtgatca gaggcgacga ggtgagacag atcgcgccag ggcagaccgg caagatcgcc 360
gactacaatt acaagctgcc tgacgacttc accggctgcg tgatcgcgtg gaactctaac 420
aatctagatt cgaaagttgg aggcaattac aattacctgt acagactgtt cagaaagagc 480
aatctgaagc ctttcgagag agacatcagc accgagatct accaggccgg cagcacaccg 540
tgtaatggcg tggagggctt caattgctac ttccctctgc agagctacgg cttccagcct 600
accaatggcg tgggctacca gccttacaga gtggtggtgc tgagcttcga gctgctgcac 660
gctcccgcta ccgtgtgcgg ccctaagaag agcaccaatc tggtgaagaa taagtgcgtg 720
aatttctag 729
<210> 7
<211> 34078
<212> DNA
<213> Artificial sequence
<400> 7
gtcgagggat gagcgaccgt taggggcggg gcgagtgacg ttttgatgac gtggttgcga 60
ggaggagcca gtttgcaagt tctcgtggga aaagtgacgt caaacgaggt gtggtttgaa 120
cacggaaata ctcaattttc ccgcgctctc tgacaggaaa tgaggtgttt ctgggcggat 180
gcaagtgaaa acgggccatt ttcgcgcgaa aactgaatga ggaagtgaaa atctgagtaa 240
tttcgcgttt atggcaggga ggagtatttg ccgagggccg agtagacttt gaccgattac 300
gtgggggttt cgattaccgt gtttttcacc taaatttccg cgtacggtgt caaagtccgg 360
tgtttttacg tacgatatca tttccccgaa aagtgccacc tgaccgtaac tataacggtc 420
ctaaggtagc gaaagctcag atctcccgat cccctatggt gcactctcag tacaatctgc 480
tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag 540
tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag 600
aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt 660
tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 720
ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 780
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 840
actttccatt gacgtcaatg ggtggactat ttacggtaaa ctgcccactt ggcagtacat 900
caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 960
tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta 1020
ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 1080
cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 1140
tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 1200
atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga 1260
gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct 1320
ggctagcgtt taaacgggcc cgcccgggct tataagccac catggtgagc aagggcgagg 1380
agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca 1440
agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt 1500
tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct 1560
acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt 1620
ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact 1680
acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga 1740
agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca 1800
acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca 1860
agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca 1920
cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg 1980
ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg 2040
ccgccgggat cactctcggc atggacgagc tgtacaagta attataagcc cgggcaagct 2100
taagtttaaa ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 2160
gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 2220
aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 2280
tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg 2340
tgggctctat ggcttctgag gcggaaagaa ccagcagatc tgcagatctg aattcatcta 2400
tgtcgggtgc ggagaaagag gtaatgaaat ggcattatgg gtattatggg tctgcattaa 2460
tgaatcggcc agatatcgac atatgctggc caccgtacat gtggcttccc atgctcgcaa 2520
gccctggccc gagttcgagc acaatgtcat gaccaggtgc aatatgcatc tggggtcccg 2580
ccgaggcatg ttcatgccct accagtgcaa cctgaattat gtgaaggtgc tgctggagcc 2640
cgatgccatg tccagagtga gcctgacggg ggtgtttgac atgaatgtgg aggtgtggaa 2700
gattctgaga tatgatgaat ccaagaccag gtgccgagcc tgcgagtgcg gagggaagca 2760
tgccaggttc cagcccgtgt gtgtggatgt gacggaggac ctgcgacccg atcatttggt 2820
gttgccctgc accgggacgg agttcggttc cagcggggaa gaatctgact agagtgagta 2880
gtgttctggg gcgggggagg acctgcatga gggccagaat aactgaaatc tgtgcttttc 2940
tgtgtgttgc agcagcatga gcggaagcgg ctcctttgag ggaggggtat tcagccctta 3000
tctgacgggg cgtctcccct cctgggcggg agtgcgtcag aatgtgatgg gatccacggt 3060
ggacggccgg cccgtgcagc ccgcgaactc ttcaaccctg acctatgcaa ccctgagctc 3120
ttcgtcgttg gacgcagctg ccgccgcagc tgctgcatct gccgccagcg ccgtgcgcgg 3180
aatggccatg ggcgccggct actacggcac tctggtggcc aactcgagtt ccaccaataa 3240
tcccgccagc ctgaacgagg agaagctgtt gctgctgatg gcccagctcg aggccttgac 3300
ccagcgcctg ggcgagctga cccagcaggt ggctcagctg caggagcaga cgcgggccgc 3360
ggttgccacg gtgaaatcca aataaaaaat gaatcaataa ataaacggag acggttgttg 3420
attttaacac agagtctgaa tctttatttg atttttcgcg cgcggtaggc cctggaccac 3480
cggtctcgat cattgagcac ccggtggatc ttttccagga cccggtagag gtgggcttgg 3540
atgttgaggt acatgggcat gagcccgtcc cgggggtgga ggtagctcca ttgcagggcc 3600
tcgtgctcgg gggtggtgtt gtaaatcacc cagtcatagc aggggcgcag ggcatggtgt 3660
tgcacaatat ctttgaggag gagactgatg gccacgggca gccctttggt gtaggtgttt 3720
acaaatctgt tgagctggga gggatgcatg cggggggaga tgaggtgcat cttggcctgg 3780
atcttgagat tggcgatgtt accgcccaga tcccgcctgg ggttcatgtt gtgcaggacc 3840
accagcacgg tgtatccggt gcacttgggg aatttatcat gcaacttgga agggaaggcg 3900
tgaaagaatt tggcgacgcc tttgtgcccg cccaggtttt ccatgcactc atccatgatg 3960
atggcgatgg gcccgtgggc ggcggcctgg gcaaagacgt ttcgggggtc ggacacatca 4020
tagttgtggt cctgggtgag gtcatcatag gccattttaa tgaatttggg gcggagggtg 4080
ccggactggg ggacaaaggt accctcgatc ccgggggcgt agttcccctc acagatctgc 4140
atctcccagg ctttgagctc ggaggggggg atcatgtcca cctgcggggc gataaagaac 4200
acggtttccg gggcggggga gatgagctgg gccgaaagca agttccggag cagctgggac 4260
ttgccgcagc cggtggggcc gtagatgacc ccgatgaccg gctgcaggtg gtagttgagg 4320
gagagacagc tgccgtcctc ccggaggagg ggggccacct cgttcatcat ctcgcgcacg 4380
tgcatgttct cgcgcaccag ttccgccagg aggcgctctc cccccaggga taggagctcc 4440
tggagcgagg cgaagttttt cagcggcttg agtccgtcgg ccatgggcat tttggagagg 4500
gtttgttgca agagttccag gcggtcccag agctcggtga tgtgctctac ggcatctcga 4560
tccagcagac ctcctcgttt cgcgggttgg gacggctgcg ggagtagggc accagacgat 4620
gggcgtccag cgcagccagg gtccggtcct tccagggtcg cagcgtccgc gtcagggtgg 4680
tctccgtcac ggtgaagggg tgcgcgccgg gctgggcgct tgcgagggtg cgcttcaggc 4740
tcatccggct ggtcgaaaac cgctcccgat cggcgccctg cgcgtcggcc aggtagcaat 4800
tgaccatgag ttcgtagttg agcgcctcgg ccgcgtggcc tttggcgcgg agcttacctt 4860
tggaagtctg cccgcaggcg ggacagagga gggacttgag ggcgtagagc ttgggggcga 4920
ggaagacgga ctcgggggcg taggcgtccg cgccgcagtg ggcgcagacg gtctcgcact 4980
ccacgagcca ggtgaggtcg ggctggtcgg ggtcaaaaac cagtttcccg ccgttctttt 5040
tgatgcgttt cttacctttg gtctccatga gctcgtgtcc ccgctgggtg acaaagaggc 5100
tgtccgtgtc cccgtagacc gactttatgg gccggtcctc gagcggtgtg ccgcggtcct 5160
cctcgtagag gaaccccgcc cactccgaga cgaaagcccg ggtccaggcc agcacgaagg 5220
aggccacgtg ggacgggtag cggtcgttgt ccaccagcgg gtccaccttt tccagggtat 5280
gcaaacacat gtccccctcg tccacatcca ggaaggtgat tggcttgtaa gtgtaggcca 5340
cgtgaccggg ggtcccggcc gggggggtat aaaagggtgc gggtccctgc tcgtcctcac 5400
tgtcttccgg atcgctgtcc aggagcgcca gctgttgggg taggtattcc ctctcgaagg 5460
cgggcatgac ctcggcactc aggttgtcag tttctagaaa cgaggaggat ttgatattga 5520
cggtgccggc ggagatgcct ttcaagagcc cctcgtccat ctggtcagaa aagacgatct 5580
ttttgttgtc gagcttggtg gcgaaggagc cgtagagggc gttggagagg agcttggcga 5640
tggagcgcat ggtctggttt ttttccttgt cggcgcgctc cttggcggcg atgttgagct 5700
gcacgtactc gcgcgccacg cacttccatt cggggaagac ggtggtcagc tcgtcgggca 5760
cgattctgac ctgccagccc cgattatgca gggtgatgag gtccacactg gtggccacct 5820
cgccgcgcag gggctcatta gtccagcaga ggcgtccgcc cttgcgcgag cagaaggggg 5880
gcagggggtc cagcatgacc tcgtcggggg ggtcggcatc gatggtgaag atgccgggca 5940
ggaggtcggg gtcaaagtag ctgatggaag tggccagatc gtccagggca gcttgccatt 6000
cgcgcacggc cagcgcgcgc tcgtagggac tgaggggcgt gccccagggc atgggatggg 6060
taagcgcgga ggcgtacatg ccgcagatgt cgtagacgta gaggggctcc tcgaggatgc 6120
cgatgtaggt ggggtagcag cgccccccgc ggatgctggc gcgcacgtag tcatacagct 6180
cgtgcgaggg ggcgaggagc cccgggccca ggttggtgcg actgggcttt tcggcgcggt 6240
agacgatctg gcggaaaatg gcatgcgagt tggaggagat ggtgggcctt tggaagatgt 6300
tgaagtgggc gtggggcagt ccgaccgagt cgcggatgaa gtgggcgtag gagtcttgca 6360
gcttggcgac gagctcggcg gtgactagga cgtccagagc gcagtagtcg agggtctcct 6420
ggatgatgtc atacttgagc tgtccctttt gtttccacag ctcgcggttg agaaggaact 6480
cttcgcggtc cttccagtac tcttcgaggg ggaacccgtc ctgatctgca cggtaagagc 6540
ctagcatgta gaactggttg acggccttgt aggcgcagca gcccttctcc acggggaggg 6600
cgtaggcctg ggcggccttg cgcagggagg tgtgcgtgag ggcgaaagtg tccctgacca 6660
tgaccttgag gaactggtgc ttgaagtcga tatcgtcgca gcccccctgc tcccagagct 6720
ggaagtccgt gcgcttcttg taggcggggt tgggcaaagc gaaagtaaca tcgttgaaga 6780
ggatcttgcc cgcgcggggc ataaagttgc gagtgatgcg gaaaggttgg ggcacctcgg 6840
cccggttgtt gatgacctgg gcggcgagca cgatctcgtc gaagccgttg atgttgtggc 6900
ccacgatgta gagttccacg aatcgcggac ggcccttgac gtggggcagt ttcttgagct 6960
cctcgtaggt gagctcgtcg gggtcgctga gcccgtgctg ctcgagcgcc cagtcggcga 7020
gatgggggtt ggcgcggagg aaggaagtcc agagatccac ggccagggcg gtttgcagac 7080
ggtcccggta ctgacggaac tgctgcccga cggccatttt ttcgggggtg acgcagtaga 7140
aggtgcgggg gtccccgtgc cagcgatccc atttgagctg gagggcgaga tcgagggcga 7200
gctcgacgag ccggtcgtcc ccggagagtt tcatgaccag catgaagggg acgagctgct 7260
tgccgaagga ccccatccag gtgtaggttt ccacatcgta ggtgaggaag agcctttcgg 7320
tgcgaggatg cgagccgatg gggaagaact ggatctcctg ccaccaattg gaggaatggc 7380
tgttgatgtg atggaagtag aaatgccgac ggcgcgccga acactcgtgc ttgtgtttat 7440
acaagcggcc acagtgctcg caacgctgca cgggatgcac gtgctgcacg agctgtacct 7500
gagttccttt gacgaggaat ttcagtggga agtggagtcg tggcgcctgc atctcgtgct 7560
gtactacgtc gtggtggtcg gcctggccct cttctgcctc gatggtggtc atgctgacga 7620
gcccgcgcgg gaggcaggtc cagacctcgg cgcgagcggg tcggagagcg aggacgaggg 7680
cgcgcaggcc ggagctgtcc agggtcctga gacgctgcgg agtcaggtca gtgggcagcg 7740
gcggcgcgcg gttgacttgc aggagttttt ccagggcgcg cgggaggtcc agatggtact 7800
tgatctccac cgcgccattg gtggcgacgt cgatggcttg cagggtcccg tgcccctggg 7860
gtgtgaccac cgtcccccgt ttcttcttgg gcggctgggg cgacgggggc ggtgcctctt 7920
ccatggttag aagcggcggc gaggacgcgc gccgggcggc aggggcggct cggggcccgg 7980
aggcaggggc ggcaggggca cgtcggcgcc gcgcgcgggt aggttctggt actgcgcccg 8040
gagaagactg gcgtgagcga cgacgcgacg gttgacgtcc tggatctgac gcctctgggt 8100
gaaggccacg ggacccgtga gtttgaacct gaaagagagt tcgacagaat caatctcggt 8160
atcgttgacg gcggcctgcc gcaggatctc ttgcacgtcg cccgagttgt cctggtaggc 8220
gatctcggtc atgaactgct cgatctcctc ctcttgaagg tctccgcggc cggcgcgctc 8280
cacggtggcc gcgaggtcgt tggagatgcg gcccatgagc tgcgagaagg cgttcatgcc 8340
cgcctcgttc cagacgcggc tgtagaccac gacgccctcg ggatcgccgg cgcgcatgac 8400
cacctgggcg aggttgagct ccacgtggcg cgtgaagacc gcgtagttgc agaggcgctg 8460
gtagaggtag ttgagcgtgg tggcgatgtg ctcggtgacg aagaaataca tgatccagcg 8520
gcggagcggc atctcgctga cgtcgcccag cgcctccaaa cgttccatgg cctcgtaaaa 8580
gtccacggcg aagttgaaaa actgggagtt gcgcgccgag acggtcaact cctcctccag 8640
aagacggatg agctcggcga tggtggcgcg cacctcgcgc tcgaaggccc ccgggagttc 8700
ctccacttcc tcttcttcct cctccactaa catctcttct acttcctcct caggcggcag 8760
tggtggcggg ggagggggcc tgcgtcgccg gcggcgcacg ggcagacggt cgatgaagcg 8820
ctcgatggtc tcgccgcgcc ggcgtcgcat ggtctcggtg acggcgcgcc cgtcctcgcg 8880
gggccgcagc gtgaagacgc cgccgcgcat ctccaggtgg ccgggggggt ccccgttggg 8940
cagggagagg gcgctgacga tgcatcttat caattgcccc gtagggactc cgcgcaagga 9000
cctgagcgtc tcgagatcca cgggatctga aaaccgctga acgaaggctt cgagccagtc 9060
gcagtcgcaa ggtaggctga gcacggtttc ttctggcggg tcatgttggt tgggagcggg 9120
gcgggcgatg ctgctggtga tgaagttgaa ataggcggtt ctgagacggc ggatggtggc 9180
gaggagcacc aggtctttgg gcccggcttg ctggatgcgc agacggtcgg ccatgcccca 9240
ggcgtggtcc tgacacctgg ccaggtcctt gtagtagtcc tgcatgagcc gctccacggg 9300
cacctcctcc tcgcccgcgc ggccgtgcat gcgcgtgagc ccgaagccgc gctggggctg 9360
gacgagcgcc aggtcggcga cgacgcgctc ggcgaggatg gcttgctgga tctgggtgag 9420
ggtggtctgg aagtcatcaa agtcgacgaa gcggtggtag gctccggtgt tgatggtgta 9480
ggagcagttg gccatgacgg accagttgac ggtctggtgg cccggacgca cgagctcgtg 9540
gtacttgagg cgcgagtagg cgcgcgtgtc gaagatgtag tcgttgcagg tgcgcaccag 9600
gtactggtag ccgatgagga agtgcggcgg cggctggcgg tagagcggcc atcgctcggt 9660
ggcgggggcg ccgggcgcga ggtcctcgag catggtgcgg tggtagccgt agatgtacct 9720
ggacatccag gtgatgccgg cggcggtggt ggaggcgcgc gggaactcgc ggacgcggtt 9780
ccagatgttg cgcagcggca ggaagtagtt catggtgggc acggtctggc ccgtgaggcg 9840
cgcgcagtcg tggatgctct atacgggcaa aaacgaaagc ggtcagcggc tcgactccgt 9900
ggcctggagg ctaagcgaac gggttgggct gcgcgtgtac cccggttcga atctcgaatc 9960
aggctggagc cgcagctaac gtggtattgg cactcccgtc tcgacccaag cctgcaccaa 10020
ccctccagga tacggaggcg ggtcgttttg caactttttt ttggaggccg gatgagacta 10080
gtaagcgcgg aaagcggccg accgcgatgg ctcgctgccg tagtctggag aagaatcgcc 10140
agggttgcgt tgcggtgtgc cccggttcga ggccggccgg attccgcggc taacgagggc 10200
gtggctgccc cgtcgtttcc aagaccccat agccagccga cttctccagt tacggagcga 10260
gcccctcttt tgttttgttt gtttttgcca gatgcatccc gtactgcggc agatgcgccc 10320
ccaccaccct ccaccgcaac aacagccccc tccacagccg gcgcttctgc ccccgcccca 10380
gcagcaactt ccagccacga ccgccgcggc cgccgtgagc ggggctggac agagttatga 10440
tcaccagctg gccttggaag agggcgaggg gctggcgcgc ctgggggcgt cgtcgccgga 10500
gcggcacccg cgcgtgcaga tgaaaaggga cgctcgcgag gcctacgtgc ccaagcagaa 10560
cctgttcaga gacaggagcg gcgaggagcc cgaggagatg cgcgcggccc ggttccacgc 10620
ggggcgggag ctgcggcgcg gcctggaccg aaagagggtg ctgagggacg aggatttcga 10680
ggcggacgag ctgacgggga tcagccccgc gcgcgcgcac gtggccgcgg ccaacctggt 10740
cacggcgtac gagcagaccg tgaaggagga gagcaacttc caaaaatcct tcaacaacca 10800
cgtgcgcacc ctgatcgcgc gcgaggaggt gaccctgggc ctgatgcacc tgtgggacct 10860
gctggaggcc atcgtgcaga accccaccag caagccgctg acggcgcagc tgttcctggt 10920
ggtgcagcat agtcgggaca acgaagcgtt cagggaggcg ctgctgaata tcaccgagcc 10980
cgagggccgc tggctcctgg acctggtgaa cattctgcag agcatcgtgg tgcaggagcg 11040
cgggctgccg ctgtccgaga agctggcggc catcaacttc tcggtgctga gtttgggcaa 11100
gtactacgct aggaagatct acaagacccc gtacgtgccc atagacaagg aggtgaagat 11160
cgacgggttt tacatgcgca tgaccctgaa agtgctgacc ctgagcgacg atctgggggt 11220
gtaccgcaac gacaggatgc accgtgcggt gagcgccagc aggcggcgcg agctgagcga 11280
ccaggagctg atgcatagtc tgcagcgggc cctgaccggg gccgggaccg agggggagag 11340
ctactttgac atgggcgcgg acctgcactg gcagcccagc cgccgggcct tggaggcggc 11400
ggcaggaccc tacgtagaag aggtggacga tgaggtggac gaggagggcg agtacctgga 11460
agactgatgg cgcgaccgta tttttgctag atgcaacaac aacagccacc tcctgatccc 11520
gcgatgcggg cggcgctgca gagccagccg tccggcatta actcctcgga cgattggacc 11580
caggccatgc aacgcatcat ggcgctgacg acccgcaacc ccgaagcctt tagacagcag 11640
ccccaggcca accggctctc ggccatcctg gaggccgtgg tgccctcgcg ctccaacccc 11700
acgcacgaga aggtcctggc catcgtgaac gcgctggtgg agaacaaggc catccgcggc 11760
gacgaggccg gcctggtgta caacgcgctg ctggagcgcg tggcccgcta caacagcacc 11820
aacgtgcaga ccaacctgga ccgcatggtg accgacgtgc gcgaggccgt ggcccagcgc 11880
gagcggttcc accgcgagtc caacctggga tccatggtgg cgctgaacgc cttcctcagc 11940
acccagcccg ccaacgtgcc ccggggccag gaggactaca ccaacttcat cagcgccctg 12000
cgcctgatgg tgaccgaggt gccccagagc gaggtgtacc agtccgggcc ggactacttc 12060
ttccagacca gtcgccaggg cttgcagacc gtgaacctga gccaggcttt caagaacttg 12120
cagggcctgt ggggcgtgca ggccccggtc ggggaccgcg cgacggtgtc gagcctgctg 12180
acgccgaact cgcgcctgct gctgctgctg gtggccccct tcacggacag cggcagcatc 12240
aaccgcaact cgtacctggg ctacctgatt aacctgtacc gcgaggccat cggccaggcg 12300
cacgtggacg agcagaccta ccaggagatc acccacgtga gccgcgccct gggccaggac 12360
gacccgggca acctggaagc caccctgaac tttttgctga ccaaccggtc gcagaagatc 12420
ccgccccagt acgcgctcag caccgaggag gagcgcatcc tgcgttacgt gcagcagagc 12480
gtgggcctgt tcctgatgca ggagggggcc acccccagcg ccgcgctcga catgaccgcg 12540
cgcaacatgg agcccagcat gtacgccagc aaccgcccgt tcatcaataa actgatggac 12600
tacttgcatc gggcggccgc catgaactct gactatttca ccaacgccat cctgaatccc 12660
cactggctcc cgccgccggg gttctacacg ggcgagtacg acatgcccga ccccaatgac 12720
gggttcctgt gggacgatgt ggacagcagc gtgttctccc cccgaccggg tgctaacgag 12780
cgccccttgt ggaagaagga aggcagcgac cgacgcccgt cctcggcgct gtccggccgc 12840
gagggtgctg ccgcggcggt gcccgaggcc gccagtcctt tcccgagctt gcccttctcg 12900
ctgaacagta tccgcagcag cgagctgggc aggatcacgc gcccgcgctt gctgggcgaa 12960
gaggagtact tgaatgactc gctgttgaga cccgagcggg agaagaactt ccccaataac 13020
gggatagaaa gcctggtgga caagatgagc cgctggaaga cgtatgcgca ggagcacagg 13080
gacgatcccc gggcgtcgca gggggccacg agccggggca gcgccgcccg taaacgccgg 13140
tggcacgaca ggcagcgggg acagatgtgg gacgatgagg actccgccga cgacagcagc 13200
gtgttggact tgggtgggag tggtaacccg ttcgctcacc tgcgcccccg tatcgggcgc 13260
atgatgtaag agaaaccgaa aataaatgat actcaccaag gccatggcga ccagcgtgcg 13320
ttcgtttctt ctctgttgtt gttgtatcta gtatgatgag gcgtgcgtac ccggagggtc 13380
ctcctccctc gtacgagagc gtgatgcagc aggcgatggc ggcggcggcg atgcagcccc 13440
cgctggaggc tccttacgtg cccccgcggt acctggcgcc tacggagggg cggaacagca 13500
ttcgttactc ggagctggca cccttgtacg ataccacccg gttgtacctg gtggacaaca 13560
agtcggcgga catcgcctcg ctgaactacc agaacgacca cagcaacttc ctgaccaccg 13620
tggtgcagaa caatgacttc acccccacgg aggccagcac ccagaccatc aactttgacg 13680
agcgctcgcg gtggggcggc cagctgaaaa ccatcatgca caccaacatg cccaacgtga 13740
acgagttcat gtacagcaac aagttcaagg cgcgggtgat ggtctcccgc aagaccccca 13800
atggggtgac agtgacagag gattatgatg gtagtcagga tgagctgaag tatgaatggg 13860
tggaatttga gctgcccgaa ggcaacttct cggtgaccat gaccatcgac ctgatgaaca 13920
acgccatcat cgacaattac ttggcggtgg ggcggcagaa cggggtgctg gagagcgaca 13980
tcggcgtgaa gttcgacact aggaacttca ggctgggctg ggaccccgtg accgagctgg 14040
tcatgcccgg ggtgtacacc aacgaggctt tccatcccga tattgtcttg ctgcccggct 14100
gcggggtgga cttcaccgag agccgcctca gcaacctgct gggcattcgc aagaggcagc 14160
ccttccagga aggcttccag atcatgtacg aggatctgga ggggggcaac atccccgcgc 14220
tcctggatgt cgacgcctat gagaaaagca aggaggatgc agcagctgaa gcaactgcag 14280
ccgtagctac cgcctctacc gaggtcaggg gcgataattt tgcaagcgcc gcagcagtgg 14340
cagcggccga ggcggctgaa accgaaagta agatagtcat tcagccggtg gagaaggata 14400
gcaagaacag gagctacaac gtactaccgg acaagataaa caccgcctac cgcagctggt 14460
acctagccta caactatggc gaccccgaga agggcgtgcg ctcctggacg ctgctcacca 14520
cctcggacgt cacctgcggc gtggagcaag tctactggtc gctgcccgac atgatgcaag 14580
acccggtcac cttccgctcc acgcgtcaag ttagcaacta cccggtggtg ggcgccgagc 14640
tcctgcccgt ctactccaag agcttcttca acgagcaggc cgtctactcg cagcagctgc 14700
gcgccttcac ctcgcttacg cacgtcttca accgcttccc cgagaaccag atcctcgtcc 14760
gcccgcccgc gcccaccatt accaccgtca gtgaaaacgt tcctgctctc acagatcacg 14820
ggaccctgcc gctgcgcagc agtatccggg gagtccagcg cgtgaccgtt actgacgcca 14880
gacgccgcac ctgcccctac gtctacaagg ccctgggcat agtcgcgccg cgcgtcctct 14940
cgagccgcac cttctaaatg tccattctca tctcgcccag taataacacc ggttggggcc 15000
tgcgcgcgcc cagcaagatg tacggaggcg ctcgccaacg ctccacgcaa caccccgtgc 15060
gcgtgcgcgg gcacttccgc gctccctggg gcgccctcaa gggccgcgtg cggtcgcgca 15120
ccaccgtcga cgacgtgatc gaccaggtgg tggccgacgc gcgcaactac acccccgccg 15180
ccgcgcccgt ctccaccgtg gacgccgtca tcgacagcgt ggtggcggac gcgcgccggt 15240
acgcccgcgc caagagccgg cggcggcgca tcgcccggcg gcaccggagc acccccgcca 15300
tgcgcgcggc gcgagccttg ctgcgcaggg ccaggcgcac gggacgcagg gccatgctca 15360
gggcggccag acgcgcggct tcaggcgcca gcgccggcag gacccggaga cgcgcggcca 15420
cggcggcggc agcggccatc gccagcatgt cccgcccgcg gcgagggaac gtgtactggg 15480
tgcgcgacgc cgccaccggt gtgcgcgtgc ccgtgcgcac ccgcccccct cgcacttgaa 15540
gatgttcact tcgcgatgtt gatgtgtccc agcggcgagg aggatgtcca agcgcaaatt 15600
caaggaagag atgctccagg tcatcgcgcc tgagatctac ggccctgcgg tggtgaagga 15660
ggaaagaaag ccccgcaaaa tcaagcgggt caaaaaggac aaaaaggaag aagaaagtga 15720
tgtggacgga ttggtggagt ttgtgcgcga gttcgccccc cggcggcgcg tgcagtggcg 15780
cgggcggaag gtgcaaccgg tgctgagacc cggcaccacc gtggtcttca cgcccggcga 15840
gcgctccggc accgcttcca agcgctccta cgacgaggtg tacggggatg atgatattct 15900
ggagcaggcg gccgagcgcc tgggcgagtt tgcttacggc aagcgcagcc gttccgcacc 15960
gaaggaagag gcggtgtcca tcccgctgga ccacggcaac cccacgccga gcctcaagcc 16020
cgtgaccttg cagcaggtgc tgccgaccgc ggcgccgcgc cgggggttca agcgcgaggg 16080
cgaggatctg taccccacca tgcagctgat ggtgcccaag cgccagaagc tggaagacgt 16140
gctggagacc atgaaggtgg acccggacgt gcagcccgag gtcaaggtgc ggcccatcaa 16200
gcaggtggcc ccgggcctgg gcgtgcagac cgtggacatc aagattccca cggagcccat 16260
ggaaacgcag accgagccca tgatcaagcc cagcaccagc accatggagg tgcagacgga 16320
tccctggatg ccatcggctc ctagtcgaag accccggcgc aagtacggcg cggccagcct 16380
gctgatgccc aactacgcgc tgcatccttc catcatcccc acgccgggct accgcggcac 16440
gcgcttctac cgcggtcata ccagcagccg ccgccgcaag accaccactc gccgccgccg 16500
tcgccgcacc gccgctgcaa ccacccctgc cgccctggtg cggagagtgt accgccgcgg 16560
ccgcgcacct ctgaccctgc cgcgcgcgcg ctaccacccg agcatcgcca tttaaacttt 16620
cgccagcttt gcagatcaat ggccctcaca tgccgccttc gcgttcccat tacgggctac 16680
cgaggaagaa aaccgcgccg tagaaggctg gcggggaacg ggatgcgtcg ccaccaccac 16740
cggcggcggc gcgccatcag caagcggttg gggggaggct tcctgcccgc gctgatcccc 16800
atcatcgccg cggcgatcgg ggcgatcccc ggcattgctt ccgtggcggt gcaggcctct 16860
cagcgccact gagacacact tggaaacatc ttgtaataaa cccatggact ctgacgctcc 16920
tggtcctgtg atgtgttttc gtagacagat ggaagacatc aatttttcgt ccctggctcc 16980
gcgacacggc acgcggccgt tcatgggcac ctggagcgac atcggcacca gccaactgaa 17040
cgggggcgcc ttcaattgga gcagtctctg gagcgggctt aagaatttcg ggtccacgct 17100
taaaacctat ggcagcaagg cgtggaacag caccacaggg caggcgctga gggataagct 17160
gaaagagcag aacttccagc agaaggtggt cgatgggctc gcctcgggca tcaacggggt 17220
ggtggacctg gccaaccagg ccgtgcagcg gcagatcaac agccgcctgg acccggtgcc 17280
gcccgccggc tccgtggaga tgccgcaggt ggaggaggag ctgcctcccc tggacaagcg 17340
gggcgagaag cgaccccgcc ccgatgcgga ggagacgctg ctgacgcaca cggacgagcc 17400
gcccccgtac gaggaggcgg tgaaactggg tctgcccacc acgcggccca tcgcgcccct 17460
ggccaccggg gtgctgaaac ccgaaaagcc cgcgaccctg gacttgcctc ctccccagcc 17520
ttcccgcccc tctacagtgg ctaagcccct gccgccggtg gccgtggccc gcgcgcgacc 17580
cgggggcacc gcccgccctc atgcgaactg gcagagcact ctgaacagca tcgtgggtct 17640
gggagtgcag agtgtgaagc gccgccgctg ctattaaacc taccgtagcg cttaacttgc 17700
ttgtctgtgt gtgtatgtat tatgtcgccg ccgccgctgt ccaccagaag gaggagtgaa 17760
gaggcgcgtc gccgagttgc aagatggcca ccccatcgat gctgccccag tgggcgtaca 17820
tgcacatcgc cggacaggac gcttcggagt acctgagtcc gggtctggtg cagtttgccc 17880
gcgccacaga cacctacttc agtctgggga acaagtttag gaaccccacg gtggcgccca 17940
cgcacgatgt gaccaccgac cgcagccagc ggctgacgct gcgcttcgtg cccgtggacc 18000
gcgaggacaa cacctactcg tacaaagtgc gctacacgct ggccgtgggc gacaaccgcg 18060
tgctggacat ggccagcacc tactttgaca tccgcggcgt gctggatcgg ggccctagct 18120
tcaaacccta ctccggcacc gcctacaaca gtctggcccc caagggagca cccaacactt 18180
gtcagtggac atataaagcc gatggtgaaa ctgccacaga aaaaacctat acatatggaa 18240
atgcacccgt gcagggcatt aacatcacaa aagatggtat tcaacttgga actgacaccg 18300
atgatcagcc aatctacgca gataaaacct atcagcctga acctcaagtg ggtgatgctg 18360
aatggcatga catcactggt actgatgaaa agtatggagg cagagctctt aagcctgata 18420
ccaaaatgaa gccttgttat ggttcttttg ccaagcctac taataaagaa ggaggtcagg 18480
caaatgtgaa aacaggaaca ggcactacta aagaatatga catagacatg gctttctttg 18540
acaacagaag tgcggctgct gctggcctag ctccagaaat tgttttgtat actgaaaatg 18600
tggatttgga aactccagat acccatattg tatacaaagc aggcacagat gacagcagct 18660
cttctattaa tttgggtcag caagccatgc ccaacagacc taactacatt ggtttcagag 18720
acaactttat cgggctcatg tactacaaca gcactggcaa tatgggggtg ctggccggtc 18780
aggcttctca gctgaatgct gtggttgact tgcaagacag aaacaccgag ctgtcctacc 18840
agctcttgct tgactctctg ggtgacagaa cccggtattt cagtatgtgg aatcaggcgg 18900
tggacagcta tgatcctgat gtgcgcatta ttgaaaatca tggtgtggag gatgaacttc 18960
ccaactattg tttccctctg gatgctgttg gcagaacaga tacttatcag ggaattaagg 19020
ctaatggaac tgatcaaacc acatggacca aagatgacag tgtcaatgat gctaatgaga 19080
taggcaaggg taatccattc gccatggaaa tcaacatcca agccaacctg tggaggaact 19140
tcctctacgc caacgtggcc ctgtacctgc ccgactctta caagtacacg ccggccaatg 19200
ttaccctgcc caccaacacc aacacctacg attacatgaa cggccgggtg gtggcgccct 19260
cgctggtgga ctcctacatc aacatcgggg cgcgctggtc gctggatccc atggacaacg 19320
tgaacccctt caaccaccac cgcaatgcgg ggctgcgcta ccgctccatg ctcctgggca 19380
acgggcgcta cgtgcccttc cacatccagg tgccccagaa atttttcgcc atcaagagcc 19440
tcctgctcct gcccgggtcc tacacctacg agtggaactt ccgcaaggac gtcaacatga 19500
tcctgcagag ctccctcggc aacgacctgc gcacggacgg ggcctccatc tccttcacca 19560
gcatcaacct ctacgccacc ttcttcccca tggcgcacaa cacggcctcc acgctcgagg 19620
ccatgctgcg caacgacacc aacgaccagt ccttcaacga ctacctctcg gcggccaaca 19680
tgctctaccc catcccggcc aacgccacca acgtgcccat ctccatcccc tcgcgcaact 19740
gggccgcctt ccgcggctgg tccttcacgc gtctcaagac caaggagacg ccctcgctgg 19800
gctccgggtt cgacccctac ttcgtctact cgggctccat cccctacctc gacggcacct 19860
tctacctcaa ccacaccttc aagaaggtct ccatcacctt cgactcctcc gtcagctggc 19920
ccggcaacga ccggctcctg acgcccaacg agttcgaaat caagcgcacc gtcgacggcg 19980
agggctacaa cgtggcccag tgcaacatga ccaaggactg gttcctggtc cagatgctgg 20040
cccactacaa catcggctac cagggcttct acgtgcccga gggctacaag gaccgcatgt 20100
actccttctt ccgcaacttc cagcccatga gccgccaggt ggtggacgag gtcaactaca 20160
aggactacca ggccgtcacc ctggcctacc agcacaacaa ctcgggcttc gtcggctacc 20220
tcgcgcccac catgcgccag ggccagccct accccgccaa ctacccctac ccgctcatcg 20280
gcaagagcgc cgtcaccagc gtcacccaga aaaagttcct ctgcgacagg gtcatgtggc 20340
gcatcccctt ctccagcaac ttcatgtcca tgggcgcgct caccgacctc ggccagaaca 20400
tgctctatgc caactccgcc cacgcgctag acatgaattt cgaagtcgac cccatggatg 20460
agtccaccct tctctatgtt gtcttcgaag tcttcgacgt cgtccgagtg caccagcccc 20520
accgcggcgt catcgaggcc gtctacctgc gcaccccctt ctcggccggt aacgccacca 20580
cctaagctct tgcttcttgc aagccatggc cgcgggctcc ggcgagcagg agctcagggc 20640
catcatccgc gacctgggct gcgggcccta cttcctgggc accttcgata agcgcttccc 20700
gggattcatg gccccgcaca agctggcctg cgccatcgtc aacacggccg gccgcgagac 20760
cgggggcgag cactggctgg ccttcgcctg gaacccgcgc tcgaacacct gctacctctt 20820
cgaccccttc gggttctcgg acgagcgcct caagcagatc taccagttcg agtacgaggg 20880
cctgctgcgc cgcagcgccc tggccaccga ggaccgctgc gtcaccctgg aaaagtccac 20940
ccagaccgtg cagggtccgc gctcggccgc ctgcgggctc ttctgctgca tgttcctgca 21000
cgccttcgtg cactggcccg accgccccat ggacaagaac cccaccatga acttgctgac 21060
gggggtgccc aacggcatgc tccagtcgcc ccaggtggaa cccaccctgc gccgcaacca 21120
ggaggcgctc taccgcttcc tcaactccca ctccgcctac tttcgctccc accgcgcgcg 21180
catcgagaag gccaccgcct tcgaccgcat gaatcaagac atgtaaaccg tgtgtgtatg 21240
ttaaatgtct ttaataaaca gcactttcat gttacacatg catctgagat gatttattta 21300
gaaatcgaaa gggttctgcc gggtctcggc atggcccgcg ggcagggaca cgttgcggaa 21360
ctggtacttg gccagccact tgaactcggg gatcagcagt ttgggcagcg gggtgtcggg 21420
gaaggagtcg gtccacagct tccgcgtcag ttgcagggcg cccagcaggt cgggcgcgga 21480
gatcttgaaa tcgcagttgg gacccgcgtt ctgcgcgcgg gagttgcggt acacggggtt 21540
gcagcactgg aacaccatca gggccgggtg cttcacgctc gccagcaccg tcgcgtcggt 21600
gatgctctcc acgtcgaggt cctcggcgtt ggccatcccg aagggggtca tcttgcaggt 21660
ctgccttccc atggtgggca cgcacccggg cttgtggttg caatcgcagt gcagggggat 21720
cagcatcatc tgggcctggt cggcgttcat ccccgggtac atggccttca tgaaagcctc 21780
caattgcctg aacgcctgct gggccttggc tccctcggtg aagaagaccc cgcaggactt 21840
gctagagaac tggttggtgg cgcacccggc gtcgtgcacg cagcagcgcg cgtcgttgtt 21900
ggccagctgc accacgctgc gcccccagcg gttctgggtg atcttggccc ggtcggggtt 21960
ctccttcagc gcgcgctgcc cgttctcgct cgccacatcc atctcgatca tgtgctcctt 22020
ctggatcatg gtggtcccgt gcaggcaccg cagcttgccc tcggcctcgg tgcacccgtg 22080
cagccacagc gcgcacccgg tgcactccca gttcttgtgg gcgatctggg aatgcgcgtg 22140
cacgaagccc tgcaggaagc ggcccatcat ggtggtcagg gtcttgttgc tagtgaaggt 22200
cagcggaatg ccgcggtgct cctcgttgat gtacaggtgg cagatgcggc ggtacacctc 22260
gccctgctcg ggcatcagct ggaagttggc tttcaggtcg gtctccacgc ggtagcggtc 22320
catcagcata gtcatgattt ccataccctt ctcccaggcc gagacgatgg gcaggctcat 22380
agggttcttc accatcatct tagcgctagc agccgcggcc agggggtcgc tctcgtccag 22440
ggtctcaaag ctccgcttgc cgtccttctc ggtgatccgc accggggggt agctgaagcc 22500
cacggccgcc agctcctcct cggcctgtct ttcgtcctcg ctgtcctggc tgacgtcctg 22560
caggaccaca tgcttggtct tgcggggttt cttcttgggc ggcagcggcg gcggagatgt 22620
tggagatggc gagggggagc gcgagttctc gctcaccact actatctctt cctcttcttg 22680
gtccgaggcc acgcggcggt aggtatgtct cttcgggggc agaggcggag gcgacgggct 22740
ctcgccgccg cgacttggcg gatggctggc agagcccctt ccgcgttcgg gggtgcgctc 22800
ccggcggcgc tctgactgac ttcctccgcg gccggccatt gtgttctcct agggaggaac 22860
aacaagcatg gagactcagc catcgccaac ctcgccatct gcccccaccg ccgacgagaa 22920
gcagcagcag cagaatgaaa gcttaaccgc cccgccgccc agccccgcca cctccgacgc 22980
ggccgtccca gacatgcaag agatggagga atccatcgag attgacctgg gctatgtgac 23040
gcccgcggag cacgaggagg agctggcagt gcgcttttca caagaagaga tacaccaaga 23100
acagccagag caggaagcag agaatgagca gagtcaggct gggctcgagc atgacggcga 23160
ctacctccac ctgagcgggg gggaggacgc gctcatcaag catctggccc ggcaggccac 23220
catcgtcaag gatgcgctgc tcgaccgcac cgaggtgccc ctcagcgtgg aggagctcag 23280
ccgcgcctac gagttgaacc tcttctcgcc gcgcgtgccc cccaagcgcc agcccaatgg 23340
cacctgcgag cccaacccgc gcctcaactt ctacccggtc ttcgcggtgc ccgaggccct 23400
ggccacctac cacatctttt tcaagaacca aaagatcccc gtctcctgcc gcgccaaccg 23460
cacccgcgcc gacgcccttt tcaacctggg tcccggcgcc cgcctacctg atatcgcctc 23520
cttggaagag gttcccaaga tcttcgaggg tctgggcagc gacgagactc gggccgcgaa 23580
cgctctgcaa ggagaaggag gagagcatga gcaccacagc gccctggtcg agttggaagg 23640
cgacaacgcg cggctggcgg tgctcaaacg cacggtcgag ctgacccatt tcgcctaccc 23700
ggctctgaac ctgcccccca aagtcatgag cgcggtcatg gaccaggtgc tcatcaagcg 23760
cgcgtcgccc atctccgagg acgagggcat gcaagactcc gaggagggca agcccgtggt 23820
cagcgacgag cagctggccc ggtggctggg tcctaatgct agtccccaga gtttggaaga 23880
gcggcgcaaa ctcatgatgg ccgtggtcct ggtgaccgtg gagctggagt gcctgcgccg 23940
cttcttcgcc gacgcggaga ccctgcgcaa ggtcgaggag aacctgcact acctcttcag 24000
gcacgggttc gtgcgccagg cctgcaagat ctccaacgtg gagctgacca acctggtctc 24060
ctacatgggc atcttgcacg agaaccgcct ggggcagaac gtgctgcaca ccaccctgcg 24120
cggggaggcc cggcgcgact acatccgcga ctgcgtctac ctctacctct gccacacctg 24180
gcagacgggc atgggcgtgt ggcagcagtg tctggaggag cagaacctga aagagctctg 24240
caagctcctg cagaagaacc tcaagggtct gtggaccggg ttcgacgagc gcaccaccgc 24300
ctcggacctg gccgacctca ttttccccga gcgcctcagg ctgacgctgc gcaacggcct 24360
gcccgacttt atgagccaaa gcatgttgca aaactttcgc tctttcatcc tcgaacgctc 24420
cggaatcctg cccgccacct gctccgcgct gccctcggac ttcgtgccgc tgaccttccg 24480
cgagtgcccc ccgccgctgt ggagccactg ctacctgctg cgcctggcca actacctggc 24540
ctaccactcg gacgtgatcg aggacgtcag cggcgagggc ctgctcgagt gccactgccg 24600
ctgcaacctc tgcacgccgc accgctccct ggcctgcaac ccccagctgc tgagcgagac 24660
ccagatcatc ggcaccttcg agttgcaagg gcccagcgaa ggcgagggtt cagccgccaa 24720
ggggggtctg aaactcaccc cggggctgtg gacctcggcc tacttgcgca agttcgtgcc 24780
cgaggactac catcccttcg agatcaggtt ctacgaggac caatcccatc cgcccaaggc 24840
cgagctgtcg gcctgcgtca tcacccaggg ggcgatcctg gcccaattgc aagccatcca 24900
gaaatcccgc caagaattct tgctgaaaaa gggccgcggg gtctacctcg acccccagac 24960
cggtgaggag ctcaaccccg gcttccccca ggatgccccg aggaaacaag aagctgaaag 25020
tggagctgcc gcccgtggag gatttggagg aagactggga gaacagcagt caggcagagg 25080
aggaggagat ggaggaagac tgggacagca ctcaggcaga ggaggacagc ctgcaagaca 25140
gtctggagga agacgaggag gaggcagagg aggaggtgga agaagcagcc gccgccagac 25200
cgtcgtcctc ggcgggggag aaagcaagca gcacggatac catctccgct ccgggtcggg 25260
gtcccgctcg accacacagt agatgggacg agaccggacg attcccgaac cccaccaccc 25320
agaccggtaa gaaggagcgg cagggataca agtcctggcg ggggcacaaa aacgccatcg 25380
tctcctgctt gcaggcctgc gggggcaaca tctccttcac ccggcgctac ctgctcttcc 25440
accgcggggt gaactttccc cgcaacatct tgcattacta ccgtcacctc cacagcccct 25500
actacttcca agaagaggca gcagcagcag aaaaagacca gcagaaaacc agcagctaga 25560
aaatccacag cggcggcagc aggtggactg aggatcgcgg cgaacgagcc ggcgcaaacc 25620
cgggagctga ggaaccggat ctttcccacc ctctatgcca tcttccagca gagtcggggg 25680
caggagcagg aactgaaagt caagaaccgt tctctgcgct cgctcacccg cagttgtctg 25740
tatcacaaga gcgaagacca acttcagcgc actctcgagg acgccgaggc tctcttcaac 25800
aagtactgcg cgctcactct taaagagtag cccgcgcccg cccagtcgca gaaaaaggcg 25860
ggaattacgt cacctgtgcc cttcgcccta gccgcctcca cccatcatca tgagcaaaga 25920
gattcccacg ccttacatgt ggagctacca gccccagatg ggcctggccg ccggtgccgc 25980
ccaggactac tccacccgca tgaattggct cagcgccggg cccgcgatga tctcacgggt 26040
gaatgacatc cgcgcccacc gaaaccagat actcctagaa cagtcagcgc tcaccgccac 26100
gccccgcaat cacctcaatc cgcgtaattg gcccgccgcc ctggtgtacc aggaaattcc 26160
ccagcccacg accgtactac ttccgcgaga cgcccaggcc gaagtccagc tgactaactc 26220
aggtgtccag ctggcgggcg gcgccaccct gtgtcgtcac cgccccgctc agggtataaa 26280
gcggctggtg atccggggca gaggcacaca gctcaacgac gaggtggtga gctcttcgct 26340
gggtctgcga cctgacggag tcttccaact cgccggatcg gggagatctt ccttcacgcc 26400
tcgtcaggcc gtcctgactt tggagagttc gtcctcgcag ccccgctcgg gtggcatcgg 26460
cactctccag ttcgtggagg agttcactcc ctcggtctac ttcaacccct tctccggctc 26520
ccccggccac tacccggacg agttcatccc gaacttcgac gccatcagcg agtcggtgga 26580
cggctacgat tgaatgtccc atggtggcgc agctgaccta gctcggcttc gacacctgga 26640
ccactgccgc cgcttccgct gcttcgctcg ggatctcgcc gagtttgcct actttgagct 26700
gcccgaggag caccctcagg gcccggccca cggagtgcgg atcgtcgtcg aagggggcct 26760
cgactcccac ctgcttcgga tcttcagcca gcgtccgatc ctggtcgagc gcgagcaagg 26820
acagaccctt ctgactctgt actgcatctg caaccacccc ggcctgcatg aaagtctttg 26880
ttgtctgctg tgtactgagt ataataaaag ctgagatcag cgactactcc ggacttccgt 26940
gtgttcctga atccatcaac cagtctttgt tcttcaccgg gaacgagacc gagctccagc 27000
tccagtgtaa gccccacaag aagtacctca cctggctgtt ccagggctcc ccgatcgccg 27060
ttgtcaacca ctgcgacaac gactatttaa atccacaata catgcccata ttagactatg 27120
aggccgagcc acagcgaccc atgctccccg ctattagtta cttcaatcta accggcggag 27180
atgactgacc cactggccaa caacaacgtc aacgaccttc tcctggacat ggacggccgc 27240
gcctcggagc agcgactcgc ccaacttcgc attcgccagc agcaggagag agccgtcaag 27300
gagctgcagg atgcggtggc catccaccag tgcaagagag gcatcttctg cctggtgaaa 27360
caggccaaga tctcctacga ggtcactcca aacgaccatc gcctctccta cgagctcctg 27420
cagcagcgcc agaagttcac ctgcctggtc ggagtcaacc ccatcgtcat cacccagcag 27480
tctggcgata ccaaggggtg catccactgc tcctgcgact cccccgactg cgtccacact 27540
ctgatcaaga ccctctgcgg cctccgcgac ctcctcccca tgaactaatc acccccttat 27600
ccagtgaaat aaagatcata ttgatgatga ttttacagaa ataaaaaata atcatttgat 27660
ttgaaataaa gatacaatca tattgatgat ttgagtttaa caaaaaaata aagaatcact 27720
tacttgaaat ctgataccag gtctctgtcc atgttttctg ccaacaccac ttcactcccc 27780
tcttcccagc tctggtactg caggccccgg cgggctgcaa acttcctcca cacgctgaag 27840
gggatgtcaa attcctcctg tccctcaatc ttcattttat cttctatcag atgtccaaaa 27900
agcgcgtccg ggtggatgat gacttcgacc ccgtctaccc ctacgatgca gacaacgcac 27960
cgaccgtgcc cttcatcaac ccccccttcg tctcttcaga tggattccaa gagaagcccc 28020
tgggggtgtt gtccctgcga ctggccgacc ccgtcaccac caagaacggg gaaatcaccc 28080
tcaagctggg agagggggtg gacctcgatt cctcgggaaa actcatctcc aacacggcca 28140
ccaaggccgc cgcccctctc agtttttcca acaacaccat ttcccttaac atggatcacc 28200
ccttttacac taaagatgga aaattatcct tacaagtttc tccaccatta aatatactga 28260
gaacaagcat tctaaacaca ctagctttag gttttggatc aggtttagga ctccgtggct 28320
ctgccttggc agtacagtta gtctctccac ttacatttga tactgatgga aacataaagc 28380
ttaccttaga cagaggtttg catgttacaa caggagatgc aattgaaagc aacataagct 28440
gggctaaagg tttaaaattt gaagatggag ccatagcaac caacattgga aatgggttag 28500
agtttggaag cagtagtaca gaaacaggtg ttgatgatgc ttacccaatc caagttaaac 28560
ttggatctgg ccttagcttt gacagtacag gagccataat ggctggtaac aaagaagacg 28620
ataaactcac tttgtggaca acacctgatc catcaccaaa ctgtcaaata ctcgcagaaa 28680
atgatgcaaa actaacactt tgcttgacta aatgtggtag tcaaatactg gccactgtgt 28740
cagtcttagt tgtaggaagt ggaaacctaa accccattac tggcaccgta agcagtgctc 28800
aggtgtttct acgttttgat gcaaacggtg ttcttttaac agaacattct acactaaaaa 28860
aatactgggg gtataggcag ggagatagca tagatggcac tccatatacc aatgctgtag 28920
gattcatgcc caatttaaaa gcttatccaa agtcacaaag ttctactact aaaaataata 28980
tagtagggca agtatacatg aatggagatg tttcaaaacc tatgcttctc actataaccc 29040
tcaatggtac tgatgacagc aacagtacat attcaatgtc attttcatac acctggacta 29100
atggaagcta tgttggagca acatttgggg ctaactctta taccttctca tacatcgccc 29160
aagaatgaac actgtatccc accctgcatg ccaacccttc ccaccccact ctgtggaaca 29220
aactctgaaa cacaaaataa aataaagttc aagtgtttta ttgattcaac agttctacat 29280
gggggtagag tcataatcgt gcatcaggat agggcggtgg tgctgcagca gcgcgcgaat 29340
aaactgctgc cgccgccgct ccgtcctgca ggaatacaac atggcagtgg tctcctcagc 29400
gatgattcgc accgcccgca gcataaggcg ccttgtcctc cgggcacagc agcgcaccct 29460
gatctcactt aaatcagcac agtaactgca gcacagcacc acaatattgt tcaaaatccc 29520
acagtgcaag gcgctgtatc caaagctcat ggcggggacc acagaaccca cgtggccatc 29580
ataccacaag cgcaggtaga ttaagtggcg acccctcata aacacgctgg acataaacat 29640
tacctctttt ggcatgttgt aattcaccac ctcccggtac catataaacc tctgattaaa 29700
catggcgcca tccaccacca tcctaaacca gctggccaaa acctgcccgc cggctataca 29760
ctgcagggaa ccgggactgg aacaatgaca gtggagagcc caggactcgt aaccatggat 29820
catcatgctc gtcatgatat caatgttggc acaacacagg cacacgtgca tacacttcct 29880
caggattaca agctcctccc gcgttagaac catatcccag ggaacaaccc attcctgaat 29940
cagcgtaaat cccacactgc agggaagacc tcgcacgtaa ctcacgttgt gcattgtcaa 30000
agtgttacat tcgggcagca gcggatgatc ctccagtatg gtagcgcggg tttctgtctc 30060
aaaaggaggt agacgatccc tactgtacgg agtgcgccga gacaaccgag atcgtgttgg 30120
tcgtagtgtc atgccaaatg gaacgccgga cgtagtcata tttcctgaag caaaaccagg 30180
tgcgggcgtg acaaacagat ctgcgtctcc ggtctcgccg cttagatcgc tctgtgtagt 30240
agttgtagta tatccactct ctcaaagcat ccaggcgccc cctggcttcg ggttctatgt 30300
aaactccttc atgcgccgct gccctgataa catccaccac cgcagaataa gccacaccca 30360
gccaacctac acattcgttc tgcgagtcac acacgggagg agcgggaaga gctggaagaa 30420
ccatgttttt tttttttatt ccaaaagatt atccaaaacc tcaaaatgaa gatctattaa 30480
gtgaacgcgc tcccctccgg tggcgtggtc aaactctaca gccaaagaac agataatggc 30540
atttgtaaga tgttgcacaa tggcttccaa aaggcaaacg gccctcacgt ccaagtggac 30600
gtaaaggcta aacccttcag ggtgaatctc ctctataaac attccagcac cttcaaccat 30660
gcccaaataa ttctcatctc gccaccttct caatatatct ctaagcaaat cccgaatatt 30720
aagtccggcc attgtaaaaa tctgctccag agcgccctcc accttcagcc tcaagcagcg 30780
aatcataaca gtcagcctta ccagtaaaaa agaaaaccta ttaaaaaaac accactcgac 30840
acggcaccag ctcaatcagt cacagtgtaa aaaagggcca agtgcagagc gagtatatat 30900
aagcttaccg agcagcagca cacaacaggc gcaagagtca gagaaaggct gagctctaac 30960
ctgtccaccc gctctctgct caatatatag cccagatcta cactgacgta aaggccaaag 31020
tctaaaaata cccgccaaat aatcacacac gcccagcaca cgcccagaaa ccggtgacac 31080
actcaaaaaa atacgcgcac ttcctcaaac gcccaaaact gccgtcattt ccgggttccc 31140
acgctacgtc atcaaaacac gactttcaaa ttccgtcgac cgttaaaaac gtcacccgcc 31200
ccgcccctaa cggtcgcccg tctctcagcc aatcagcgcc ccgcatcccc aaattcaaac 31260
acctcatttg catattaacg cgcacaaaaa gtttgaggta tattattgat gatggttaat 31320
taagaattca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca 31380
acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg 31440
caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggcgcc tgatgcggta 31500
ttttctcctt acgcatctgt gcggtatttc acaccgcata tggtgcactc tcagtacaat 31560
ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc 31620
ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag 31680
ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt 31740
gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg 31800
cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa 31860
tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa 31920
gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct 31980
tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg 32040
tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg 32100
ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt 32160
atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga 32220
cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga 32280
attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac 32340
gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg 32400
ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac 32460
gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct 32520
agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct 32580
gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg 32640
gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat 32700
ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg 32760
tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat 32820
tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct 32880
catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa 32940
gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa 33000
aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc 33060
gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta 33120
gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct 33180
gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg 33240
atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag 33300
cttggagcga acgacctaca ccgaactgag atacctacag cgtgagcttt gagaaagcgc 33360
cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg 33420
agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt 33480
tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg 33540
gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca 33600
catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg 33660
agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc 33720
ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag 33780
ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag 33840
ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg 33900
tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa 33960
gcttgcatgc ctgcaggttt aaacttaatt aaccatcttc aataatatac ctcaaacttt 34020
ttgtgcgcgt taatatgcaa atgaggcgtt tgaatttggg gaggaagggc ggtgattg 34078
<210> 8
<211> 825
<212> DNA
<213> Artificial sequence
<400> 8
gccaccatgc tgcgcggact gtgctgcgtg ctgctactgt gcggcgccgt gttcgtgagc 60
cccagccagg agatccacgc ccgattcagg agaggagcca gaggacgcgt gcagcccacc 120
gagagcatcg tgcgcttccc caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 180
gccacccgct tcgccagcgt gtacgcctgg aaccgcaagc gcatcagcaa ctgcgtggcc 240
gactacagcg tgctgtacaa cagcgccagc ttcagcacct tcaagtgcta cggcgtgagc 300
cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatccgc 360
ggcgacgagg tgcgccagat cgcccccggc cagaccggca agatcgccga ctacaactac 420
aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaacaa cctggacagc 480
aaggtgggcg gcaactacaa ctacctgtac cgcctgttcc gcaagagcaa cctgaagccc 540
ttcgagcgcg acatcagcac cgagatctac caggccggca gcaccccctg caacggcgtg 600
gagggcttca actgctactt ccccctgcag agctacggct tccagcccac caacggcgtg 660
ggctaccagc cctaccgcgt ggtggtgctg agcttcgagc tgctgcacgc ccccgccacc 720
gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttctggagc 780
cacccccagt tcgagaagga ctacaaggac gacgacgaca agtaa 825

Claims (10)

1. A recombinant adenovirus which is (a1) or (a2) or (a3) or (a 4):
(a1) expressing the recombinant adenovirus of the protein shown in the sequence 3 of the sequence table;
(a2) expressing the recombinant adenovirus of the protein shown in the sequence 5 of the sequence table;
(a3) expressing the recombinant adenovirus of the protein shown in the sequence 1 of the sequence table;
(a4) expressing the recombinant adenovirus of the protein consisting of the 14 th to 236 th amino acid residues in the sequence 3 of the sequence table.
2. A protein which is (b1) or (b2) or (b3) as follows:
(b1) protein shown in a sequence 3 in a sequence table;
(b2) protein shown in a sequence 5 in a sequence table;
(b3) and (b) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of (b1) or (b 2).
3. A nucleic acid molecule encoding the protein of claim 2.
4. A recombinant plasmid which is (d1) or (d2) or (d3) or (d4) as follows:
(d1) inserting a DNA molecule of a protein shown in a sequence 3 of a coding sequence table into a chimpanzee adenovirus vector to obtain a recombinant plasmid;
(d2) inserting a DNA molecule of a protein shown in a sequence 5 of a coding sequence table into a chimpanzee adenovirus vector to obtain a recombinant plasmid;
(d3) inserting a DNA molecule of a protein shown in a sequence 1 of a coding sequence table into a chimpanzee adenovirus vector to obtain a recombinant plasmid;
(d4) the recombinant plasmid is obtained by inserting DNA molecules of protein consisting of 14 th to 236 th amino acid residues in a sequence 3 of a coding sequence table into a chimpanzee adenovirus vector.
5. A recombinant adenovirus obtained by transfecting the recombinant plasmid according to claim 4 into an adenovirus packaging cell and then culturing the cell.
6. A kit for preparing a recombinant adenovirus comprising the recombinant plasmid of claim 4 and an adenovirus packaging cell.
7. A product, the active ingredient of which is the recombinant adenovirus according to claim 1 or 5 or the protein according to claim 2 or the nucleic acid molecule according to claim 3 or the recombinant plasmid according to claim 4;
the application of the product is (e1) or (e 2):
(e1) as a novel coronavirus vaccine;
(e2) can be used as an anti-novel coronavirus drug.
8. The recombinant adenovirus according to claim 1 or 5, which is (f1) or (f 2):
(e1) preparing a novel coronavirus vaccine;
(e2) preparing the medicine for resisting the novel coronavirus.
9. The protein of claim 2, the nucleic acid molecule of claim 3, or the recombinant plasmid of claim 4, wherein the protein of claim 2 or the nucleic acid molecule of claim 3 or the recombinant plasmid of claim 4 is (f1) or (f 2):
(e1) preparing a novel coronavirus vaccine;
(e2) preparing the medicine for resisting the novel coronavirus.
10. The use of the kit of claim 6, which is (f1) or (f 2):
(e1) preparing a novel coronavirus vaccine;
(e2) preparing the medicine for resisting the novel coronavirus.
CN202010369075.2A 2020-05-01 2020-05-01 3 kinds of recombinant adenovirus, RBD of SARS-CoV-2 Spike protein and their application Pending CN113583978A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
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