CN117165538A - Varicella zoster virus vaccine and application thereof - Google Patents

Varicella zoster virus vaccine and application thereof Download PDF

Info

Publication number
CN117165538A
CN117165538A CN202210480448.2A CN202210480448A CN117165538A CN 117165538 A CN117165538 A CN 117165538A CN 202210480448 A CN202210480448 A CN 202210480448A CN 117165538 A CN117165538 A CN 117165538A
Authority
CN
China
Prior art keywords
varicella zoster
virus
coding gene
zoster virus
adc68xy3
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210480448.2A
Other languages
Chinese (zh)
Inventor
周东明
杨茜
邢嫚
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Xiangyi Biotechnology Co ltd
Original Assignee
Suzhou Xiangyi Biotechnology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Xiangyi Biotechnology Co ltd filed Critical Suzhou Xiangyi Biotechnology Co ltd
Priority to CN202210480448.2A priority Critical patent/CN117165538A/en
Publication of CN117165538A publication Critical patent/CN117165538A/en
Pending legal-status Critical Current

Links

Landscapes

  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention discloses varicella zoster virus vaccine and application thereof. The invention successfully constructs and identifies 8 recombinant chimpanzee adenoviruses by using a replication-defective chimpanzee adenovirus vector AdC68XY 3. After 8 recombinant chimpanzee adenoviruses were tested for immunization using the mouse model, a T cell response with specific VZVgE and an antibody response with serum-specific VZVgE were elicited, and the results showed that: the 8 recombinant chimpanzee adenoviruses prepared by the invention can induce stronger antiviral specific immune response. More importantly, unlike the live attenuated vaccine and recombinant protein vaccine which are on the market and can only induce the antiviral CD4T cell response, the 8 recombinant chimpanzee adenovirus prepared by the invention can induce the strong antiviral CD4T cell response and simultaneously induce the extremely strong antiviral CD8T cell response. Therefore, the 8 recombinant chimpanzee adenovirus of the invention can be used as a novel varicella zoster vaccine candidate.

Description

Varicella zoster virus vaccine and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a varicella zoster virus vaccine and application thereof, in particular to a novel varicella zoster virus vaccine based on chimpanzee adenovirus vector AdC68XY3 and application thereof.
Background
Varicella zoster virus (Varicella Zoster Virus, VZV) is the causative agent of varicella (Varicella or Chickenpox) and shingles (Zoster or Shingles). VZV is a ubiquitous and specifically infected human alpha-herpes virus, and does not infect or is highly restricted in other animals, which also makes the study of the pathogenesis of VZV infection very challenging. Epidemiological evidence suggests that VZV primarily infects human T lymphocytes, epithelial cells and neuronal cells, and primarily spreads among cells, CD4 and CD 8T lymphocytes being more important for host antiviral defenses than antibodies. Primary infections with VZV cause varicella, especially severe varicella herpes, either locally or systemically, in infants or immunocompromised individuals. When the VZV is infected for the first time, the VZV particles can be transported to ganglion neurons at different positions along the reverse axon through T cell viremia or skin replication parts, and latent infection is established and cannot be thoroughly cleared until the individual is recovered. With the age of the individual, or the immune function of the immunocompromised individual, the cellular immunity (Cell-Mediated Immunity, CMI) against VZV is weakened and VZV that is latent in neurons is reactivated to develop shingles. Antiviral CMI studies in adult varicella patients indicate that disease severity is positively correlated with viral load and negatively correlated with antiviral specific cellular response of T lymphocytes.
VZV is extremely infectious and can be transmitted by the air route. The skin herpes vesicles of varicella and zoster patients contain highly infectious viruses, and the infected skin cells and VZV (Cell-free) are frequently shed, which are considered to be the main sources of airborne viruses. Varicella is prevalent worldwide each year, particularly in children but not exclusively adults, and its infectious incidence is generally about 13-16% in winter and spring. As CMI against VZV decreases with age, both the incidence and severity of shingles increase. Herpes zoster can lead to a range of complications including chronic post-herpetic neuralgia (Postherpetic Neuralgia, PHN), meningitis, myelitis, cranial nerve palsy, vascular disease, keratitis and retinopathy among other serious neurological and ocular diseases, as well as ulcers, hepatitis and pancreatitis among various visceral or gastrointestinal diseases. PHN is the most serious complication of shingles, and about 15% of shingles patients are afflicted with pain over a long period of time. Age is also an important risk factor for PHN, especially in people over 50 years of age. About 1/3 of the adults are susceptible to shingles before the shingles vaccine is marketed.
The development of preventive varicella vaccine was traced back to 1970, and attenuated live vaccine strain (vOka) was successfully developed by Japanese scientist high-bridge institute (Michiaki Takahashi), on the basis of which freeze-dried varicella attenuated live vaccine is now used. Today varicella vaccines greatly improve the quality of life of people, and children cannot be hospitalized with severe disease complications caused by infection of varicella ill and lessons or infection. Adults susceptible to varicella can also benefit from vaccination with varicella vaccines. The effective rate of varicella vaccine was found to be 65.4% (one dose) and 94.9% (two doses) in a recent study of random control.
Early-stage herpes zoster vaccine development was based on varicella vaccine, and Zostavax, which was obtained from the company moesand in 2006 in the U.S. was likewise an attenuated live vaccine with a PFU (plaque forming unit) at least 12 times that of varicella vaccine. The vaccine has an effective rate of 69.8% in the 50-59 year old population and 64% in the 60-69 year old population; the efficacy in preventing PHN was 66.5% in people over 60 years old and 37% in people over 70 years old. However, since the vaccine is an attenuated live virus, there is a certain increased risk of illness after vaccination, particularly for people with immunodeficiency or low immunity, zostavax is temporarily not available in China. In addition, the shintrix, developed by the company glanin smith, and marketed in 2017, is a VZVgE recombinant protein vaccine (AS 01B adjuvant) with higher efficacy than the attenuated vaccine, 96.6% in the 50-59 year old population, 97.4% in the 60-69 year old population, 90% in the 70-79 year old population, and 89.2% in the above 80 year old population.
Disclosure of Invention
The invention aims to provide a novel varicella zoster virus vaccine based on chimpanzee adenovirus vector AdC68XY3 and application thereof.
In order to achieve the above object, the present invention provides, first, a recombinant virus which is any one of the following A1) to A8):
a1 A recombinant adenovirus expressing varicella zoster virus gE protein obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene into the genomic DNA of an AdC68XY3-empty virus;
a2 A recombinant adenovirus expressing varicella zoster virus gE protein obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene and CPG sequence into the genomic DNA of an AdC68XY3-empty virus;
a3 A recombinant adenovirus expressing varicella zoster virus gE protein and cytokine IL21 obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene and cytokine IL21 coding gene into the genome DNA of an AdC68XY3-empty virus;
a4 A recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein encoding gene and a T cell costimulatory molecule CD40L encoding gene into the genome DNA of an AdC68XY3-empty virus, and expressing the varicella zoster virus gE protein and the T cell costimulatory molecule CD 40L;
A5 A recombinant adenovirus obtained by inserting a DNA molecule containing a gene encoding the gE protein of varicella zoster virus and a DNA molecule containing a gene encoding the ProB protein of trimeric porin into the genomic DNA of AdC68XY3-empty virus, expressing the gE protein of varicella zoster virus and the ProB protein of trimeric porin;
a6 A recombinant adenovirus obtained by inserting a DNA molecule comprising a varicella zoster virus gE protein encoding gene, a varicella zoster virus Orf9 protein encoding gene and a varicella zoster virus Orf63 protein encoding gene into genomic DNA of an AdC68XY3-empty virus, expressing varicella zoster virus gE protein, varicella zoster virus Orf9 protein and varicella zoster virus Orf63 protein;
a7 A recombinant adenovirus expressing varicella zoster virus gE protein and varicella zoster virus gH protein obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene and varicella zoster virus gH protein coding gene into the genome DNA of an AdC68XY3-empty virus;
a8 A recombinant adenovirus expressing varicella zoster virus gE protein and varicella zoster virus gB protein obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene and varicella zoster virus gB protein coding gene into the genome DNA of an AdC68XY3-empty virus;
The nucleotide sequence of the genomic DNA of the AdC68XY3-empty virus is shown as a sequence 1.
In the recombinant virus, the amino acid sequence of the varicella zoster virus gE protein is shown as a sequence 4.
The amino acid sequence of the cytokine IL21 is shown in sequence 10.
The amino acid sequence of the T cell costimulatory molecule CD40L is shown in SEQ ID NO. 12.
The amino acid sequence of the trimeric porin ProB is shown in sequence 15.
The amino acid sequence of the varicella zoster virus Orf9 protein is shown as sequence 18.
The amino acid sequence of the varicella zoster virus Orf63 protein is shown as sequence 21.
The amino acid sequence of the varicella zoster virus gH protein is shown as sequence 23.
The amino acid sequence of the varicella zoster virus gB protein is shown as sequence 25.
Further, the varicella zoster virus gE protein coding gene is shown in a sequence 3 (containing a terminator) or a sequence 3 (without the terminator) from 1 to 1869. Specifically, in the recombinant viruses A1) and A2) above, the varicella zoster virus gE protein encoding gene is shown as a sequence 3, and in the recombinant viruses A3) to A8) above, the varicella zoster virus gE protein encoding gene is shown as a sequence 3 at positions 1-1869.
The cytokine IL21 encoding gene is shown in sequence 9.
The gene encoding the T cell costimulatory molecule CD40L is shown in sequence 11.
The encoding gene of the trimeric porin ProB is shown in a sequence 14.
The varicella zoster virus Orf9 protein encoding gene is shown in sequence 17.
The varicella zoster virus Orf63 protein encoding gene is shown in sequence 20.
The varicella zoster virus gH protein coding gene is shown in a sequence 22.
The varicella zoster virus gB protein coding gene is shown in a sequence 24.
The CPG sequence is shown in SEQ ID NO. 6. The CPG sequence may be 2-10 consecutive repeats of the CPG sequence.
Still further, in the A1), the DNA molecule containing the varicella zoster virus gE protein coding gene comprises a CMV promoter a, the varicella zoster virus gE protein coding gene and a BGH polyA signal a in sequence.
In the A2), the DNA molecule containing the varicella zoster virus gE protein coding gene and the CPG sequence sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a DNA molecule containing 4 continuous repeated CPG sequences and a BGH polyA signal a.
In the A3), the DNA molecule containing the varicella zoster virus gE protein coding gene and the cytokine IL21 coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a connector p2a sequence, the cytokine IL21 coding gene and a BGH polyA signal a.
In the A4), the DNA molecule containing the varicella zoster virus gE protein coding gene and the T cell costimulatory molecule CD40L coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a linker p2a sequence, the T cell costimulatory molecule CD40L coding gene and a BGH polyA signal a.
In the A5), the DNA molecule containing the varicella zoster virus gE protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene and a BGH polyA signal a; the DNA molecule containing the trimeric porin ProB coding gene sequentially comprises a CMV promoter b, the trimeric porin ProB coding gene and a BGH polyA signal b.
In the A6), the DNA molecule containing varicella zoster virus gE protein encoding gene, varicella zoster virus Orf9 protein encoding gene and varicella zoster virus Orf63 protein encoding gene comprises CMV promoter a, varicella zoster virus gE protein encoding gene, linker p2a sequence, varicella zoster virus Orf9 protein encoding gene, linker f2a sequence, varicella zoster virus Orf63 protein encoding gene and BGH polyA signal a in sequence.
In the A7), the DNA molecule containing the varicella zoster virus gE protein coding gene and the varicella zoster virus gH protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a linker p2a sequence, the varicella zoster virus gH protein coding gene and a BGH polyA signal a.
In the A8), the DNA molecule containing the varicella zoster virus gE protein coding gene and the varicella zoster virus gB protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a linker p2a sequence, the varicella zoster virus gB protein coding gene and a BGH polyA signal a.
The nucleotide sequence of any of the CMV promoters a described above is shown in SEQ ID NO. 2.
The nucleotide sequence of any one of the BGH polyA signals a is shown in the sequence 5.
The nucleotide sequence of any of the above DNA molecules containing 4 consecutive repeats of CPG sequence is shown in SEQ ID NO. 7.
The sequence of any of the above-mentioned linkers p2a is shown in SEQ ID NO. 8.
The sequence of any of the above linkers f2a is shown as sequence 19.
The nucleotide sequence of any of the CMV promoters b described above is shown in SEQ ID NO 13.
The nucleotide sequence of any of the above BGH polyA signals b is shown in SEQ ID NO. 16.
Still further, in the A1), the DNA molecule containing the varicella zoster virus gE protein encoding gene is composed of a CMV promoter a, the varicella zoster virus gE protein encoding gene and a BGH polyA signal a in this order;
in the A2), the DNA molecule containing the varicella zoster virus gE protein coding gene and the CPG sequence sequentially consists of a CMV promoter a, the varicella zoster virus gE protein coding gene, a DNA molecule containing 4 continuous repeated CPG sequences and a BGH polyA signal a;
in the A3), the DNA molecule containing the varicella zoster virus gE protein coding gene and the cytokine IL21 coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a connector p2a sequence, the cytokine IL21 coding gene and a BGH polyA signal a;
in the A4), the DNA molecule containing varicella zoster virus gE protein coding gene and T cell costimulatory molecule CD40L coding gene sequentially comprises CMV promoter a, varicella zoster virus gE protein coding gene, linker p2a sequence, T cell costimulatory molecule CD40L coding gene and BGH polyA signal a;
in the A5), the DNA molecule containing the varicella zoster virus gE protein coding gene sequentially consists of a CMV promoter a, the varicella zoster virus gE protein coding gene and a BGH polyA signal a; the DNA molecule containing the trimeric porin ProB coding gene sequentially comprises a CMV promoter b, the trimeric porin ProB coding gene and a BGH polyA signal b;
In the A6), the DNA molecule containing varicella zoster virus gE protein coding gene, varicella zoster virus Orf9 protein coding gene and varicella zoster virus Orf63 protein coding gene is sequentially composed of CMV promoter a, varicella zoster virus gE protein coding gene, linker p2a sequence, varicella zoster virus Orf9 protein coding gene, linker f2a sequence, varicella zoster virus Orf63 protein coding gene and BGH polyA signal a;
in the A7), the DNA molecule containing the varicella zoster virus gE protein coding gene and the varicella zoster virus gH protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a linker p2a sequence, the varicella zoster virus gH protein coding gene and a BGH polyA signal a;
in the A8), the DNA molecule containing the varicella zoster virus gE protein coding gene and the varicella zoster virus gB protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a linker p2a sequence, the varicella zoster virus gB protein coding gene and a BGH polyA signal a.
Further, in the A1), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene into an E1 deletion region of an AdC68XY3-empty virus;
In the A2), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene and CPG sequence into an E1 deletion region of an AdC68XY3-empty vector virus;
in the A3), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene and a cytokine IL21 coding gene into an E1 deletion region of an AdC68XY3-empty virus;
in the A4), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene and a T cell costimulatory molecule CD40L coding gene into an E1 deletion region of an AdC68XY3-empty virus;
in the A5), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene into an E1 deletion region of an AdC68XY3-empty virus, and inserting a DNA molecule containing a trimeric porin ProB coding gene into an E3 deletion region of the AdC68XY3-empty virus;
in the A6), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene, varicella zoster virus Orf9 protein coding gene and varicella zoster virus Orf63 protein coding gene into an E1 deletion region of an AdC68XY3-empty virus;
In the A7), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene and a varicella zoster virus gH protein coding gene into an E1 deletion region of an AdC68XY3-empty virus;
in the A8), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene and a varicella zoster virus gB protein coding gene into an E1 deletion region of an AdC68XY3-empty virus.
Any of the E1 deleted regions described above may be as shown at positions 518-624 of sequence 1.
Any of the above described E3 deleted regions may be as shown in sequence 1 at positions 25317-25318.
Any of the above insertions is a broad-sense insertion, and may be inserted between any two adjacent nucleotides in the E1 deletion region or the E3 deletion region, or may be inserted between any two non-adjacent nucleotides in the E1 deletion region or the E3 deletion region (i.e., a partial nucleotide substitution).
In a specific embodiment of the present invention, in the A1), the recombinant virus is a recombinant adenovirus obtained by replacing the DNA molecule shown in positions 518-624 of the AdC68XY3-empty virus with a DNA molecule containing the gene encoding the varicella zoster virus gE protein;
In the A2), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th site of an AdC68XY3-empty virus with a DNA molecule containing varicella zoster virus gE protein coding gene and CPG sequence;
in the A3), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th site of an AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene and a cytokine IL21 coding gene;
in the A4), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th site of an AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene and a T cell costimulatory molecule CD40L coding gene;
in the A5), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th site of the AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene, and inserting a DNA molecule containing a trimeric porin ProB coding gene between 25317-25318 th site of the AdC68XY3-empty virus;
in the A6), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th position of an AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene, a varicella zoster virus Orf9 protein coding gene and a varicella zoster virus Orf63 protein coding gene;
In the A7), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th position of an AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene and a varicella zoster virus gH protein coding gene;
in the A8), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th position of an AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene and a varicella zoster virus gB protein coding gene.
In order to achieve the above purpose, the invention also provides a new application of the recombinant virus.
The invention provides the use of the recombinant virus described above in any one of the following X1) -X5):
x1) as varicella zoster vaccine;
x2) as a varicella zoster virus drug;
x3) preparing varicella zoster vaccine;
x4) preparing a varicella zoster virus drug;
x5) preparing a product for preventing and/or treating varicella or zoster.
In order to achieve the above object, the present invention finally provides a product, the active ingredient of which is the recombinant virus described above; the use of the product is as follows Z1) or Z2):
Z1) varicella zoster virus;
z2) preventing and/or treating varicella or zoster.
In any of the above applications or products, the product may be a vaccine or a medicament. The vaccine may be a prophylactic vaccine or a therapeutic vaccine.
The vaccine can be administered at an immunizing dose of (0.1-10) x 10 10 vp。
The subject to be immunized with the vaccine may be a mammal, including mice, monkeys, and humans.
The number of immunizations of the vaccine may be one or two or more.
The immunization mode of the vaccine can be intramuscular injection, subcutaneous injection or nasal spray.
In a specific embodiment of the invention, the vaccine is administered to a subject in the form of a mouse, in particular a female Balb/c mouse of 6-8 weeks of age, at an immunizing dose of 5X 10 10 vp, the number of immunization times is one, and the immunization mode is intramuscular injection.
Adjuvants or immunopotentiators or immunomodulators or other vaccines may also be added to the above vaccines, if desired.
Pharmaceutically acceptable carriers can also be added to the above drugs, if necessary.
The invention successfully constructs and identifies 8 recombinant chimpanzee adenoviruses AdC68XY3-gE, adC68XY3-gE-CPG, adC68XY3-gE-IL21, adC68XY3-gE-CD40L, adC68XY3-gE-ProB, adC68XY3-gE-Orf9-Orf63, adC68XY3-gE-gH and AdC68XY3-gE-gB by using replication defective chimpanzee adenovirus vectors AdC68XY 3. After 8 recombinant chimpanzee adenoviruses were tested for immunization using the mouse model, a T cell response with specific VZVgE and an antibody response with serum-specific VZVgE were elicited, and the results showed that: the 8 recombinant chimpanzee adenoviruses prepared by the invention can induce stronger antiviral specific immune response. More importantly, unlike the attenuated live vaccine (Zostavax) and the recombinant protein vaccine (shintrix) which are on the market and can only induce the antiviral CD 4T cell response, the 8 recombinant chimpanzee adenovirus prepared by the invention can induce the strong antiviral CD 4T cell response and simultaneously induce the extremely strong antiviral CD 8T cell response. Therefore, the 8 recombinant chimpanzee adenovirus of the invention can be used as a novel varicella zoster vaccine candidate.
Drawings
FIG. 1 shows basic information of 8 novel varicella zoster vaccine candidates with recombinant chimpanzee adenovirus AdC68XY3 as a vector.
FIG. 2 is a pAdC68XY3-empty plasmid map and identification thereof. A is pAdC68XY3-empty plasmid map; b is a simulated image of pAdC68XY3-empty electrophoresis using ApaI, bglII, xhoI enzyme; c is a real electropherogram of pAdC68XY3-empty cut using ApaI, bglII, xhoI.
FIG. 3 is a map of the recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE and its identification. A is pAdC68XY3-gE plasmid map; b is a simulated image of the electrophoresis of pAdC68XY3-gE cut with ApaI, bglII, mfeI; c is a true electropherogram of pAdC68XY3-gE cut with ApaI, bglII, mfeI.
FIG. 4 is a map of the recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-CPG and its identification. A is pAdC68XY3-gE-CPG plasmid map; b is a simulated chart of the electrophoresis of pAdC68XY3-gE-CPG cut by using ApaI, bglII, mfeI; c is a true electropherogram of pAdC68XY3-gE-CPG cut with ApaI, bglII, mfeI.
FIG. 5 is a map of recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-IL21 and its identification. A is pAdC68XY3-gE-IL21 plasmid map; b is an electrophoresis simulation of pAdC68XY3-gE-IL21 cut by using ApaI, mfeI, xhoI; c is a true electropherogram of pAdC68XY3-gE-IL21 cut with ApaI, mfeI, xhoI.
FIG. 6 is a map of the recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-CD40L and its identification. A is pAdC68XY3-gE-CD40L plasmid map; b is a simulated image of the electrophoresis of pAdC68XY3-gE-CD40L cut with ApaI, bglII, mfeI; c is a true electropherogram of pAdC68XY3-gE-CD40L cut with ApaI, bglII, mfeI.
FIG. 7 is a graph of the recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-ProB and its identification. A is pAdC68XY3-gE-ProB plasmid map; b is a simulated chart of the electrophoresis of pAdC68XY3-gE-ProB cut by using ApaI, bglII, mfeI; c is a real electropherogram of pAdC68XY3-gE-ProB cut using ApaI, bglII, mfeI.
FIG. 8 is a map of the recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-Orf9-Orf63 and identification thereof. A is pAdC68XY3-gE-Orf9-Orf63 plasmid map; b is a simulated electrophoresis chart of pAdC68XY3-gE-Orf9-Orf63 cut by using ApaI, mfeI, xhoI; c is a true electropherogram of pAdC68XY3-gE-Orf9-Orf63 cut with ApaI, mfeI, xhoI.
FIG. 9 is a graph of the recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-gH and its identification. A is pAdC68XY3-gE-gH plasmid map; b is a simulated chart of pAdC68XY3-gE-gH electrophoresis cut by using ApaI, xhoI, mfeI; c is a true electropherogram of pAdC68XY3-gE-gH cut using ApaI, xhoI, mfeI.
FIG. 10 is a map of the recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-gB and its identification. A is pAdC68XY3-gE-gB plasmid map; b is a simulated chart of pAdC68XY3-gE-gB electrophoresis cut using ApaI, mfeI, xhoI; c is a true electropherogram of pAdC68XY3-gE-gB cut using ApaI, mfeI, xhoI.
FIG. 11 is a map of the genomic DNA cleavage assay of recombinant chimpanzee adenoviruses. A is a recombinant chimpanzee adenovirus AdC68XY3-empty genome DNA, and a ApaI, bglII, xhoI enzyme digestion identification map is obtained; b is a recombinant chimpanzee adenovirus AdC68XY3-gE genome DNA, and a ApaI, bglII, mfeI enzyme digestion identification map is obtained; c is a recombinant chimpanzee adenovirus AdC68XY3-gE-CPG genome DNA, and a ApaI, bglII, mfeI enzyme digestion identification map is obtained; d is a recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 genome DNA, and the DNA is subjected to ApaI, mfeI, xhoI enzyme digestion identification map; e is a recombinant chimpanzee adenovirus AdC68XY3-gE-CD40L genome DNA, and a BglII, mfeI, xhoI enzyme digestion identification map is obtained; f is a recombinant chimpanzee adenovirus AdC68XY3-gE-ProB genome DNA, and a ApaI, bglII, mfeI enzyme digestion identification map is obtained; g is a recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63 genome DNA, and the genome DNA is subjected to ApaI, mfeI, xhoI enzyme digestion identification map; h is a recombinant chimpanzee adenovirus AdC68XY3-gE-gH genome DNA, and a ApaI, mfeI, xhoI enzyme digestion identification map is obtained; i is a recombinant chimpanzee adenovirus AdC68XY3-gE-gB genome DNA, and the recombinant chimpanzee adenovirus AdC68XY3-gE-gB genome DNA is subjected to ApaI, bglII, xhoI enzyme digestion identification map.
FIG. 12 shows Western blotting for detecting VZVGE protein expression. A is the immunoblotting identification of expressed VZVGE protein after HEK293 cells are infected by different doses of AdC68XY3-gE recombinant chimpanzee adenovirus; b is the western blotting identification of expressing VZVGE after the HEK293 cells are infected by different doses of AdC68XY3-gE-CPG recombinant chimpanzee adenovirus; c is the expression VZVGE western blotting identification after the HEK293 cells are infected by the different doses of AdC68XY3-gE-IL21 recombinant chimpanzee adenovirus; d is the expression VZVGE protein immunoblotting identification after the HEK293 cells are infected by different doses of AdC68XY3-gE-CD40L recombinant chimpanzee adenovirus; e is expressed VZVGE immunoblotting identification after HEK293 cells are infected by different doses of AdC68XY3-gE-ProB recombinant chimpanzee adenovirus; f is the immunoblotting identification of expressing VZVGE protein, VZVOrf9 protein and VZVOrf63 protein after the HEK293 cells are infected by different doses of AdC68XY3-gE-Orf9-Orf63 recombinant chimpanzee adenovirus; g is the immunoblotting identification of expressed VZVGE protein and VZVGH protein after the HEK293 cells are infected by different doses of AdC68XY3-gE-gH recombinant chimpanzee adenovirus; h is the immunoblotting identification of expressed VZVGE protein and VZVGE protein after the HEK293 cells are infected by different doses of AdC68XY3-gE-gB recombinant chimpanzee adenovirus.
FIG. 13 is a protocol for a recombinant chimpanzee adenovirus immunized mouse.
FIG. 14 is a graph of flow cytometry gag strategy analysis of recombinant chimpanzee adenovirus AdC68XY3-gE immunized mice.
Figure 15 elicits an antigen VZVgE specific CD 4T cell response in recombinant chimpanzee adenovirus immunized mice. A is expression of IFN gamma + Cell occupancy of CD4 + T cell ratio; b is expression of IL2 + Cell occupancy of CD4 + T cell ratio; c is expression of TNF alpha + Cell occupancy of CD4 + T cell ratio; d is expression of IFNgamma + ，IL2 + And/or TNF alpha + Cell occupancy of CD4 by at least one cytokine + T cell ratio; e is analysis of CD4 using Boolean Combination Gates + Three cells among T cellsCombined expression pattern of factors. The significance analysis method used was one-way ANOVA and the data was expressed as geometric mean with 95% confidence interval (n=6).
FIG. 16 elicits antigen VZVGE-specific CD 8T cell responses in recombinant chimpanzee adenovirus immunized mice. A is expression of IFN gamma + Cell occupancy of CD8 + T cell ratio; b is expression of IL2 + Cell occupancy of CD8 + T cell ratio; c is expression of TNF alpha + Cell occupancy of CD8 + T cell ratio; d is expression of IFNgamma + ，IL2 + And/or TNF alpha + Cell occupancy of CD8 by at least one cytokine + T cell ratio; e is analysis of CD8 using Boolean Combination Gates + Combined expression pattern of three cytokines in T cells. The significance analysis method used was one-way ANOVA and the data was expressed as geometric mean with 95% confidence interval (n=6).
Figure 17 elicits a serum antigen VZVgE specific antibody response for recombinant chimpanzee adenovirus immunized mice. After A is a recombinant chimpanzee adenovirus immunized mouse, taking serum at week 2, week 4, week 6, week 8 and week 13, and detecting the titer level of an antigen VZVGE specific antibody IgG; b is the titer level of gE-specific antibody IgG at week 6 after mouse immunization; c is the expression level of the gE specific antibody IgG2a in the serum of the 6 th week after the immunization of the mice; d is the expression level of the gE specific antibody IgG2b in the serum of the 6 th week after the immunization of the mice; e is the expression level of the gE-specific antibody IgG1 in the serum of week 6 after immunization of the mice. The significance analysis method used was one-way ANOVA and the data was expressed as geometric mean with 95% confidence interval (n=5).
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
The main reagents and sources involved in the following examples are as follows: a pool of 150 peptides (15 amino acids each and overlapping every 10 amino acids) against VZVgE antigen (gold sri); cell co-stimulatory factor Anti-mouse CD28, anti-mouse CD49d, anti-mouse CD16/CD32, cytokine secretion blocker GolgiPlug TM ， Cytofix/Cytoperm TM Fixation/Permeabilization Kit et al (BD); streaming antibody AF488 Anti-CD3 (bioleged, 100210), BV785 Anti-CD4 (bioleged, 100552), APC Anti-CD8 (bioleged, 100712), LIVE/DEAD TM Fixable Aqua dead cell stain (Invitrogen, L34957), PE Anti-IFNgamma (bioleged, 505808), PE-Cy7 Anti-IL2 (bioleged, 503832), BV650 Anti-TNFalpha (bioleged, 506333), flow cytometer LSRFortessa (BD); lipofectamine 2000 (Invitrogen), DNeasy Blood&Tissue Kit (Qiagen), recombinant VZVGE protein (Abcam, ab 43050), anti-VZVGE protein antibodies (Abcam, ab 272686), HRP-labeled Anti-mouse and Anti-human IgG antibodies (Sigma-Aldrich), western blotting-related reagents (New Saimei organism).
Example 1 preparation and identification of recombinant chimpanzee adenoviruses
1. Construction and identification of recombinant chimpanzee adenovirus plasmids
According to the Human herpesvirus strain VZVs/Shanggai.CHN/42.11/1 glycoprotein E gene (Genebank: KC 820942.1) searched on NCBI, namely gene orf68 (CDS region, 1872bp, containing stop codon) encoding the VZVGE protein was optimized according to the human codon, and EcoRI cleavage site (gaattc) and Kozak sequence (gccacc) were added at the 5 'end, hindIII cleavage site (aagctt) was added at the 3' end, and codon optimization and synthesis were completed by the Optimago. Then, target genes such as VZVGE and the like are inserted into pShuttle plasmid containing CMV promoter and BGH ployA through restriction enzyme ligation or DNA recombination technology, and then the whole expression elements are cloned into pAdC68XY3-empty plasmid respectively by utilizing rare enzymes I-Ceu I and PI-Sce I, so that the following 8 recombinant chimpanzee adenovirus plasmids are constructed: pAdC68XY3-gE, pAdC68XY3-gE-CPG, pAdC68XY3-gE-IL21, pAdC68XY3-gE-CD40L, pAdC XY3-gE-ProB, pAdC68XY3-gE-Orf9-Orf63, pAdC68XY3-gE-gH and pAdC68XY3-gE-gB. The basic information of pAdC68XY3-empty plasmid and 8 recombinant chimpanzee adenovirus plasmids is shown in FIG. 1. The detailed information is as follows:
The pAdC68XY3-empty plasmid map is shown in FIG. 2A. The pAdC68XY3-empty plasmid was subjected to cleavage assay using ApaI, bglII, xhoI, and the cleavage assay results are shown in FIGS. 2B and 2C.
The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE is shown in FIG. 3A. The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE was subjected to cleavage identification using ApaI, bglII, mfeI, and the cleavage identification results are shown in FIGS. 3B and 3C.
The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-CPG is shown in FIG. 4A. The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-CPG was subjected to cleavage assay using ApaI, bglII, mfeI, and the cleavage assay results are shown in FIGS. 4B and 4C.
The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-IL21 is shown in FIG. 5A. The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-IL21 was digested with ApaI, mfeI, xhoI, and the results of the digestion are shown in FIGS. 5B and 5C.
The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-CD40L is shown in FIG. 6A. The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-CD40L was subjected to cleavage assay using ApaI, bglII, mfeI, and the cleavage assay results are shown in FIGS. 6B and 6C.
The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-ProB is shown in FIG. 7A. The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-ProB was subjected to cleavage assay using ApaI, bglII, mfeI, and the cleavage assay results are shown in FIGS. 7B and 7C.
The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-Orf9-Orf63 is depicted in FIG. 8A. The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-Orf9-Orf63 was subjected to cleavage assay using ApaI, mfeI, xhoI, and the results of the cleavage assay are shown in FIGS. 8B and 8C.
The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-gH is shown in FIG. 9A. The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-gH was subjected to cleavage assay using ApaI, xhoI, mfeI, and the cleavage assay results are shown in FIGS. 9B and 9C.
The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-gB is shown in FIG. 10A. The recombinant chimpanzee adenovirus plasmid pAdC68XY3-gE-gB was subjected to cleavage assay using ApaI, mfeI, xhoI, and the cleavage assay results are shown in FIGS. 10B and 10C.
The results of the enzyme digestion identification of the plasmids show that the simulated electrophoresis patterns are completely consistent with the real electrophoresis patterns, which shows that the invention successfully constructs 8 recombinant chimpanzee adenovirus plasmids.
2. Packaging and amplification of recombinant chimpanzee adenoviruses
1. Packaging of recombinant chimpanzee adenoviruses
The pAdC68XY3-empty plasmid and 8 recombinant chimpanzee adenovirus plasmids were digested singly with Pac I to give linearized DNA, which was transfected into HEK293 cells (Shanghai department of China) with Lipofectamine 2000 (Invitrogen), respectively. Before transfection, the cell culture medium was changed to serum-free DMEM, then DNA/Lipofectamine 2000 liposome complex mixed in 1:3 was added, and after 4-6 hours of cell transfection, the cell culture medium was changed to DMEM medium containing 5% fbs. Cytopathy is observed under a microscope every day after transfection, and when cell plaques reach 60% -80%, rescue virus cell suspensions (virus liquid) are collected.
Linearized DNA of recombinant chimpanzee adenovirus plasmids pAdC68XY3-gE, pAdC68XY3-gE-CPG, pAdC68XY3-gE-IL21, pAdC68XY3-gE-CD40L, pAdC XY3-gE-ProB, pAdC68XY3-gE-Orf9-Orf63, pAdC68XY3-gE-gH and pAdC68XY3-gE-gB were packaged by HEK293 cells to obtain recombinant chimpanzee adenoviruses AdC68XY3-gE, adC68XY3-gE-CPG, adC68XY3-gE-IL21, adC68XY3-gE-CD40L, adC XY3-gE-ProB, adC68XY3-gE-Orf9-Orf63, adC68XY3-gE-gH and AdC68XY3-gE-gB, respectively.
Linearized DNA of pAdC68XY3-empty vector was packaged by HEK293 cells to give control adenovirus AdC68XY3-empty (AdC 68XY3-empty vector).
The nucleotide sequence of the control adenovirus AdC68XY3-empty (AdC 68XY 3-empty) is shown as sequence 1, and is a novel chimpanzee adenovirus vector obtained by modifying the genome DNA of chimpanzee adenovirus AdC68, and in the modified vector, the E1 region is completely deleted, the E3 region is partially deleted, and the E4 region is partially deleted and replaced by human adenovirus type 5 ORF6/7. The E1 deletion region in the vector is shown in 518 th-624 th of sequence 1, and the E3 deletion region is shown in 25317 th-25318 th of sequence 1.
The recombinant chimpanzee adenovirus AdC68XY3-gE is obtained by replacing DNA molecules shown in 518-624 th sites of AdC68XY3-empty virus with a VZVGE expression element and keeping other sequences of the AdC68XY3-empty virus unchanged. Wherein the VZVGE expression element sequentially comprises a CMV promoter a (sequence 2), a varicella zoster virus VZVGE codon optimized sequence (sequence 3) and a BGH polyA signal a (sequence 5). The recombinant chimpanzee adenovirus AdC68XY3-gE expresses a VZVGE protein, the amino acid sequence of which is shown in sequence 4.
The recombinant chimpanzee adenovirus AdC68XY3-gE-CPG is obtained by replacing DNA molecules shown in 518-624 th sites of AdC68XY3-empty vector with a VZVGE-CPG4 expression element and keeping other sequences of the AdC68XY3-empty vector unchanged. Wherein the VZVGE-CPG4 expression element sequentially comprises a CMV promoter a (sequence 2), a varicella zoster virus VZVGE codon optimized sequence (sequence 3), a DNA molecule (sequence 7) containing 4 continuous repeated CPG sequences and a BGH polyA signal a (sequence 5). The recombinant chimpanzee adenovirus AdC68XY3-gE-CPG expresses the VZVGE protein, the amino acid sequence of which is shown in sequence 4.
The recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 is obtained by replacing DNA molecules shown in 518-624 th sites of AdC68XY3-empty vector with a VZVGE-p2a-IL21 (human) expression element and keeping other sequences of pAdC68XY3-empty vector unchanged. Wherein, the expression element of the VZVGE-p2a-IL21 (human) sequentially comprises a CMV promoter a (sequence 2), a varicella zoster virus VZVGE codon optimized sequence (1 st to 1869 th bit in sequence 3), a connector p2a sequence (sequence 8), a cytokine IL21 coding gene sequence (human) (sequence 9) and a BGH polyA signal a (sequence 5). The recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 expresses the VZVGE protein and the cytokine IL21, the amino acid sequence of the VZVGE protein is shown in a sequence 4, and the amino acid sequence of the cytokine IL21 is shown in a sequence 10.
The recombinant chimpanzee adenovirus AdC68XY3-gE-CD40L is obtained by replacing DNA molecules shown in 518-624 th sites of AdC68XY3-empty vector with VZVGE-p2a-CD40L (human) expression elements and keeping other sequences of the AdC68XY3-empty vector unchanged. Wherein the expression element of the VZVGE-p2a-CD40L (human) sequentially comprises a CMV promoter a (sequence 2), a varicella zoster virus VZVGE codon optimized sequence (1 st to 1869 th bit in sequence 3), a connector p2a sequence (sequence 8), a T cell co-stimulatory molecule CD40L coding gene sequence (human) (sequence 11) and a BGH polyA signal a (sequence 5). The recombinant chimpanzee adenovirus AdC68XY3-gE-CD40L expresses the VZVGE protein and the T cell costimulatory molecule CD40L, the amino acid sequence of which is shown in sequence 4, and the amino acid sequence of which is shown in sequence 12.
The recombinant chimpanzee adenovirus AdC68XY3-gE-ProB is obtained by replacing DNA molecules shown in 518-624 th sites of AdC68XY3-empty vector with VZVGE expression elements, inserting ProB expression elements between 25317-25318 th sites of AdC68XY3-empty vector, and keeping other sequences of the AdC68XY3-empty vector unchanged. Wherein the VZVGE expression element sequentially comprises a CMV promoter a (sequence 2), a varicella zoster virus VZVGE codon optimized sequence (sequence 3) and a BGH polyA signal a (sequence 5); the ProB expression element consisted of CMV promoter b (SEQ ID NO: 13), the trimeric porin ProB sequence (Neisseria meningitidis) (SEQ ID NO: 14) and the BGH polyA signal b (SEQ ID NO: 16) in sequence. The recombinant chimpanzee adenovirus AdC68XY3-gE-ProB expresses the VZVGE protein and the trimeric porin ProB, the amino acid sequence of which is shown in sequence 4, and the amino acid sequence of which is shown in sequence 15.
The recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63 is obtained by replacing DNA molecules shown at 518-624 th sites of AdC68XY3-empty virus with expression elements of VZVGE-p2a-VZVOrf9-p2a-VZVOrf63 and keeping other sequences of the AdC68XY3-empty virus unchanged. Wherein, the VZVGE-p2a-VZVOrf9-f2a-VZVOrf63 expression element is composed of a CMV promoter a (sequence 2), a varicella zoster virus VZVGE codon optimized sequence (1 st to 1869 th bit in sequence 3), a linker p2a sequence (sequence 8), a varicella zoster virus VZVOrf9 sequence (sequence 17), a linker f2a sequence (sequence 19), a varicella zoster virus VZVOrf63 sequence (sequence 20) and a BGH polyA signal a (sequence 5) in sequence. Recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63 expresses the VZVGE protein, the VZVOrf9 protein and the VZVOrf63 protein, the amino acid sequence of the VZVGE protein is shown in sequence 4, the amino acid sequence of the VZVOrf9 protein is shown in sequence 18, and the amino acid sequence of the VZVOrf63 protein is shown in sequence 21.
The recombinant chimpanzee adenovirus AdC68XY3-gE-gH is obtained by replacing DNA molecules shown in 518-624 th sites of AdC68XY3-empty vector with VZVGE-p2a-VZVGH expression elements and keeping other sequences of the AdC68XY3-empty vector unchanged. Wherein the VZVGE-p2a-VZVGH expression element consists of a CMV promoter a (sequence 2), a varicella zoster virus VZVGE codon optimized sequence (1 st to 1869 th bit in sequence 3), a linker p2a sequence (sequence 8), a varicella zoster virus VZVVGH sequence (sequence 22) and a BGH polyA signal a (sequence 5) in sequence. The recombinant chimpanzee adenovirus AdC68XY3-gE-gH expresses the VZVGE protein and the VZVGH protein, the amino acid sequence of the VZVGE protein is shown as a sequence 4, and the amino acid sequence of the VZVGH protein is shown as a sequence 23.
The recombinant chimpanzee adenovirus AdC68XY3-gE-gB is obtained by replacing DNA molecules shown in 518-624 th sites of AdC68XY3-empty vector with VZVGE-p2a-VZVgB expression elements and keeping other sequences of the AdC68XY3-empty vector unchanged. Wherein the VZVGE-p2 a-VZVGG expression element sequentially comprises a CMV promoter a (sequence 2), a varicella zoster virus VZVGE codon optimized sequence (1 st to 1869 th bit in sequence 3), a linker p2a sequence (sequence 8), varicella zoster virus VZVVGB (sequence 24) and a BGH polyA signal a (sequence 5). The recombinant chimpanzee adenovirus AdC68XY3-gE-gB expresses the VZVGE protein and the VZVGE protein, the amino acid sequence of the VZVGE protein is shown as a sequence 4, and the amino acid sequence of the VZVGE protein is shown as a sequence 25.
2. Amplification of recombinant chimpanzee adenoviruses
The 8 recombinant chimpanzee adenoviruses and the control adenovirus AdC68XY3-empty were amplified respectively as follows: repeatedly freezing and thawing the collected virus liquid for 3 times at room temperature and-80 ℃, centrifuging to collect supernatant, infecting a proper amount of HEK293 cells, infecting 24 hours of cytopathy, and harvesting virus cell suspension according to the method. After 3-4 amplifications of recombinant chimpanzee adenoviruses in a large number of HEK293 cells, approximately 500-600mL of viral cell suspension was finally harvested, cells were collected by centrifugation at the last time and resuspended in 10 mL serum-free DMEM.
3. Recombinant chimpanzee adenovirus concentration purification and titer detection
The titers of the 8 recombinant chimpanzee adenoviruses and the control adenovirus AdC68XY3-empty were purified and detected, respectively, as follows: concentrating recombinant chimpanzee adenovirus by using 1.2g/mL and 1.4g/mL cesium chloride density gradient centrifugation, desalting the concentrated virus by liquid chromatography, removing cesium chloride, replacing buffer with PBS buffer to obtain about 2mL virus solution, adding 10% glycerol, separating into small parts, freezing at-80deg.C for use, measuring absorbance of 9 recombinant chimpanzee adenovirus strains at A260 by Nanodrop, and multiplying the absorbance by 1.1X10 12 Thus obtaining the titer (vp/mL) of each recombinant chimpanzee adenovirus.
The titers of the 8 recombinant chimpanzee adenoviruses and the control adenoviruses AdC68XY3-empty obtained after amplification and purification are shown in Table 1.
TABLE 1 viral titre
Recombinant chimpanzee adenoviruses Titer (vp/mL)
AdC68XY3-empty 1.42×10 13
AdC68XY3-gE 9.9×10 12
AdC68XY3-gE-CPG 3.1×10 12
AdC68XY3-gE-IL21 4.73×10 12
AdC68XY3-gE-CD40L 8.58×10 12
AdC68XY3-gE-ProB 5.3×10 12
AdC68XY3-gE-Orf9-Orf63 6.93×10 12
AdC68XY3-gE-gH 5.8×10 12
AdC68XY3-gE-gB 3.6×10 12
4. Recombinant chimpanzee adenovirus genome restriction enzyme assay
A small aliquot of the frozen 8 recombinant chimpanzee adenoviruses AdC68XY3-gE, adC68XY3-gE-CPG, adC68XY3-gE-IL21, adC68XY3-gE-CD40L, adC XY3-gE-ProB, adC68XY3-gE-Orf9-Orf63, adC68XY3-gE-gH, adC68XY3-gE-gB and control adenovirus pAdC68XY3-empty were taken, and after complete thawing of the virus at room temperature, DNeasy Blood & Tissue Kit was used to extract the viral genomic DNA and 50-100. Mu.L deionized water was used to elute the genomic DNA. Each viral genome was then identified by restriction enzyme using ApaI, bglII, xhoI or MfeI.
The results of the cleavage assay are shown in FIG. 11. As can be seen from the figures: all the electrophoresis bands are clearly visible, which indicates that the virus obtained by concentration and purification can be used for the subsequent animal immunization experiments.
5. Western blotting detection of VZVGE protein expression
8 recombinant chimpanzee adenoviruses AdC68XY3-gE, adC68XY3-gE-CPG, adC68XY3-gE-IL21, adC68XY3-gE-CD40L, adC XY3-gE-ProB, adC68XY3-gE-Orf9-Orf63, adC68XY3-gE-gH and AdC68XY3-gE-gB and control adenoviruses pAdC68XY3-empty were used to infect HEK293 cells, 2X 10 6 The virus infection dose of each cell/hole is 10 respectively 8 vp/well, 10 9 vp/well 10 10 vp/well, cells were lysed on ice using protein lysate (containing protease inhibitors) and centrifuged to obtain protein supernatant, which was used to detect VZVgE protein expression using western blot 24 hours after infection. The specific detection steps are as follows: mixing proper amount of protein supernatant with 5 times of loading buffer solution, boiling to prepare sample, performing SDS-PAGE electrophoresis, transferring PVDF membrane, sealing and the like. Then adding Anti-VZVGE protein antibody or Anti-beta-action or Anti-GAPDH antibody and incubating at 4deg.C overnight. And incubating the HRP-labeled Anti-mouse or Anti-human secondary antibody after membrane washing every other day, and finally obtaining a development result of western blotting.
The results are shown in FIG. 12, 10 10 The expression of any VZVGE protein cannot be detected completely by the control adenovirus AdC68XY3-empty infected HEK293 cells of vp; and 10 (10) 9 vp and 10 10 The expression of the VZVGE protein can be detected obviously by infecting HEK293 cells with vp recombinant chimpanzee adenovirus AdC68XY3-gE (FIG. 12A); 10 8 vp、10 9 vp and 10 10 The expression of VZVGE protein can be obviously detected by infecting HEK293 cells with vp recombinant chimpanzee adenovirus AdC68XY3-gE-CPG (FIG. 12B), 10 9 vp and 10 10 The expression of VZVGE protein can be obviously detected by infecting HEK293 cells with vp recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 (FIG. 12C), 10 9 vp and 10 10 The expression of VZVGE protein can be obviously detected by infecting HEK293 cells with vp recombinant chimpanzee adenovirus AdC68XY3-gE-CDL(FIG. 12D), 10 8 vp、10 9 vp and 10 10 The expression of VZVGE protein can be obviously detected by infecting HEK293 cells with vp recombinant chimpanzee adenovirus AdC68XY3-gE-ProB (FIG. 12E), 10 9 vp and 10 10 The expression of VZVGE, VZVOrf9 and VZVOrf63 proteins (FIG. 12F), 10 can be detected obviously by infecting HEK293 cells with vp recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63 9 vp and 10 10 The expression of VZVGE and VZVGH proteins can be obviously detected by infecting HEK293 cells with vp recombinant chimpanzee adenovirus AdC68XY3-gE-gH (FIG. 12G), 10 9 vp and 10 10 The expression of the VZVGE and VZVGE proteins can be obviously detected by infecting HEK293 cells with vp recombinant chimpanzee adenovirus AdC68XY3-gE-gB (FIG. 12H), which shows that the invention successfully obtains 8 recombinant chimpanzee adenoviruses which express the VZVGE proteins at the cellular level.
EXAMPLE 2 immunization effect of recombinant chimpanzee adenovirus as varicella zoster vaccine
1. Immunization method
The experimental protocol for recombinant chimpanzee adenovirus immunized mice is shown in figure 13. The method comprises the following steps:
female mice (Bai Chart) of 6-8 weeks old were divided into 9 groups as follows:
group1: an immune control adenovirus AdC68XY3-empty;
group2: immunizing a recombinant chimpanzee adenovirus AdC68XY3-gE;
group3: immunizing recombinant chimpanzee adenovirus AdC68XY3-gE-CPG;
group4: immunization of recombinant chimpanzee adenovirus AdC68XY3-gE-IL21;
group5: immunization of recombinant chimpanzee adenovirus AdC68XY3-gE-CD40L;
group6: immunizing a recombinant chimpanzee adenovirus AdC68XY3-gE-ProB;
group7: immunization of recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63;
group8: immunizing a recombinant chimpanzee adenovirus AdC68XY3-gE-gH;
group9: immunization of recombinant chimpanzee adenovirus AdC68XY3-gE-gB.
The immunization protocol for each group was as follows: week 0 muscle immunization aboveRecombinant chimpanzee adenovirus (5X 10) 10 vp/100 μl/mouse, 50 μl each for the left and right legs), 5-6 mice per group. Spleens of mice (6 in each group) were taken at week 2 post immunization, and spleen cells were isolated to detect antigen VZVgE-specific T cell responses; mice were collected at week 2, week 4, week 6, week 8, week 13 (5 animals per group) under the jaw after immunization, respectively, and serum was isolated to detect antigen VZVgE-specific antibody responses.
The specific steps for detecting the VZVGE specific T cell response by the mouse spleen cell flow cytometry are as follows: mice were sacrificed and were whole body sterilized with 75% alcohol and spleens were removed. Cells were obtained by grinding the spleen and sieving with a 40 μm sieve, then red blood cells were lysed on ice, centrifuged and the spleen cells were resuspended and counted. Taking 5×10 6 Spleen cells were resuspended in 200. Mu.L of RPMI 1640 complete medium (10% FBS) containing VZVGE peptide pool (final peptide pool concentration of 1.25. Mu.g/mL/peptide), T cell co-stimulatory factor Anti-mouse CD49d (antibody dilution ratio 1:500) and Anti-mouse CD28 (antibody dilution ratio 1:500), 96 well cell plates were placed at 37℃in 5% CO 2 Stimulation was performed for 2 hours. BD GolgiPlug (dilution ratio 1:1000) was then added to each sample well and the cell plates were returned to 37℃with 5% CO 2 Culture was continued overnight. The following day, cells were stained with Anti-mouse CD16/CD32 (antibody dilution ratio 1:500) to block nonspecific binding of cell surface Fc receptor, then stained with cell surface antibody Anti-CD3, anti-CD4, anti-CD8 (antibody dilution ratio 1:200) and LIVE/DEAD DEAD cells (antibody dilution ratio 1:1000), cells were fixed by Cytofix/Cytoperm rupture, intracellular factors Anti-IFN-gamma, anti-IL-2 and Anti-TNF-alpha (antibody dilution ratio 1:200), and finally resuspended with 350-450. Mu.L of flow buffer. The flow cytometric analyzer measures antigen VZVgE specific T cell responses. The flow cytometry Gating strategy (fig. 14) is specifically as follows: 1) Firstly, selecting se:Sub>A cell main group through SSC-A and FSC-A, then selecting se:Sub>A single cell group through FSC-A and FSC-W twice, and finally selecting se:Sub>A living cell group through Live/read expression negative; 2) By CD3 + CD4 + Selecting CD 4T cells, selecting CD4 respectively + IFNγ + 、CD4 + IL2 + 、CD4 + TNFα + An expressed cell; 3) By CD3 + CD8 + Selecting CD 8T cells, selecting CD8 respectively + IFNγ + 、CD8 + IL2 + 、CD8 + TNFα + And (3) expressing the cells.
The specific steps for detecting the VZVGE specific antibody reaction by the mouse serum ELISA are as follows: recombinant VZVgE protein was coated with coating dilution at 100 ng/well and ELISA plates incubated overnight at 4 ℃. The next day, PBST (PBS buffer containing 0.5% Tween-20) was washed 1 time. Then 200. Mu.L/well of 5% skim milk (w/v in PBST) was added and blocked at 37℃for 2 hours, and the plates were washed 3 times with PBST. The test mice serum was diluted 1:100 using 0.5% skim milk (w/v, dissolved in PBST) and 2-fold diluted, 100. Mu.L/well of the diluted sample was added and ELISA plates were incubated at 37℃for 2 hours. PBST plates were washed 5 times, 100. Mu.L/well of HRP (horseradish peroxidase) -labeled Anti-mouse IgG was added as a secondary enzyme-labeled antibody (1:10000 diluted in PBST), and incubated at 37℃for 1 hour. Plates were washed 7 times with PBST. Adding TMB color development liquid, placing at room temperature, developing for 3-5 min in dark place, adding 2M H after the color development is completed 2 SO 4 The reaction was terminated and absorbance was measured at OD450 using a multifunctional microplate reader.
2. Detection result of VZVGE specific CD 4T cell response after mice are immunized with recombinant chimpanzee adenovirus
The results of the antigen VZVgE specific CD 4T cell response flow assay are shown in fig. 14 and 15. FIG. 14 is a graph showing the results of streaming data for one of the mice in the recombinant chimpanzee adenovirus AdC68XY3-gE immunized group.
8 recombinant chimpanzee adenovirus immunosets secreted IFNγ compared to control adenovirus AdC68XY3-empty + CD4 + T cells occupy CD4 + The ratio of T cells was significantly increased (FIG. 15A), wherein the recombinant chimpanzee adenovirus AdC68XY3-gE was 6.3 fold higher than the control adenovirus AdC68XY3-empty (p<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-CPG is improved by 6.2 times (p) compared with the control adenovirus AdC68XY3-empty<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 was 4.8 fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-CD40L was 4.2 fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 Recombinant chimpanzee adenovirus AdC68XY3-gE-ProB ratio)The control adenovirus AdC68XY3-empty was 4.3-fold higher (p<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63 increased 3.6 fold (p) over the control adenovirus AdC68XY3-empty<0.01 The recombinant chimpanzee adenovirus AdC68XY3-gE-gH was 3.3 fold higher than the control adenovirus AdC68XY3-empty (p) <0.01 The recombinant chimpanzee adenovirus AdC68XY3-gE-gB was 4.4-fold higher than the control adenovirus AdC68XY3-empty (p)< 0.001)。
Compared with the control adenovirus AdC68XY3-empty, the 6 recombinant chimpanzee adenovirus immune group secretes IL2 + CD4 + T cells occupy CD4 + The proportion of T cells was significantly increased (FIG. 15B), wherein the recombinant chimpanzee adenovirus AdC68XY3-gE was increased 4.3-fold over the control adenovirus AdC68XY3-empty (p<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-CPG is 4.1 times higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 increased 3.4 fold (p) over the control adenovirus AdC68XY3-empty<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-CD40L was 2.9-fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-ProB was 2.7 fold higher than the control adenovirus AdC68XY3-empty (p)<0.01 The recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63 increased 2.5 fold (p) over the control adenovirus AdC68XY3-empty<0.01)。
Compared with the control adenovirus AdC68XY3-empty, 7 recombinant chimpanzee adenovirus immune group secretes TNF alpha + CD4 + T cells occupy CD4 + The proportion of T cells was significantly increased (FIG. 15C), wherein the recombinant chimpanzee adenovirus AdC68XY3-gE was increased 5.5-fold over the control adenovirus AdC68XY3-empty (p <0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-CPG is improved by 5.4 times (p) compared with the control adenovirus AdC68XY3-empty<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 was 4.1-fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-CD40L was 3.6 fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-ProB was 4.0 fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63 increased 2.8-fold (p) over the control adenovirus AdC68XY3-empty<0.01)，The recombinant chimpanzee adenovirus AdC68XY3-gE-gB was 3.1 fold higher than the control adenovirus AdC68XY3-empty (p<0.001)。
By Boolean Gates analysis, the 8 recombinant chimpanzee adenovirus immunoset of the invention secretes IFNgamma compared with the control adenovirus AdC68XY3-empty + ，IL2 + And/or TNF alpha + Cell occupancy of CD4 by at least one cytokine + The proportion of T cells was also significantly increased (FIG. 15D), wherein the recombinant chimpanzee adenovirus AdC68XY3-gE was increased 3.2-fold over the control adenovirus AdC68XY3-empty (p<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-CPG was 3-fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 increased 2.5-fold (p) over the control adenovirus AdC68XY3-empty <0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-CD40L was 2.3 fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-ProB was 2.3 fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63 increased 2-fold (p) over the control adenovirus AdC68XY3-empty<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-gH was 1.7 fold higher than the control adenovirus AdC68XY3-empty (p)<0.05 The recombinant chimpanzee adenovirus AdC68XY3-gE-gB was 2.1 fold higher than the control adenovirus AdC68XY3-empty (p)<0.001)。
To determine whether 8 recombinant chimpanzee adenoviruses of the invention would affect the versatility of T cells, the invention analyzed CD4 using Boolean Combination Gates + The combined expression pattern of three cytokine secretion in T cells shows that CD4 expresses one, two or three cytokines simultaneously + An overall view of T cell ratios (FIG. 15E) illustrating the 8 recombinant chimpanzee adenovirus induced CD4 of the invention + T-cytokines have a balance.
3. Detection result of VZVGE specific CD 8T cell response after mice are immunized with recombinant chimpanzee adenovirus
The results of the antigen VZVgE specific CD 8T cell response flow assay are shown in fig. 14 and fig. 16.
6 recombinant chimpanzees compared to control adenovirus AdC68XY3-emptySecretion of ifnγ by the chimpanzee adenovirus group + CD8 + T cells occupy CD8 + The ratio of T cells was significantly increased (FIG. 16A), wherein the recombinant chimpanzee adenovirus AdC68XY3-gE was 44.6-fold higher than the control adenovirus AdC68XY3-empty (p<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-CPG was 50-fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 was 36.5 fold higher than the control adenovirus AdC68XY3-empty (p)<0.01 The recombinant chimpanzee adenovirus AdC68XY3-gE-ProB was 50.4 fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63 increased 32.1 fold (p) over the control adenovirus AdC68XY3-empty<0.05 The recombinant chimpanzee adenovirus AdC68XY3-gE-gB was 38.1 fold higher than the control adenovirus AdC68XY3-empty (p)< 0.01)。
8 recombinant chimpanzee adenovirus immunosets secreted IL2 compared to control adenovirus AdC68XY3-empty + CD8 + T cells occupy CD8 + The proportion of T cells did not change significantly (fig. 16B).
Compared with control adenovirus AdC68XY3-empty, 5 recombinant chimpanzee adenovirus immune group secretes TNFa + CD8 + T cells occupy CD8 + The proportion of T cells was significantly increased (FIG. 16C), wherein the recombinant chimpanzee adenovirus AdC68XY3-gE was 13.9-fold higher than the control adenovirus AdC68XY3-empty (p <0.01 The recombinant chimpanzee adenovirus AdC68XY3-gE-CPG was 16.7 fold higher than the control adenovirus AdC68XY3-empty (p)<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 increased 12.2-fold (p) over the control adenovirus AdC68XY3-empty<0.05 The recombinant chimpanzee adenovirus AdC68XY3-gE-ProB increased 17.7 fold (p) over the control adenovirus AdC68XY3-empty<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63 increased 12.3 fold (p) over the control adenovirus AdC68XY3-empty<0.05)。
By Boolean Gates analysis, the 8 recombinant chimpanzee adenovirus immune group of the invention secretes and expresses FN gamma compared with control adenovirus AdC68XY3-empty + ，IL2 + And/or TNF alpha + Cell occupancy of CD8 by at least one cytokine + The proportion of T cells was also significantly increased (FIG. 16D), in whichThe recombinant chimpanzee adenovirus AdC68XY3-gE was 11.2 fold higher than the control adenovirus AdC68XY3-empty (p<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-CPG is improved by 12.4 times (p) compared with the control adenovirus AdC68XY3-empty<0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 was 9.4 fold higher than the control adenovirus AdC68XY3-empty (p)<0.01 The recombinant chimpanzee adenovirus AdC68XY3-gE-ProB was 12.5 fold higher than the control adenovirus AdC68XY3-empty (p) <0.001 The recombinant chimpanzee adenovirus AdC68XY3-gE-Orf9-Orf63 increased 8.7 fold (p) over the control adenovirus AdC68XY3-empty<0.05 The recombinant chimpanzee adenovirus AdC68XY3-gE-gB was 9.7 fold higher than the control adenovirus AdC68XY3-empty (p)<0.01)。
To determine whether the 8 recombinant chimpanzee adenoviruses of the invention would affect the versatility of T cells, the invention analyzed CD8 using Boolean Combination Gates + The combined expression pattern of three cytokine secretion in T cells shows that CD8 simultaneously expresses one, two or three cytokines + An overall view of the T cell scale (FIG. 16E), illustrating the 8 recombinant chimpanzee adenovirus induced CD8 in the present invention + T-cytokine responses are mainly ifnγ and tnfα, with little induction of IL2.
In conclusion, 8 recombinant chimpanzee adenoviruses developed by the invention can induce strong CD 8T cell response while inducing CD 4T cell response as varicella zoster candidate vaccine. This is in marked contrast to the fact that two varicella zoster vaccines which are currently marketed are only able to induce a CD 4T cell response.
4. Detection of VZVGE-specific antibody response by mouse serum ELISA
The results of the antigen VZVgE specific IgG antibody reaction ELISA assay are shown in figure 17.
The 8 recombinant chimpanzee adenoviruses successfully induced a stronger VZVgE-specific IgG antibody immune response (both AdC68XY3-gE-gH and AdC68XY3-gE-gB not shown) after 2 weeks of immunization of mice compared to the control adenovirus, adC68XY3-empty, and remained stable after week 4, and at higher levels upon week 13 detection (fig. 17A). Compared with the control adenovirus AdC68XY3-empty, the titers of VZVGE specific IgG antibodies in serum of the 6 recombinant chimpanzee adenovirus mice are significantly improved after 6 weeks of immunization (FIG. 17B), wherein the titers of the VZVGE specific IgG antibodies of the recombinant chimpanzee adenovirus AdC68XY3-gE are improved by 318.6 times (p < 0.001) compared with the control adenovirus AdC68XY3-empty, the titers of the recombinant chimpanzee adenovirus AdC68XY3-gE-CPG are improved by 301.6 times (p < 0.001) compared with the control adenovirus AdC68XY3-empty, the titers of the recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 are improved by 228.9 times (p < 0.001) compared with the control adenovirus AdC68XY3-empty, the titers of the recombinant chimpanzee adenovirus AdC68XY3-gE are improved by 241.7 times (p < 0.001), and the titers of the recombinant chimpanzee adenovirus AdC68XY3-gE-CD40L are improved by p < 3.8 times (p < 8.001), the titers of the recombinant chimpanzee adenovirus AdC68XY 3-gE-3-empty are improved by 301.6 times (p < 0.001) compared with the control adenovirus AdC68XY3-empty, and the recombinant chimpanzee AdC68XY3-empty is improved by 228.9 times (p < 0.001). These results all indicate that the recombinant chimpanzee adenovirus of the invention can strongly trigger the immune response of antigen-specific IgG antibodies as varicella zoster vaccine candidate.
To further analyze the typed IgGs immune response of immunized mice, the present invention also separately examined VZVgE-specific IgG2a, igG2b and IgG1 antibody responses. The results show that: the VZVGE-specific IgG2a antibody response induced after 6 weeks immunization of the 5 recombinant chimpanzee adenovirus compared to the control adenovirus AdC68XY3-empty was significantly improved (FIG. 17C), wherein the recombinant chimpanzee adenovirus AdC68XY3-gE was improved 30.0-fold (p < 0.001) over the control adenovirus AdC68XY3-empty, the recombinant chimpanzee adenovirus AdC68XY3-gE-CPG was improved 38.7-fold (p < 0.001) over the control adenovirus AdC68XY3-empty, the recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 was improved 23.1-fold (p < 0.01) over the control adenovirus AdC68XY3-gE-ProB by 31.1-fold (p < 0.001), and the recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 was improved 23.1-fold (p < 0.001) over the control adenovirus AdC68XY 3-empty.
The VZVGE-specific IgG2b antibody response induced by 6 recombinant chimpanzee adenovirus was significantly increased 6 weeks after immunization with control adenovirus AdC68XY3-empty (FIG. 17D), wherein the recombinant chimpanzee adenovirus AdC68XY3-gE was increased 61.0-fold (p < 0.001) over control adenovirus AdC68XY3-empty, the recombinant chimpanzee adenovirus AdC68XY3-gE-CPG was increased 55.8-fold (p < 0.001) over control adenovirus AdC68XY3-empty, the recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 was increased 58.0-fold (p < 0.001) over control adenovirus AdC68XY3-gE-CD40L, the recombinant chimpanzee adenovirus AdC68XY 3-gpt was increased 59.1-fold (p < 0.001), the recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 was increased 58.0-fold (p < 0.001) over control adenovirus AdC68XY3-empty, the recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 was increased 58.0-fold (p < 0.1).
The VZVGE-specific IgG1 antibody response induced after 6 weeks immunization of the 5 recombinant chimpanzee adenovirus compared to the control adenovirus AdC68XY3-empty was significantly improved (FIG. 17E), wherein the recombinant chimpanzee adenovirus AdC68XY3-gE was 16.5-fold improved (p < 0.001) over the control adenovirus AdC68XY3-empty, the recombinant chimpanzee adenovirus AdC68XY3-gE-CPG was 16.0-fold improved (p < 0.001) over the control adenovirus AdC68XY3-empty, the recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 was 10.3-fold improved (p < 0.05) over the control adenovirus AdC68XY3-gE-CD40L, and the recombinant chimpanzee adenovirus AdC68XY 3-gE-3-empty was 13.5-fold improved (p < 0.001) over the control adenovirus AdC68XY3-empty, and the recombinant chimpanzee adenovirus AdC68XY3-gE-IL21 was 10.3-fold improved (p < 0.01).
In conclusion, 8 recombinant chimpanzee adenoviruses developed by the application can be used as varicella zoster candidate vaccine to strongly excite balanced antigen-specific antibody response.
The present application is described in detail above. It will be apparent to those skilled in the art that the present application can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the application and without undue experimentation. While the application has been described with respect to specific embodiments, it will be appreciated that the application may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
Sequence listing
<110> Suzhou gaming biotechnology Co., ltd
<120> varicella zoster virus vaccine and use thereof
<160> 25
<170> PatentIn version 3.5
<210> 1
<211> 30510
<212> DNA
<213> Artificial Sequence
<400> 1
ccatcttcaa taatatacct caaacttttt gtgcgcgtta atatgcaaat gaggcgtttg 60
aatttgggga ggaagggcgg tgattggtcg agggatgagc gaccgttagg ggcggggcga 120
gtgacgtttt gatgacgtgg ttgcgaggag gagccagttt gcaagttctc gtgggaaaag 180
tgacgtcaaa cgaggtgtgg tttgaacacg gaaatactca attttcccgc gctctctgac 240
aggaaatgag gtgtttctgg gcggatgcaa gtgaaaacgg gccattttcg cgcgaaaact 300
gaatgaggaa gtgaaaatct gagtaatttc gcgtttatgg cagggaggag tatttgccga 360
gggccgagta gactttgacc gattacgtgg gggtttcgat taccgtgttt ttcacctaaa 420
tttccgcgta cggtgtcaaa gtccggtgtt tttacgtacg atatcatttc cccgaaagtg 480
ccacctgacc gtaactataa cggtcctaag gtagcgagag acccaagctg gctagcgttt 540
aaacgggccc tctagactcg agcggccgcc actgtgctgg atgatccgag ctcggtacca 600
agcttaagtt taaaccgctg atcaatctat gtcgggtgcg gagaaagagg taatgaaatg 660
gtattatggg tattatgggt ctgcattaat gaatcggtca gatatcgaca tatgctggcc 720
accgtacatg tggcttccca tgctcgcaag ccctggcccg agttcgagca caatgtcatg 780
accaggtgca atatgcatct ggggtcccgc cgaggcatgt tcatgcccta ccagtgcaac 840
ctgaattatg tgaaggtgct gctggagccc gatgccatgt ccagagtgag cctgacgggg 900
gtgtttgaca tgaatgtgga ggtgtggaag attctgagat atgatgaatc caagaccagg 960
tgccgagcct gcgagtgcgg agggaagcat gccaggttcc agcccgtgtg tgtggatgtg 1020
acggaggacc tgcgacccga tcatttggtg ttgccctgca ccgggacgga gttcggttcc 1080
agcggggaag aatctgacta gagtgagtag tgttctgggg cgggggagga cctgcatgag 1140
ggccagaata actgaaatct gtgcttttct gtgtgttgca gcagcatgag cggaagcggc 1200
tcctttgagg gaggggtatt cagcccttat ctgacggggc gtctcccctc ctgggcggga 1260
gtgcgtcaga atgtgatggg atccacggtg gacggccggc ccgtgcagcc cgcgaactct 1320
tcaaccctga cctatgcaac cctgagctct tcgtcgttgg acgcagctgc cgccgcagct 1380
gctgcatctg ccgccagcgc cgtgcgcgga atggccatgg gcgccggcta ctacggcact 1440
ctggtggcca actcgagttc caccaataat cccgccagcc tgaacgagga gaagctgttg 1500
ctgctgatgg cccagctcga ggccttgacc cagcgcctgg gcgagctgac ccagcaggtg 1560
gctcagctgc aggagcagac gcgggccgcg gttgccacgg tgaaatccaa ataaaaaatg 1620
aatcaataaa taaacggaga cggttgttga ttttaacaca gagtctgaat ctttatttga 1680
tttttcgcgc gcggtaggcc ctggaccacc ggtctcgatc attgagcacc cggtggatct 1740
tttccaggac ccggtagagg tgggcttgga tgttgaggta catgggcatg agcccgtccc 1800
gggggtggag gtagctccat tgcagggcct cgtgctcggg ggtggtgttg taaatcaccc 1860
agtcatagca ggggcgcagg gcatggtgtt gcacaatatc tttgaggagg agactgatgg 1920
ccacgggcag ccctttggtg taggtgttta caaatctgtt gagctgggag ggatgcatgc 1980
ggggggagat gaggtgcatc ttggcctgga tcttgagatt ggcgatgtta ccgcccagat 2040
cccgcctggg gttcatgttg tgcaggacca ccagcacggt gtatccggtg cacttgggga 2100
atttatcatg caacttggaa gggaaggcgt gaaagaattt ggcgacgcct ttgtgcccgc 2160
ccaggttttc catgcactca tccatgatga tggcgatggg cccgtgggcg gcggcctggg 2220
caaagacgtt tcgggggtcg gacacatcat agttgtggtc ctgggtgagg tcatcatagg 2280
ccattttaat gaatttgggg cggagggtgc cggactgggg gacaaaggta ccctcgatcc 2340
cgggggcgta gttcccctca cagatctgca tctcccaggc tttgagctcg gaggggggga 2400
tcatgtccac ctgcggggcg ataaagaaca cggtttccgg ggcgggggag atgagctggg 2460
ccgaaagcaa gttccggagc agctgggact tgccgcagcc ggtggggccg tagatgaccc 2520
cgatgaccgg ctgcaggtgg tagttgaggg agagacagct gccgtcctcc cggaggaggg 2580
gggccacctc gttcatcatc tcgcgcacgt gcatgttctc gcgcaccagt tccgccagga 2640
ggcgctctcc ccccagggat aggagctcct ggagcgaggc gaagtttttc agcggcttga 2700
gtccgtcggc catgggcatt ttggagaggg tttgttgcaa gagttccagg cggtcccaga 2760
gctcggtgat gtgctctacg gcatctcgat ccagcagacc tcctcgtttc gcgggttggg 2820
acggctgcgg gagtagggca ccagacgatg ggcgtccagc gcagccaggg tccggtcctt 2880
ccagggtcgc agcgtccgcg tcagggtggt ctccgtcacg gtgaaggggt gcgcgccggg 2940
ctgggcgctt gcgagggtgc gcttcaggct catccggctg gtcgaaaacc gctcccgatc 3000
ggcgccctgc gcgtcggcca ggtagcaatt gaccatgagt tcgtagttga gcgcctcggc 3060
cgcgtggcct ttggcgcgga gcttaccttt ggaagtctgc ccgcaggcgg gacagaggag 3120
ggacttgagg gcgtagagct tgggggcgag gaagacggac tcgggggcgt aggcgtccgc 3180
gccgcagtgg gcgcagacgg tctcgcactc cacgagccag gtgaggtcgg gctggtcggg 3240
gtcaaaaacc agtttcccgc cgttcttttt gatgcgtttc ttacctttgg tctccatgag 3300
ctcgtgtccc cgctgggtga caaagaggct gtccgtgtcc ccgtagaccg actttatggg 3360
ccggtcctcg agcggtgtgc cgcggtcctc ctcgtagagg aaccccgccc actccgagac 3420
gaaagcccgg gtccaggcca gcacgaagga ggccacgtgg gacgggtagc ggtcgttgtc 3480
caccagcggg tccacctttt ccagggtatg caaacacatg tccccctcgt ccacatccag 3540
gaaggtgatt ggcttgtaag tgtaggccac gtgaccgggg gtcccggccg ggggggtata 3600
aaagggtgcg ggtccctgct cgtcctcact gtcttccgga tcgctgtcca ggagcgccag 3660
ctgttggggt aggtattccc tctcgaaggc gggcatgacc tcggcactca ggttgtcagt 3720
ttctagaaac gaggaggatt tgatattgac ggtgccggcg gagatgcctt tcaagagccc 3780
ctcgtccatc tggtcagaaa agacgatctt tttgttgtcg agcttggtgg cgaaggagcc 3840
gtagagggcg ttggagagga gcttggcgat ggagcgcatg gtctggtttt tttccttgtc 3900
ggcgcgctcc ttggcggcga tgttgagctg cacgtactcg cgcgccacgc acttccattc 3960
ggggaagacg gtggtcagct cgtcgggcac gattctgacc tgccagcccc gattatgcag 4020
ggtgatgagg tccacactgg tggccacctc gccgcgcagg ggctcattag tccagcagag 4080
gcgtccgccc ttgcgcgagc agaagggggg cagggggtcc agcatgacct cgtcgggggg 4140
gtcggcatcg atggtgaaga tgccgggcag gaggtcgggg tcaaagtagc tgatggaagt 4200
ggccagatcg tccagggcag cttgccattc gcgcacggcc agcgcgcgct cgtagggact 4260
gaggggcgtg ccccagggca tgggatgggt aagcgcggag gcgtacatgc cgcagatgtc 4320
gtagacgtag aggggctcct cgaggatgcc gatgtaggtg gggtagcagc gccccccgcg 4380
gatgctggcg cgcacgtagt catacagctc gtgcgagggg gcgaggagcc ccgggcccag 4440
gttggtgcga ctgggctttt cggcgcggta gacgatctgg cggaaaatgg catgcgagtt 4500
ggaggagatg gtgggccttt ggaagatgtt gaagtgggcg tggggcagtc cgaccgagtc 4560
gcggatgaag tgggcgtagg agtcttgcag cttggcgacg agctcggcgg tgactaggac 4620
gtccagagcg cagtagtcga gggtctcctg gatgatgtca tacttgagct gtcccttttg 4680
tttccacagc tcgcggttga gaaggaactc ttcgcggtcc ttccagtact cttcgagggg 4740
gaacccgtcc tgatctgcac ggtaagagcc tagcatgtag aactggttga cggccttgta 4800
ggcgcagcag cccttctcca cggggagggc gtaggcctgg gcggccttgc gcagggaggt 4860
gtgcgtgagg gcgaaagtgt ccctgaccat gaccttgagg aactggtgct tgaagtcgat 4920
atcgtcgcag cccccctgct cccagagctg gaagtccgtg cgcttcttgt aggcggggtt 4980
gggcaaagcg aaagtaacat cgttgaagag gatcttgccc gcgcggggca taaagttgcg 5040
agtgatgcgg aaaggttggg gcacctcggc ccggttgttg atgacctggg cggcgagcac 5100
gatctcgtcg aagccgttga tgttgtggcc cacgatgtag agttccacga atcgcggacg 5160
gcccttgacg tggggcagtt tcttgagctc ctcgtaggtg agctcgtcgg ggtcgctgag 5220
cccgtgctgc tcgagcgccc agtcggcgag atgggggttg gcgcggagga aggaagtcca 5280
gagatccacg gccagggcgg tttgcagacg gtcccggtac tgacggaact gctgcccgac 5340
ggccattttt tcgggggtga cgcagtagaa ggtgcggggg tccccgtgcc agcgatccca 5400
tttgagctgg agggcgagat cgagggcgag ctcgacgagc cggtcgtccc cggagagttt 5460
catgaccagc atgaagggga cgagctgctt gccgaaggac cccatccagg tgtaggtttc 5520
cacatcgtag gtgaggaaga gcctttcggt gcgaggatgc gagccgatgg ggaagaactg 5580
gatctcctgc caccaattgg aggaatggct gttgatgtga tggaagtaga aatgccgacg 5640
gcgcgccgaa cactcgtgct tgtgtttata caagcggcca cagtgctcgc aacgctgcac 5700
gggatgcacg tgctgcacga gctgtacctg agttcctttg acgaggaatt tcagtgggaa 5760
gtggagtcgt ggcgcctgca tctcgtgctg tactacgtcg tggtggtcgg cctggccctc 5820
ttctgcctcg atggtggtca tgctgacgag cccgcgcggg aggcaggtcc agacctcggc 5880
gcgagcgggt cggagagcga ggacgagggc gcgcaggccg gagctgtcca gggtcctgag 5940
acgctgcgga gtcaggtcag tgggcagcgg cggcgcgcgg ttgacttgca ggagtttttc 6000
cagggcgcgc gggaggtcca gatggtactt gatctccacc gcgccattgg tggcgacgtc 6060
gatggcttgc agggtcccgt gcccctgggg tgtgaccacc gtcccccgtt tcttcttggg 6120
cggctggggc gacgggggcg gtgcctcttc catggttaga agcggcggcg aggacgcgcg 6180
ccgggcggca ggggcggctc ggggcccgga ggcaggggcg gcaggggcac gtcggcgccg 6240
cgcgcgggta ggttctggta ctgcgcccgg agaagactgg cgtgagcgac gacgcgacgg 6300
ttgacgtcct ggatctgacg cctctgggtg aaggccacgg gacccgtgag tttgaacctg 6360
aaagagagtt cgacagaatc aatctcggta tcgttgacgg cggcctgccg caggatctct 6420
tgcacgtcgc ccgagttgtc ctggtaggcg atctcggtca tgaactgctc gatctcctcc 6480
tcttgaaggt ctccgcggcc ggcgcgctcc acggtggccg cgaggtcgtt ggagatgcgg 6540
cccatgagct gcgagaaggc gttcatgccc gcctcgttcc agacgcggct gtagaccacg 6600
acgccctcgg gatcgcgggc gcgcatgacc acctgggcga ggttgagctc cacgtggcgc 6660
gtgaagaccg cgtagttgca gaggcgctgg tagaggtagt tgagcgtggt ggcgatgtgc 6720
tcggtgacga agaaatacat gatccagcgg cggagcggca tctcgctgac gtcgcccagc 6780
gcctccaaac gttccatggc ctcgtaaaag tccacggcga agttgaaaaa ctgggagttg 6840
cgcgccgaga cggtcaactc ctcctccaga agacggatga gctcggcgat ggtggcgcgc 6900
acctcgcgct cgaaggcccc cgggagttcc tccacttcct cttcttcctc ctccactaac 6960
atctcttcta cttcctcctc aggcggcagt ggtggcgggg gagggggcct gcgtcgccgg 7020
cggcgcacgg gcagacggtc gatgaagcgc tcgatggtct cgccgcgccg gcgtcgcatg 7080
gtctcggtga cggcgcgccc gtcctcgcgg ggccgcagcg tgaagacgcc gccgcgcatc 7140
tccaggtggc cgggggggtc cccgttgggc agggagaggg cgctgacgat gcatcttatc 7200
aattgccccg tagggactcc gcgcaaggac ctgagcgtct cgagatccac gggatctgaa 7260
aaccgctgaa cgaaggcttc gagccagtcg cagtcgcaag gtaggctgag cacggtttct 7320
tctggcgggt catgttggtt gggagcgggg cgggcgatgc tgctggtgat gaagttgaaa 7380
taggcggttc tgagacggcg gatggtggcg aggagcacca ggtctttggg cccggcttgc 7440
tggatgcgca gacggtcggc catgccccag gcgtggtcct gacacctggc caggtccttg 7500
tagtagtcct gcatgagccg ctccacgggc acctcctcct cgcccgcgcg gccgtgcatg 7560
cgcgtgagcc cgaagccgcg ctggggctgg acgagcgcca ggtcggcgac gacgcgctcg 7620
gcgaggatgg cttgctggat ctgggtgagg gtggtctgga agtcatcaaa gtcgacgaag 7680
cggtggtagg ctccggtgtt gatggtgtag gagcagttgg ccatgacgga ccagttgacg 7740
gtctggtggc ccggacgcac gagctcgtgg tacttgaggc gcgagtaggc gcgcgtgtcg 7800
aagatgtagt cgttgcaggt gcgcaccagg tactggtagc cgatgaggaa gtgcggcggc 7860
ggctggcggt agagcggcca tcgctcggtg gcgggggcgc cgggcgcgag gtcctcgagc 7920
atggtgcggt ggtagccgta gatgtacctg gacatccagg tgatgccggc ggcggtggtg 7980
gaggcgcgcg ggaactcgcg gacgcggttc cagatgttgc gcagcggcag gaagtagttc 8040
atggtgggca cggtctggcc cgtgaggcgc gcgcagtcgt ggatgctcta tacgggcaaa 8100
aacgaaagcg gtcagcggct cgactccgtg gcctggaggc taagcgaacg ggttgggctg 8160
cgcgtgtacc ccggttcgaa tctcgaatca ggctggagcc gcagctaacg tggtattggc 8220
actcccgtct cgacccaagc ctgcaccaac cctccaggat acggaggcgg gtcgttttgc 8280
aacttttttt tggaggccgg atgagactag taagcgcgga aagcggccga ccgcgatggc 8340
tcgctgccgt agtctggaga agaatcgcca gggttgcgtt gcggtgtgcc ccggttcgag 8400
gccggccgga ttccgcggct aacgagggcg tggctgcccc gtcgtttcca agaccccata 8460
gccagccgac ttctccagtt acggagcgag cccctctttt gttttgtttg tttttgccag 8520
atgcatcccg tactgcggca gatgcgcccc caccaccctc caccgcaaca acagccccct 8580
ccacagccgg cgcttctgcc cccgccccag cagcaacttc cagccacgac cgccgcggcc 8640
gccgtgagcg gggctggaca gagttatgat caccagctgg ccttggaaga gggcgagggg 8700
ctggcgcgcc tgggggcgtc gtcgccggag cggcacccgc gcgtgcagat gaaaagggac 8760
gctcgcgagg cctacgtgcc caagcagaac ctgttcagag acaggagcgg cgaggagccc 8820
gaggagatgc gcgcggcccg gttccacgcg gggcgggagc tgcggcgcgg cctggaccga 8880
aagagggtgc tgagggacga ggatttcgag gcggacgagc tgacggggat cagccccgcg 8940
cgcgcgcacg tggccgcggc caacctggtc acggcgtacg agcagaccgt gaaggaggag 9000
agcaacttcc aaaaatcctt caacaaccac gtgcgcaccc tgatcgcgcg cgaggaggtg 9060
accctgggcc tgatgcacct gtgggacctg ctggaggcca tcgtgcagaa ccccaccagc 9120
aagccgctga cggcgcagct gttcctggtg gtgcagcata gtcgggacaa cgaagcgttc 9180
agggaggcgc tgctgaatat caccgagccc gagggccgct ggctcctgga cctggtgaac 9240
attctgcaga gcatcgtggt gcaggagcgc gggctgccgc tgtccgagaa gctggcggcc 9300
atcaacttct cggtgctgag tttgggcaag tactacgcta ggaagatcta caagaccccg 9360
tacgtgccca tagacaagga ggtgaagatc gacgggtttt acatgcgcat gaccctgaaa 9420
gtgctgaccc tgagcgacga tctgggggtg taccgcaacg acaggatgca ccgtgcggtg 9480
agcgccagca ggcggcgcga gctgagcgac caggagctga tgcatagtct gcagcgggcc 9540
ctgaccgggg ccgggaccga gggggagagc tactttgaca tgggcgcgga cctgcactgg 9600
cagcccagcc gccgggcctt ggaggcggcg gcaggaccct acgtagaaga ggtggacgat 9660
gaggtggacg aggagggcga gtacctggaa gactgatggc gcgaccgtat ttttgctaga 9720
tgcaacaaca acagccacct cctgatcccg cgatgcgggc ggcgctgcag agccagccgt 9780
ccggcattaa ctcctcggac gattggaccc aggccatgca acgcatcatg gcgctgacga 9840
cccgcaaccc cgaagccttt agacagcagc cccaggccaa ccggctctcg gccatcctgg 9900
aggccgtggt gccctcgcgc tccaacccca cgcacgagaa ggtcctggcc atcgtgaacg 9960
cgctggtgga gaacaaggcc atccgcggcg acgaggccgg cctggtgtac aacgcgctgc 10020
tggagcgcgt ggcccgctac aacagcacca acgtgcagac caacctggac cgcatggtga 10080
ccgacgtgcg cgaggccgtg gcccagcgcg agcggttcca ccgcgagtcc aacctgggat 10140
ccatggtggc gctgaacgcc ttcctcagca cccagcccgc caacgtgccc cggggccagg 10200
aggactacac caacttcatc agcgccctgc gcctgatggt gaccgaggtg ccccagagcg 10260
aggtgtacca gtccgggccg gactacttct tccagaccag tcgccagggc ttgcagaccg 10320
tgaacctgag ccaggctttc aagaacttgc agggcctgtg gggcgtgcag gccccggtcg 10380
gggaccgcgc gacggtgtcg agcctgctga cgccgaactc gcgcctgctg ctgctgctgg 10440
tggccccctt cacggacagc ggcagcatca accgcaactc gtacctgggc tacctgatta 10500
acctgtaccg cgaggccatc ggccaggcgc acgtggacga gcagacctac caggagatca 10560
cccacgtgag ccgcgccctg ggccaggacg acccgggcaa cctggaagcc accctgaact 10620
ttttgctgac caaccggtcg cagaagatcc cgccccagta cgcgctcagc accgaggagg 10680
agcgcatcct gcgttacgtg cagcagagcg tgggcctgtt cctgatgcag gagggggcca 10740
cccccagcgc cgcgctcgac atgaccgcgc gcaacatgga gcccagcatg tacgccagca 10800
accgcccgtt catcaataaa ctgatggact acttgcatcg ggcggccgcc atgaactctg 10860
actatttcac caacgccatc ctgaatcccc actggctccc gccgccgggg ttctacacgg 10920
gcgagtacga catgcccgac cccaatgacg ggttcctgtg ggacgatgtg gacagcagcg 10980
tgttctcccc ccgaccgggt gctaacgagc gccccttgtg gaagaaggaa ggcagcgacc 11040
gacgcccgtc ctcggcgctg tccggccgcg agggtgctgc cgcggcggtg cccgaggccg 11100
ccagtccttt cccgagcttg cccttctcgc tgaacagtat ccgcagcagc gagctgggca 11160
ggatcacgcg cccgcgcttg ctgggcgaag aggagtactt gaatgactcg ctgttgagac 11220
ccgagcggga gaagaacttc cccaataacg ggatagaaag cctggtggac aagatgagcc 11280
gctggaagac gtatgcgcag gagcacaggg acgatccccg ggcgtcgcag ggggccacga 11340
gccggggcag cgccgcccgt aaacgccggt ggcacgacag gcagcgggga cagatgtggg 11400
acgatgagga ctccgccgac gacagcagcg tgttggactt gggtgggagt ggtaacccgt 11460
tcgctcacct gcgcccccgt atcgggcgca tgatgtaaga gaaaccgaaa ataaatgata 11520
ctcaccaagg ccatggcgac cagcgtgcgt tcgtttcttc tctgttgttg ttgtatctag 11580
tatgatgagg cgtgcgtacc cggagggtcc tcctccctcg tacgagagcg tgatgcagca 11640
ggcgatggcg gcggcggcga tgcagccccc gctggaggct ccttacgtgc ccccgcggta 11700
cctggcgcct acggaggggc ggaacagcat tcgttactcg gagctggcac ccttgtacga 11760
taccacccgg ttgtacctgg tggacaacaa gtcggcggac atcgcctcgc tgaactacca 11820
gaacgaccac agcaacttcc tgaccaccgt ggtgcagaac aatgacttca cccccacgga 11880
ggccagcacc cagaccatca actttgacga gcgctcgcgg tggggcggcc agctgaaaac 11940
catcatgcac accaacatgc ccaacgtgaa cgagttcatg tacagcaaca agttcaaggc 12000
gcgggtgatg gtctcccgca agacccccaa tggggtgaca gtgacagagg attatgatgg 12060
tagtcaggat gagctgaagt atgaatgggt ggaatttgag ctgcccgaag gcaacttctc 12120
ggtgaccatg accatcgacc tgatgaacaa cgccatcatc gacaattact tggcggtggg 12180
gcggcagaac ggggtgctgg agagcgacat cggcgtgaag ttcgacacta ggaacttcag 12240
gctgggctgg gaccccgtga ccgagctggt catgcccggg gtgtacacca acgaggcttt 12300
ccatcccgat attgtcttgc tgcccggctg cggggtggac ttcaccgaga gccgcctcag 12360
caacctgctg ggcattcgca agaggcagcc cttccaggaa ggcttccaga tcatgtacga 12420
ggatctggag gggggcaaca tccccgcgct cctggatgtc gacgcctatg agaaaagcaa 12480
ggaggatgca gcagctgaag caactgcagc cgtagctacc gcctctaccg aggtcagggg 12540
cgataatttt gcaagcgccg cagcagtggc agcggccgag gcggctgaaa ccgaaagtaa 12600
gatagtcatt cagccggtgg agaaggatag caagaacagg agctacaacg tactaccgga 12660
caagataaac accgcctacc gcagctggta cctagcctac aactatggcg accccgagaa 12720
gggcgtgcgc tcctggacgc tgctcaccac ctcggacgtc acctgcggcg tggagcaagt 12780
ctactggtcg ctgcccgaca tgatgcaaga cccggtcacc ttccgctcca cgcgtcaagt 12840
tagcaactac ccggtggtgg gcgccgagct cctgcccgtc tactccaaga gcttcttcaa 12900
cgagcaggcc gtctactcgc agcagctgcg cgccttcacc tcgcttacgc acgtcttcaa 12960
ccgcttcccc gagaaccaga tcctcgtccg cccgcccgcg cccaccatta ccaccgtcag 13020
tgaaaacgtt cctgctctca cagatcacgg gaccctgccg ctgcgcagca gtatccgggg 13080
agtccagcgc gtgaccgtta ctgacgccag acgccgcacc tgcccctacg tctacaaggc 13140
cctgggcata gtcgcgccgc gcgtcctctc gagccgcacc ttctaaatgt ccattctcat 13200
ctcgcccagt aataacaccg gttggggcct gcgcgcgccc agcaagatgt acggaggcgc 13260
tcgccaacgc tccacgcaac accccgtgcg cgtgcgcggg cacttccgcg ctccctgggg 13320
cgccctcaag ggccgcgtgc ggtcgcgcac caccgtcgac gacgtgatcg accaggtggt 13380
ggccgacgcg cgcaactaca cccccgccgc cgcgcccgtc tccaccgtgg acgccgtcat 13440
cgacagcgtg gtggccgacg cgcgccggta cgcccgcgcc aagagccggc ggcggcgcat 13500
cgcccggcgg caccggagca cccccgccat gcgcgcggcg cgagccttgc tgcgcagggc 13560
caggcgcacg ggacgcaggg ccatgctcag ggcggccaga cgcgcggctt caggcgccag 13620
cgccggcagg acccggagac gcgcggccac ggcggcggca gcggccatcg ccagcatgtc 13680
ccgcccgcgg cgagggaacg tgtactgggt gcgcgacgcc gccaccggtg tgcgcgtgcc 13740
cgtgcgcacc cgcccccctc gcacttgaag atgttcactt cgcgatgttg atgtgtccca 13800
gcggcgagga ggatgtccaa gcgcaaattc aaggaagaga tgctccaggt catcgcgcct 13860
gagatctacg gccctgcggt ggtgaaggag gaaagaaagc cccgcaaaat caagcgggtc 13920
aaaaaggaca aaaaggaaga agaaagtgat gtggacggat tggtggagtt tgtgcgcgag 13980
ttcgcccccc ggcggcgcgt gcagtggcgc gggcggaagg tgcaaccggt gctgagaccc 14040
ggcaccaccg tggtcttcac gcccggcgag cgctccggca ccgcttccaa gcgctcctac 14100
gacgaggtgt acggggatga tgatattctg gagcaggcgg ccgagcgcct gggcgagttt 14160
gcttacggca agcgcagccg ttccgcaccg aaggaagagg cggtgtccat cccgctggac 14220
cacggcaacc ccacgccgag cctcaagccc gtgaccttgc agcaggtgct gccgaccgcg 14280
gcgccgcgcc gggggttcaa gcgcgagggc gaggatctgt accccaccat gcagctgatg 14340
gtgcccaagc gccagaagct ggaagacgtg ctggagacca tgaaggtgga cccggacgtg 14400
cagcccgagg tcaaggtgcg gcccatcaag caggtggccc cgggcctggg cgtgcagacc 14460
gtggacatca agattcccac ggagcccatg gaaacgcaga ccgagcccat gatcaagccc 14520
agcaccagca ccatggaggt gcagacggat ccctggatgc catcggctcc tagtcgaaga 14580
ccccggcgca agtacggcgc ggccagcctg ctgatgccca actacgcgct gcatccttcc 14640
atcatcccca cgccgggcta ccgcggcacg cgcttctacc gcggtcatac cagcagccgc 14700
cgccgcaaga ccaccactcg ccgccgccgt cgccgcaccg ccgctgcaac cacccctgcc 14760
gccctggtgc ggagagtgta ccgccgcggc cgcgcacctc tgaccctgcc gcgcgcgcgc 14820
taccacccga gcatcgccat ttaaactttc gcctgctttg cagatcaatg gccctcacat 14880
gccgccttcg cgttcccatt acgggctacc gaggaagaaa accgcgccgt agaaggctgg 14940
cggggaacgg gatgcgtcgc caccaccacc ggcggcggcg cgccatcagc aagcggttgg 15000
ggggaggctt cctgcccgcg ctgatcccca tcatcgccgc ggcgatcggg gcgatccccg 15060
gcattgcttc cgtggcggtg caggcctctc agcgccactg agacacactt ggaaacatct 15120
tgtaataaac caatggactc tgacgctcct ggtcctgtga tgtgttttcg tagacagatg 15180
gaagacatca atttttcgtc cctggctccg cgacacggca cgcggccgtt catgggcacc 15240
tggagcgaca tcggcaccag ccaactgaac gggggcgcct tcaattggag cagtctctgg 15300
agcgggctta agaatttcgg gtccacgctt aaaacctatg gcagcaaggc gtggaacagc 15360
accacagggc aggcgctgag ggataagctg aaagagcaga acttccagca gaaggtggtc 15420
gatgggctcg cctcgggcat caacggggtg gtggacctgg ccaaccaggc cgtgcagcgg 15480
cagatcaaca gccgcctgga cccggtgccg cccgccggct ccgtggagat gccgcaggtg 15540
gaggaggagc tgcctcccct ggacaagcgg ggcgagaagc gaccccgccc cgatgcggag 15600
gagacgctgc tgacgcacac ggacgagccg cccccgtacg aggaggcggt gaaactgggt 15660
ctgcccacca cgcggcccat cgcgcccctg gccaccgggg tgctgaaacc cgaaaagccc 15720
gcgaccctgg acttgcctcc tccccagcct tcccgcccct ctacagtggc taagcccctg 15780
ccgccggtgg ccgtggcccg cgcgcgaccc gggggcaccg cccgccctca tgcgaactgg 15840
cagagcactc tgaacagcat cgtgggtctg ggagtgcaga gtgtgaagcg ccgccgctgc 15900
tattaaacct accgtagcgc ttaacttgct tgtctgtgtg tgtatgtatt atgtcgccgc 15960
cgccgctgtc caccagaagg aggagtgaag aggcgcgtcg ccgagttgca agatggccac 16020
cccatcgatg ctgccccagt gggcgtacat gcacatcgcc ggacaggacg cttcggagta 16080
cctgagtccg ggtctggtgc agtttgcccg cgccacagac acctacttca gtctggggaa 16140
caagtttagg aaccccacgg tggcgcccac gcacgatgtg accaccgacc gcagccagcg 16200
gctgacgctg cgcttcgtgc ccgtggaccg cgaggacaac acctactcgt acaaagtgcg 16260
ctacacgctg gccgtgggcg acaaccgcgt gctggacatg gccagcacct actttgacat 16320
ccgcggcgtg ctggatcggg gccctagctt caaaccctac tccggcaccg cctacaacag 16380
tctggccccc aagggagcac ccaacacttg tcagtggaca tataaagccg atggtgaaac 16440
tgccacagaa aaaacctata catatggaaa tgcacccgtg cagggcatta acatcacaaa 16500
agatggtatt caacttggaa ctgacaccga tgatcagcca atctacgcag ataaaaccta 16560
tcagcctgaa cctcaagtgg gtgatgctga atggcatgac atcactggta ctgatgaaaa 16620
gtatggaggc agagctctta agcctgatac caaaatgaag ccttgttatg gttcttttgc 16680
caagcctact aataaagaag gaggtcaggc aaatgtgaaa acaggaacag gcactactaa 16740
agaatatgac atagacatgg ctttctttga caacagaagt gcggctgctg ctggcctagc 16800
tccagaaatt gttttgtata ctgaaaatgt ggatttggaa actccagata cccatattgt 16860
atacaaagca ggcacagatg acagcagctc ttctattaat ttgggtcagc aagccatgcc 16920
caacagacct aactacattg gtttcagaga caactttatc gggctcatgt actacaacag 16980
cactggcaat atgggggtgc tggccggtca ggcttctcag ctgaatgctg tggttgactt 17040
gcaagacaga aacaccgagc tgtcctacca gctcttgctt gactctctgg gtgacagaac 17100
ccggtatttc agtatgtgga atcaggcggt ggacagctat gatcctgatg tgcgcattat 17160
tgaaaatcat ggtgtggagg atgaacttcc caactattgt ttccctctgg atgctgttgg 17220
cagaacagat acttatcagg gaattaaggc taatggaact gatcaaacca catggaccaa 17280
agatgacagt gtcaatgatg ctaatgagat aggcaagggt aatccattcg ccatggaaat 17340
caacatccaa gccaacctgt ggaggaactt cctctacgcc aacgtggccc tgtacctgcc 17400
cgactcttac aagtacacgc cggccaatgt taccctgccc accaacacca acacctacga 17460
ttacatgaac ggccgggtgg tggcgccctc gctggtggac tcctacatca acatcggggc 17520
gcgctggtcg ctggatccca tggacaacgt gaaccccttc aaccaccacc gcaatgcggg 17580
gctgcgctac cgctccatgc tcctgggcaa cgggcgctac gtgcccttcc acatccaggt 17640
gccccagaaa tttttcgcca tcaagagcct cctgctcctg cccgggtcct acacctacga 17700
gtggaacttc cgcaaggacg tcaacatgat cctgcagagc tccctcggca acgacctgcg 17760
cacggacggg gcctccatct ccttcaccag catcaacctc tacgccacct tcttccccat 17820
ggcgcacaac acggcctcca cgctcgaggc catgctgcgc aacgacacca acgaccagtc 17880
cttcaacgac tacctctcgg cggccaacat gctctacccc atcccggcca acgccaccaa 17940
cgtgcccatc tccatcccct cgcgcaactg ggccgccttc cgcggctggt ccttcacgcg 18000
tctcaagacc aaggagacgc cctcgctggg ctccgggttc gacccctact tcgtctactc 18060
gggctccatc ccctacctcg acggcacctt ctacctcaac cacaccttca agaaggtctc 18120
catcaccttc gactcctccg tcagctggcc cggcaacgac cggctcctga cgcccaacga 18180
gttcgaaatc aagcgcaccg tcgacggcga gggctacaac gtggcccagt gcaacatgac 18240
caaggactgg ttcctggtcc agatgctggc ccactacaac atcggctacc agggcttcta 18300
cgtgcccgag ggctacaagg accgcatgta ctccttcttc cgcaacttcc agcccatgag 18360
ccgccaggtg gtggacgagg tcaactacaa ggactaccag gccgtcaccc tggcctacca 18420
gcacaacaac tcgggcttcg tcggctacct cgcgcccacc atgcgccagg gccagcccta 18480
ccccgccaac tacccctacc cgctcatcgg caagagcgcc gtcaccagcg tcacccagaa 18540
aaagttcctc tgcgacaggg tcatgtggcg catccccttc tccagcaact tcatgtccat 18600
gggcgcgctc accgacctcg gccagaacat gctctatgcc aactccgccc acgcgctaga 18660
catgaatttc gaagtcgacc ccatggatga gtccaccctt ctctatgttg tcttcgaagt 18720
cttcgacgtc gtccgagtgc accagcccca ccgcggcgtc atcgaggccg tctacctgcg 18780
cacccccttc tcggccggta acgccaccac ctaagctctt gcttcttgca agccatggcc 18840
gcgggctccg gcgagcagga gctcagggcc atcatccgcg acctgggctg cgggccctac 18900
ttcctgggca ccttcgataa gcgcttcccg ggattcatgg ccccgcacaa gctggcctgc 18960
gccatcgtca acacggccgg ccgcgagacc gggggcgagc actggctggc cttcgcctgg 19020
aacccgcgct cgaacacctg ctacctcttc gaccccttcg ggttctcgga cgagcgcctc 19080
aagcagatct accagttcga gtacgagggc ctgctgcgcc gcagcgccct ggccaccgag 19140
gaccgctgcg tcaccctgga aaagtccacc cagaccgtgc agggtccgcg ctcggccgcc 19200
tgcgggctct tctgctgcat gttcctgcac gccttcgtgc actggcccga ccgccccatg 19260
gacaagaacc ccaccatgaa cttgctgacg ggggtgccca acggcatgct ccagtcgccc 19320
caggtggaac ccaccctgcg ccgcaaccag gaggcgctct accgcttcct caactcccac 19380
tccgcctact ttcgctccca ccgcgcgcgc atcgagaagg ccaccgcctt cgaccgcatg 19440
aatcaagaca tgtaaaccgt gtgtgtatgt taaatgtctt taataaacag cactttcatg 19500
ttacacatgc atctgagatg atttatttag aaatcgaaag ggttctgccg ggtctcggca 19560
tggcccgcgg gcagggacac gttgcggaac tggtacttgg ccagccactt gaactcgggg 19620
atcagcagtt tgggcagcgg ggtgtcgggg aaggagtcgg tccacagctt ccgcgtcagt 19680
tgcagggcgc ccagcaggtc gggcgcggag atcttgaaat cgcagttggg acccgcgttc 19740
tgcgcgcggg agttgcggta cacggggttg cagcactgga acaccatcag ggccgggtgc 19800
ttcacgctcg ccagcaccgt cgcgtcggtg atgctctcca cgtcgaggtc ctcggcgttg 19860
gccatcccga agggggtcat cttgcaggtc tgccttccca tggtgggcac gcacccgggc 19920
ttgtggttgc aatcgcagtg cagggggatc agcatcatct gggcctggtc ggcgttcatc 19980
cccgggtaca tggccttcat gaaagcctcc aattgcctga acgcctgctg ggccttggct 20040
ccctcggtga agaagacccc gcaggacttg ctagagaact ggttggtggc gcacccggcg 20100
tcgtgcacgc agcagcgcgc gtcgttgttg gccagctgca ccacgctgcg cccccagcgg 20160
ttctgggtga tcttggcccg gtcggggttc tccttcagcg cgcgctgccc gttctcgctc 20220
gccacatcca tctcgatcat gtgctccttc tggatcatgg tggtcccgtg caggcaccgc 20280
agcttgccct cggcctcggt gcacccgtgc agccacagcg cgcacccggt gcactcccag 20340
ttcttgtggg cgatctggga atgcgcgtgc acgaagccct gcaggaagcg gcccatcatg 20400
gtggtcaggg tcttgttgct agtgaaggtc agcggaatgc cgcggtgctc ctcgttgatg 20460
tacaggtggc agatgcggcg gtacacctcg ccctgctcgg gcatcagctg gaagttggct 20520
ttcaggtcgg tctccacgcg gtagcggtcc atcagcatag tcatgatttc catacccttc 20580
tcccaggccg agacgatggg caggctcata gggttcttca ccatcatctt agcgctagca 20640
gccgcggcca gggggtcgct ctcgtccagg gtctcaaagc tccgcttgcc gtccttctcg 20700
gtgatccgca ccggggggta gctgaagccc acggccgcca gctcctcctc ggcctgtctt 20760
tcgtcctcgc tgtcctggct gacgtcctgc aggaccacat gcttggtctt gcggggtttc 20820
ttcttgggcg gcagcggcgg cggagatgtt ggagatggcg agggggagcg cgagttctcg 20880
ctcaccacta ctatctcttc ctcttcttgg tccgaggcca cgcggcggta ggtatgtctc 20940
ttcgggggca gaggcggagg cgacgggctc tcgccgccgc gacttggcgg atggctggca 21000
gagccccttc cgcgttcggg ggtgcgctcc cggcggcgct ctgactgact tcctccgcgg 21060
ccggccattg tgttctccta gggaggaaca acaagcatgg agactcagcc atcgccaacc 21120
tcgccatctg cccccaccgc cgacgagaag cagcagcagc agaatgaaag cttaaccgcc 21180
ccgccgccca gccccgccac ctccgacgcg gccgtcccag acatgcaaga gatggaggaa 21240
tccatcgaga ttgacctggg ctatgtgacg cccgcggagc acgaggagga gctggcagtg 21300
cgcttttcac aagaagagat acaccaagaa cagccagagc aggaagcaga gaatgagcag 21360
agtcaggctg ggctcgagca tgacggcgac tacctccacc tgagcggggg ggaggacgcg 21420
ctcatcaagc atctggcccg gcaggccacc atcgtcaagg atgcgctgct cgaccgcacc 21480
gaggtgcccc tcagcgtgga ggagctcagc cgcgcctacg agttgaacct cttctcgccg 21540
cgcgtgcccc ccaagcgcca gcccaatggc acctgcgagc ccaacccgcg cctcaacttc 21600
tacccggtct tcgcggtgcc cgaggccctg gccacctacc acatcttttt caagaaccaa 21660
aagatccccg tctcctgccg cgccaaccgc acccgcgccg acgccctttt caacctgggt 21720
cccggcgccc gcctacctga tatcgcctcc ttggaagagg ttcccaagat cttcgagggt 21780
ctgggcagcg acgagactcg ggccgcgaac gctctgcaag gagaaggagg agagcatgag 21840
caccacagcg ccctggtcga gttggaaggc gacaacgcgc ggctggcggt gctcaaacgc 21900
acggtcgagc tgacccattt cgcctacccg gctctgaacc tgccccccaa agtcatgagc 21960
gcggtcatgg accaggtgct catcaagcgc gcgtcgccca tctccgagga cgagggcatg 22020
caagactccg aggagggcaa gcccgtggtc agcgacgagc agctggcccg gtggctgggt 22080
cctaatgcta gtccccagag tttggaagag cggcgcaaac tcatgatggc cgtggtcctg 22140
gtgaccgtgg agctggagtg cctgcgccgc ttcttcgccg acgcggagac cctgcgcaag 22200
gtcgaggaga acctgcacta cctcttcagg cacgggttcg tgcgccaggc ctgcaagatc 22260
tccaacgtgg agctgaccaa cctggtctcc tacatgggca tcttgcacga gaaccgcctg 22320
gggcagaacg tgctgcacac caccctgcgc ggggaggccc ggcgcgacta catccgcgac 22380
tgcgtctacc tctacctctg ccacacctgg cagacgggca tgggcgtgtg gcagcagtgt 22440
ctggaggagc agaacctgaa agagctctgc aagctcctgc agaagaacct caagggtctg 22500
tggaccgggt tcgacgagcg caccaccgcc tcggacctgg ccgacctcat tttccccgag 22560
cgcctcaggc tgacgctgcg caacggcctg cccgacttta tgagccaaag catgttgcaa 22620
aactttcgct ctttcatcct cgaacgctcc ggaatcctgc ccgccacctg ctccgcgctg 22680
ccctcggact tcgtgccgct gaccttccgc gagtgccccc cgccgctgtg gagccactgc 22740
tacctgctgc gcctggccaa ctacctggcc taccactcgg acgtgatcga ggacgtcagc 22800
ggcgagggcc tgctcgagtg ccactgccgc tgcaacctct gcacgccgca ccgctccctg 22860
gcctgcaacc cccagctgct gagcgagacc cagatcatcg gcaccttcga gttgcaaggg 22920
cccagcgaag gcgagggttc agccgccaag gggggtctga aactcacccc ggggctgtgg 22980
acctcggcct acttgcgcaa gttcgtgccc gaggactacc atcccttcga gatcaggttc 23040
tacgaggacc aatcccatcc gcccaaggcc gagctgtcgg cctgcgtcat cacccagggg 23100
gcgatcctgg cccaattgca agccatccag aaatcccgcc aagaattctt gctgaaaaag 23160
ggccgcgggg tctacctcga cccccagacc ggtgaggagc tcaaccccgg cttcccccag 23220
gatgccccga ggaaacaaga agctgaaagt ggagctgccg cccgtggagg atttggagga 23280
agactgggag aacagcagtc aggcagagga ggaggagatg gaggaagact gggacagcac 23340
tcaggcagag gaggacagcc tgcaagacag tctggaggaa gacgaggagg aggcagagga 23400
ggaggtggaa gaagcagccg ccgccagacc gtcgtcctcg gcgggggaga aagcaagcag 23460
cacggatacc atctccgctc cgggtcgggg tcccgctcga ccacacagta gatgggacga 23520
gaccggacga ttcccgaacc ccaccaccca gaccggtaag aaggagcggc agggatacaa 23580
gtcctggcgg gggcacaaaa acgccatcgt ctcctgcttg caggcctgcg ggggcaacat 23640
ctccttcacc cggcgctacc tgctcttcca ccgcggggtg aactttcccc gcaacatctt 23700
gcattactac cgtcacctcc acagccccta ctacttccaa gaagaggcag cagcagcaga 23760
aaaagaccag cagaaaacca gcagctagaa aatccacagc ggcggcagca ggtggactga 23820
ggatcgcggc gaacgagccg gcgcaaaccc gggagctgag gaaccggatc tttcccaccc 23880
tctatgccat cttccagcag agtcgggggc aggagcagga actgaaagtc aagaaccgtt 23940
ctctgcgctc gctcacccgc agttgtctgt atcacaagag cgaagaccaa cttcagcgca 24000
ctctcgagga cgccgaggct ctcttcaaca agtactgcgc gctcactctt aaagagtagc 24060
ccgcgcccgc ccagtcgcag aaaaaggcgg gaattacgtc acctgtgccc ttcgccctag 24120
ccgcctccac ccatcatcat gagcaaagag attcccacgc cttacatgtg gagctaccag 24180
ccccagatgg gcctggccgc cggtgccgcc caggactact ccacccgcat gaattggctc 24240
agcgccgggc ccgcgatgat ctcacgggtg aatgacatcc gcgcccaccg aaaccagata 24300
ctcctagaac agtcagcgct caccgccacg ccccgcaatc acctcaatcc gcgtaattgg 24360
cccgccgccc tggtgtacca ggaaattccc cagcccacga ccgtactact tccgcgagac 24420
gcccaggccg aagtccagct gactaactca ggtgtccagc tggcgggcgg cgccaccctg 24480
tgtcgtcacc gccccgctca gggtataaag cggctggtga tccggggcag aggcacacag 24540
ctcaacgacg aggtggtgag ctcttcgctg ggtctgcgac ctgacggagt cttccaactc 24600
gccggatcgg ggagatcttc cttcacgcct cgtcaggccg tcctgacttt ggagagttcg 24660
tcctcgcagc cccgctcggg tggcatcggc actctccagt tcgtggagga gttcactccc 24720
tcggtctact tcaacccctt ctccggctcc cccggccact acccggacga gttcatcccg 24780
aacttcgacg ccatcagcga gtcggtggac ggctacgatt gaatgtccca tggtggcgca 24840
gctgacctag ctcggcttcg acacctggac cactgccgcc gcttccgctg cttcgctcgg 24900
gatctcgccg agtttgccta ctttgagctg cccgaggagc accctcaggg cccggcccac 24960
ggagtgcgga tcgtcgtcga agggggcctc gactcccacc tgcttcggat cttcagccag 25020
cgtccgatcc tggtcgagcg cgagcaagga cagacccttc tgactctgta ctgcatctgc 25080
aaccaccccg gcctgcatga aagtctttgt tgtctgctgt gtactgagta taataaaagc 25140
tgagatcagc gactactccg gacttccgtg tgttcctgaa tccatcaacc agtctttgtt 25200
cttcaccggg aacgagaccg agctccagct ccagtgtaag ccccacaaga agtacctcac 25260
ctggctgttc cagggctccc cgatcgccgt tgtcaaccac tgcgacaacg actatttaaa 25320
tccacaatac atgcccatat tagactatga ggccgagcca cagcgaccca tgctccccgc 25380
tattagttac ttcaatctaa ccggcggaga tgactgaccc actggccaac aacaacgtca 25440
acgaccttct cctggacatg gacggccgcg cctcggagca gcgactcgcc caacttcgca 25500
ttcgccagca gcaggagaga gccgtcaagg agctgcagga tgcggtggcc atccaccagt 25560
gcaagagagg catcttctgc ctggtgaaac aggccaagat ctcctacgag gtcactccaa 25620
acgaccatcg cctctcctac gagctcctgc agcagcgcca gaagttcacc tgcctggtcg 25680
gagtcaaccc catcgtcatc acccagcagt ctggcgatac caaggggtgc atccactgct 25740
cctgcgactc ccccgactgc gtccacactc tgatcaagac cctctgcggc ctccgcgacc 25800
tcctccccat gaactaatca cccccttatc cagtgaaata aagatcatat tgatgatgat 25860
tttacagaaa taaaaaataa tcatttgatt tgaaataaag atacaatcat attgatgatt 25920
tgagtttaac aaaaaaataa agaatcactt acttgaaatc tgataccagg tctctgtcca 25980
tgttttctgc caacaccact tcactcccct cttcccagct ctggtactgc aggccccggc 26040
gggctgcaaa cttcctccac acgctgaagg ggatgtcaaa ttcctcctgt ccctcaatct 26100
tcattttatc ttctatcaga tgtccaaaaa gcgcgtccgg gtggatgatg acttcgaccc 26160
cgtctacccc tacgatgcag acaacgcacc gaccgtgccc ttcatcaacc cccccttcgt 26220
ctcttcagat ggattccaag agaagcccct gggggtgttg tccctgcgac tggccgaccc 26280
cgtcaccacc aagaacgggg aaatcaccct caagctggga gagggggtgg acctcgattc 26340
ctcgggaaaa ctcatctcca acacggccac caaggccgcc gcccctctca gtttttccaa 26400
caacaccatt tcccttaaca tggatcaccc cttttacact aaagatggaa aattatcctt 26460
acaagtttct ccaccattaa atatactgag aacaagcatt ctaaacacac tagctttagg 26520
ttttggatca ggtttaggac tccgtggctc tgccttggca gtacagttag tctctccact 26580
tacatttgat actgatggaa acataaagct taccttagac agaggtttgc atgttacaac 26640
aggagatgca attgaaagca acataagctg ggctaaaggt ttaaaatttg aagatggagc 26700
catagcaacc aacattggaa atgggttaga gtttggaagc agtagtacag aaacaggtgt 26760
tgatgatgct tacccaatcc aagttaaact tggatctggc cttagctttg acagtacagg 26820
agccataatg gctggtaaca aagaagacga taaactcact ttgtggacaa cacctgatcc 26880
atcaccaaac tgtcaaatac tcgcagaaaa tgatgcaaaa ctaacacttt gcttgactaa 26940
atgtggtagt caaatactgg ccactgtgtc agtcttagtt gtaggaagtg gaaacctaaa 27000
ccccattact ggcaccgtaa gcagtgctca ggtgtttcta cgttttgatg caaacggtgt 27060
tcttttaaca gaacattcta cactaaaaaa atactggggg tataggcagg gagatagcat 27120
agatggcact ccatatacca atgctgtagg attcatgccc aatttaaaag cttatccaaa 27180
gtcacaaagt tctactacta aaaataatat agtagggcaa gtatacatga atggagatgt 27240
ttcaaaacct atgcttctca ctataaccct caatggtact gatgacagca acagtacata 27300
ttcaatgtca ttttcataca cctggactaa tggaagctat gttggagcaa catttggggc 27360
taactcttat accttctcat acatcgccca agaatgaaca ctgtatccca ccctgcatgc 27420
caacccttcc caccccactc tgtggaacaa actctgaaac acaaaataaa ataaagttca 27480
agtgttttat tgattcaaca gtttcacaga accctagtat tcaacctgcc acctccctcc 27540
caacacacag agtacacagt cctttctccc cggctggcct taaaaagcat catatcatgg 27600
gtaacagaca tattcttagg tgttatattc cacacggttt cctgtcgagc caaacgctca 27660
tcagtgatat taataaactc cccgggcagc tcacttaagt tcatgtcgct gtccagctgc 27720
tgagccacag gctgctgtcc aacttgcggt tgcttaacgg gcggcgaagg agaagtccac 27780
gcctacatgg gggtagagtc ataatcgtgc atcaggatag ggcggtggtg ctgcagcagc 27840
gcgcgaataa actgctgccg ccgccgctcc gtcctgcagg aatacaacat ggcagtggtc 27900
tcctcagcga tgattcgcac cgcccgcagc ataaggcgcc ttgtcctccg ggcacagcag 27960
cgcaccctga tctcacttaa atcagcacag taactgcagc acagcaccac aatattgttc 28020
aaaatcccac agtgcaaggc gctgtatcca aagctcatgg cggggaccac agaacccacg 28080
tggccatcat accacaagcg caggtagatt aagtggcgac ccctcataaa cacgctggac 28140
ataaacatta cctcttttgg catgttgtaa ttcaccacct cccggtacca tataaacctc 28200
tgattaaaca tggcgccatc caccaccatc ctaaaccagc tggccaaaac ctgcccgccg 28260
gctatacact gcagggaacc gggactggaa caatgacagt ggagagccca ggactcgtaa 28320
ccatggatca tcatgctcgt catgatatca atgttggcac aacacaggca cacgtgcata 28380
cacttcctca ggattacaag ctcctcccgc gttagaacca tatcccaggg aacaacccat 28440
tcctgaatca gcgtaaatcc cacactgcag ggaagacctc gcacgtaact cacgttgtgc 28500
attgtcaaag tgttacattc gggcagcagc ggatgatcct ccagtatggt agcgcgggtt 28560
tctgtctcaa aaggaggtag acgatcccta ctgtacggag tgcgccgaga caaccgagat 28620
cgtgttggtc gtagtgtcat gccaaatgga acgccggacg tagtcatttt cgtacttgct 28680
gtagcagaac ctggtccggg cgctgcacac cgatcgccgg cggcggtctc ggcgcttgga 28740
acgctcggtg ttgaaattgt aaaacagcca ctctctcaga ccgtgcagca gatctagggc 28800
ctcaggagtg atgaagatcc catcatgcct gatggctctg atcacatcga ccaccgtgga 28860
atgggccaga cccagccaga tgatgcaatt ttgttgggtt tcggtgacgg cgggggaggg 28920
aagaacagga agaaccatga ttaactttta atccaaacgg tctcggagta cttcaaaatg 28980
aagatcgcgg agatggcacc tctcgccccc gctgtgttgg tggaaaataa cagccaggtc 29040
aaaggtgata cggttctcga gatgttccac ggtggcttcc agcaaagcct ccacgcgcac 29100
atccagaaac aagacaatag cgaaagcggg agggttctct aattcctcaa tcatcatgtt 29160
acactcctgc accatcccca gataattttc atttttccag ccttgaatga ttcgaactag 29220
ttcctgaggt aaatccaagc cagccatgat aaagagctcg cgcagagcgc cctccaccgg 29280
cattcttaag cacaccctca taattccaag atattctgct cctggttcac ctgcagcaga 29340
ttgacaagcg gaatatcaaa atctctgccg cgatccctga gctcctccct cagcaataac 29400
tgtaagtact ctttcatatc ctctccgaaa tttttagcca taggaccacc aggaataaga 29460
ttagggcaag ccacagtaca gataaaccga agtcctcccc agtgagcatt gccaaatgca 29520
agactgctat aagcatgctg gctagacccg gtgatatctt ccagataact ggacagaaaa 29580
tcgcccaggc aatttttaag aaaatcaaca aaagaaaaat cctccaggtg gacgtttaga 29640
gcctcgggaa caacgatgaa gtaaatgcaa gcggtgcgtt ccagcatggt tagttagctg 29700
atctgtagaa aaaacaaaaa tgaacattaa accatgctag cctggcgaac aggtgggtaa 29760
atcgttctct ccagcaccag gcaggccacg gggtctccgg cgcgaccctc gtaaaaattg 29820
tcgctatgat tgaaaaccat cacagagaga cgttcccggt ggccggcgtg aatgattcga 29880
caagatgaat acacccccgg aacattggcg tccgcgagtg aaaaaaagcg cccgaggaag 29940
caataaggca ctacaatgct cagtctcaag tccagcaaag cgatgccatg cggatgaagc 30000
acaaaattct caggtacaaa atgtaattac tcccctcctg cacaggcagc aaagcccccg 30060
atccctccag gtacacatac aaagcctcag cgtccatagc ttaccgagca gcagcacaca 30120
acaggcgcaa gagtcagaga aaggctgagc tctaacctgt ccacccgctc tctgctcaat 30180
atatagccca gatctacact gacgtaaagg ccaaagtcta aaaatacccg ccaaataatc 30240
acacacgccc agcacacgcc cagaaaccgg tgacacactc aaaaaaatac gcgcacttcc 30300
tcaaacgccc aaaactgccg tcatttccgg gttcccacgc tacgtcatca aaacacgact 30360
ttcaaattcc gtcgaccgtt aaaaacgtca cccgccccgc ccctaacggt cgcccgtctc 30420
tcagccaatc agcgccccgc atccccaaat tcaaacacct catttgcata ttaacgcgca 30480
caaaaagttt gaggtatatt attgatgatg 30510
<210> 2
<211> 915
<212> DNA
<213> Artificial Sequence
<400> 2
aagctcagat ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca 60
tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc 120
aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg 180
gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta 240
ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 300
ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 360
ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 420
cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 480
atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 540
cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 600
attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca 660
cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat 720
caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg 780
cgtgtacggt gggaggtcta tataagcaga gctctctggc taactagaga acccactgct 840
tactggctta tcgaaattaa tacgactcac tatagggaga cccaagctgg ctagcgttta 900
aacgggcccg ccacc 915
<210> 3
<211> 1872
<212> DNA
<213> Artificial Sequence
<400> 3
atgggcaccg tgaacaagcc cgtggtgggc gtgctgatgg ggttcggcat catcaccggc 60
accctgagaa tcaccaaccc cgtgagagcc agcgtgctga gatacgacga cttccacatc 120
gatgaagata aactggacac caatagtgtg tacgaacctt actaccacag cgaccatgcc 180
gaaagcagct gggtgaacag aggagagagc agtagaaagg cctacgacca caacagcccc 240
tacatttggc ccagaaacga ctacgatggc tttctggaga acgcccacga gcaccacgga 300
gtgtacaacc agggacgggg aatcgacagc ggagaaagac tgatgcagcc aacacagatg 360
agcgctcagg aagacctggg agacgacacc ggcatccacg tgatcccaac cctgaacggc 420
gacgacagac acaaaatcgt gaacgtggac cagagacagt acggcgacgt gttcaagggc 480
gacctgaacc ccaaaccaca gggccagaga ctgatcgagg tgtccgtgga agagaaccac 540
cctttcaccc tgcgcgcccc cattcagaga atctacggcg tgagatacac cgagacatgg 600
agcttcctgc ccagcctgac ttgcaccggc gacgccgctc ctgccatcca gcacatctgc 660
ctgaaacaca ccacctgctt ccaggacgtg gtggtggatg tggactgcgc cgaaaacacc 720
aaggaggacc agctggccga gatctcttat agatttcagg gcaaaaagga ggccgaccag 780
ccctggatcg tggtgaacac ctcaaccctg ttcgacgagc tggagctgga tccacccgag 840
atcgagcccg gcgtgctgaa ggtgctgaga accgagaagc agtacctggg agtgtacatc 900
tggaacatga gaggcagcga cggcaccagc acatatgcca cctttctggt gacctggaag 960
ggagacgaga agacccggaa ccccacaccc gccgtgaccc ctcagcctag gggcgctgag 1020
ttccacatgt ggaactacca cagccacgtg ttcagcgtgg gcgacacctt cagcctggct 1080
atgcacctgc agtacaaaat ccacgaagcc cccttcgacc tgctgctgga gtggctgtac 1140
gtgcccatcg atcccacatg ccagcccatg agactgtact ccacctgcct gtaccacccc 1200
aacgcccccc agtgcctgtc ccacatgaac agcggatgca ccttcacctc cccccacctg 1260
gcccagagag tggccagcac cgtgtaccag aactgcgagc acgccgacaa ctacaccgcc 1320
tactgcctgg gcatcagcca catggaaccc agctttggcc tgatcctgca cgacggcggc 1380
accacactga aattcgtgga cacccccgaa agcctgagcg gactgtacgt gttcgtggtg 1440
tacttcaacg gccacgtgga ggccgtggcc tacacagtgg tgtccaccgt ggaccacttc 1500
gtgaacgcca ttgaggagag aggcttcccc cccaccgctg gccagcctcc tgctactacc 1560
aagcccaaag agattacccc cgtgaacccc ggcacctccc ccctgctgag atacgccgcc 1620
tggaccggcg gcctggctgc tgtggtgctg ctgtgcctgg tgatcttcct gatctgcacc 1680
gccaaaagaa tgagagtgaa ggcctacagg gtggacaaaa gcccttacaa ccagtccatg 1740
tactacgctg gcctgcccgt ggatgacttc gaggactctg agagcaccga caccgaagaa 1800
gagttcggca acgccatcgg aggctcacat ggcggcagct cctacaccgt gtacatcgac 1860
aagaccagat ga 1872
<210> 4
<211> 623
<212> PRT
<213> Artificial Sequence
<400> 4
Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly
1 5 10 15
Ile Ile Thr Gly Thr Leu Arg Ile Thr Asn Pro Val Arg Ala Ser Val
20 25 30
Leu Arg Tyr Asp Asp Phe His Ile Asp Glu Asp Lys Leu Asp Thr Asn
35 40 45
Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp
50 55 60
Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro
65 70 75 80
Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His
85 90 95
Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu
100 105 110
Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp
115 120 125
Asp Thr Gly Ile His Val Ile Pro Thr Leu Asn Gly Asp Asp Arg His
130 135 140
Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly
145 150 155 160
Asp Leu Asn Pro Lys Pro Gln Gly Gln Arg Leu Ile Glu Val Ser Val
165 170 175
Glu Glu Asn His Pro Phe Thr Leu Arg Ala Pro Ile Gln Arg Ile Tyr
180 185 190
Gly Val Arg Tyr Thr Glu Thr Trp Ser Phe Leu Pro Ser Leu Thr Cys
195 200 205
Thr Gly Asp Ala Ala Pro Ala Ile Gln His Ile Cys Leu Lys His Thr
210 215 220
Thr Cys Phe Gln Asp Val Val Val Asp Val Asp Cys Ala Glu Asn Thr
225 230 235 240
Lys Glu Asp Gln Leu Ala Glu Ile Ser Tyr Arg Phe Gln Gly Lys Lys
245 250 255
Glu Ala Asp Gln Pro Trp Ile Val Val Asn Thr Ser Thr Leu Phe Asp
260 265 270
Glu Leu Glu Leu Asp Pro Pro Glu Ile Glu Pro Gly Val Leu Lys Val
275 280 285
Leu Arg Thr Glu Lys Gln Tyr Leu Gly Val Tyr Ile Trp Asn Met Arg
290 295 300
Gly Ser Asp Gly Thr Ser Thr Tyr Ala Thr Phe Leu Val Thr Trp Lys
305 310 315 320
Gly Asp Glu Lys Thr Arg Asn Pro Thr Pro Ala Val Thr Pro Gln Pro
325 330 335
Arg Gly Ala Glu Phe His Met Trp Asn Tyr His Ser His Val Phe Ser
340 345 350
Val Gly Asp Thr Phe Ser Leu Ala Met His Leu Gln Tyr Lys Ile His
355 360 365
Glu Ala Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp
370 375 380
Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro
385 390 395 400
Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr
405 410 415
Ser Pro His Leu Ala Gln Arg Val Ala Ser Thr Val Tyr Gln Asn Cys
420 425 430
Glu His Ala Asp Asn Tyr Thr Ala Tyr Cys Leu Gly Ile Ser His Met
435 440 445
Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys
450 455 460
Phe Val Asp Thr Pro Glu Ser Leu Ser Gly Leu Tyr Val Phe Val Val
465 470 475 480
Tyr Phe Asn Gly His Val Glu Ala Val Ala Tyr Thr Val Val Ser Thr
485 490 495
Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr
500 505 510
Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val
515 520 525
Asn Pro Gly Thr Ser Pro Leu Leu Arg Tyr Ala Ala Trp Thr Gly Gly
530 535 540
Leu Ala Ala Val Val Leu Leu Cys Leu Val Ile Phe Leu Ile Cys Thr
545 550 555 560
Ala Lys Arg Met Arg Val Lys Ala Tyr Arg Val Asp Lys Ser Pro Tyr
565 570 575
Asn Gln Ser Met Tyr Tyr Ala Gly Leu Pro Val Asp Asp Phe Glu Asp
580 585 590
Ser Glu Ser Thr Asp Thr Glu Glu Glu Phe Gly Asn Ala Ile Gly Gly
595 600 605
Ser His Gly Gly Ser Ser Tyr Thr Val Tyr Ile Asp Lys Thr Arg
610 615 620
<210> 5
<211> 300
<212> DNA
<213> Artificial Sequence
<400> 5
aagcttaagt ttaaaccgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 60
tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 120
ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 180
tggggtgggg caggacagca agggggagga ttgggaagac aatagcaggc atgctgggga 240
tgcggtgggc tctatggctt ctgaggcgga aagaaccagc agatctgcag atctgaattc 300
<210> 6
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 6
tcgtcgtttt cggcgcgcgc cg 22
<210> 7
<211> 106
<212> DNA
<213> Artificial Sequence
<400> 7
cacagaagat gatcgtcgtt ttcggcgcgc gccgtcgtcg ttttcggcgc gcgccgtcgt 60
cgttttcggc gcgcgccgtc gtcgttttcg gcgcgcgccg ggtacc 106
<210> 8
<211> 66
<212> DNA
<213> Artificial Sequence
<400> 8
ggaagcggag ctactaactt cagcctgctg aagcaggctg gagacgtgga ggagaaccct 60
ggacct 66
<210> 9
<211> 489
<212> DNA
<213> Artificial Sequence
<400> 9
atgagaagca gccccgggaa catggagaga atcgtgatct gcctgatggt gatcttcctg 60
ggcaccctgg tgcacaagag cagcagccag ggccaggaca gacatatgat ccggatgaga 120
cagctgatcg acatcgtgga ccagctgaag aattacgtga acgacctggt gcctgaattc 180
ctgcccgcac ccgaggacgt ggagaccaac tgcgaatgga gcgcctttag ctgcttccag 240
aaggcccagc tgaagagcgc aaacacaggc aacaatgaga gaatcatcaa cgtgagcatc 300
aaaaagctga agagaaagcc acccagcacc aacgcaggaa gaagacagaa gcacagactg 360
acctgcccca gctgcgacag ctacgaaaaa aagcccccca aggagttcct ggagagattc 420
aagagcctgc tgcagaagat gatccaccag cacctgagca gcagaaccca cggcagcgag 480
gacagctga 489
<210> 10
<211> 162
<212> PRT
<213> Artificial Sequence
<400> 10
Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met
1 5 10 15
Val Ile Phe Leu Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln
20 25 30
Asp Arg His Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln
35 40 45
Leu Lys Asn Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro
50 55 60
Glu Asp Val Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln
65 70 75 80
Lys Ala Gln Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile
85 90 95
Asn Val Ser Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala
100 105 110
Gly Arg Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr
115 120 125
Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu
130 135 140
Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly Ser Glu
145 150 155 160
Asp Ser
<210> 11
<211> 786
<212> DNA
<213> Artificial Sequence
<400> 11
atgatcgaga cctacaacca gaccagcccc agaagcgccg ctacaggact gcctatcagc 60
atgaagatct tcatgtacct gctgaccgtg ttcctgatca cccagatgat cggcagcgcc 120
ctgttcgctg tgtacctgca tagaagactg gacaagatcg aggacgagag aaacctgcac 180
gaggactttg tgttcatgaa gaccattcag agatgcaaca ccggcgagag aagcctgagt 240
ctgctgaatt gcgaagagat caaatcccag ttcgagggat tcgtgaagga cattatgctg 300
aacaaggagg agaccaagaa agaaaacagc ttcgaaatgc agaagggaga ccagaacccc 360
cagatcgccg ctcatgtgat cagcgaggct agcagtaaga ccacctccgt gctgcagtgg 420
gccgaaaagg gatactacac aatgagcaac aacctggtga ccctggagaa cggcaaacag 480
ctgaccgtga agagacaggg cctgtactac atctacgccc aggtgacttt ctgcagcaac 540
agagaggcca gcagccaggc tccattcatc gcttctctgt gcctgaagtc tcccggcaga 600
ttcgagagaa tcctgctgcg cgccgccaac acacattcaa gtgccaagcc ctgcggccag 660
cagtcaatcc atctgggagg cgtgttcgag ctgcagcctg gagcttctgt gttcgtgaac 720
gtgaccgacc cctcccaggt gagtcacgga acaggattca cttccttcgg cctgctgaag 780
ctgtga 786
<210> 12
<211> 261
<212> PRT
<213> Artificial Sequence
<400> 12
Met Ile Glu Thr Tyr Asn Gln Thr Ser Pro Arg Ser Ala Ala Thr Gly
1 5 10 15
Leu Pro Ile Ser Met Lys Ile Phe Met Tyr Leu Leu Thr Val Phe Leu
20 25 30
Ile Thr Gln Met Ile Gly Ser Ala Leu Phe Ala Val Tyr Leu His Arg
35 40 45
Arg Leu Asp Lys Ile Glu Asp Glu Arg Asn Leu His Glu Asp Phe Val
50 55 60
Phe Met Lys Thr Ile Gln Arg Cys Asn Thr Gly Glu Arg Ser Leu Ser
65 70 75 80
Leu Leu Asn Cys Glu Glu Ile Lys Ser Gln Phe Glu Gly Phe Val Lys
85 90 95
Asp Ile Met Leu Asn Lys Glu Glu Thr Lys Lys Glu Asn Ser Phe Glu
100 105 110
Met Gln Lys Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser
115 120 125
Glu Ala Ser Ser Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly
130 135 140
Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln
145 150 155 160
Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr
165 170 175
Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser
180 185 190
Leu Cys Leu Lys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala
195 200 205
Ala Asn Thr His Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His
210 215 220
Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn
225 230 235 240
Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe
245 250 255
Gly Leu Leu Lys Leu
260
<210> 13
<211> 698
<212> DNA
<213> Artificial Sequence
<400> 13
cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc 60
atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac 120
cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa 180
tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag 240
tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc 300
ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct 360
acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg 420
gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt 480
tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga 540
cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctctggctaa 600
ctagagaacc cactgcttac tggcttatcg aaattaatac gactcactat agggagaccc 660
aagctggcta gcgtttaaac gggccctcta gagccacc 698
<210> 14
<211> 996
<212> DNA
<213> Artificial Sequence
<400> 14
atgaagaaga gcctgatcgc cctgaccctg gccgctctgc ctgtggctgc tatggctgat 60
gtgaccctgt acggcaccat caaggccgga gtggagacct ctaggagcgt gttccaccag 120
aacggacagg tgaccgaagt gaccaccgcc acaggaatcg tggacctggg atctaagatt 180
ggctttaagg gacaggagga cctgggaaat ggactgaagg ccatctggca ggtggagcag 240
aaggccagta tcgccggaac cgacagcgga tggggaaata gacagagctt tatcgggctg 300
aaggggggat tcggcaagct gagagtggga agactgaaca gcgtgctgaa ggacaccggg 360
gacatcaacc cttgggacag caagagcgac tacctgggag tgaacaagat cgctgaacca 420
gaggccagac tgatctccgt gagatatgac tctccagaat tcgccggcct gagcggaagc 480
gtgcagtatg ctctgaacga caacgctggc aggcacaata gcgagagcta ccacgccggc 540
ttcaactaca agaacggagg attcttcgtg cagtacggcg gagcttacaa aagacaccat 600
caggtgcagg agggcctgaa catcgagaaa taccagatcc acagactggt gagcggatac 660
gacaacgacg ccctgtacgc ctccgtggct gtgcagcagc aggatgctaa gctgaccgac 720
gcctccaaca gccacaacag ccagaccgag gtggccgcta cactggctta cagattcgga 780
aacgtgaccc ccagagtgag ctacgcccat ggattcaagg ggctggtgga cgacgccgat 840
atcggaaacg agtacgacca ggtggtggtg ggagccgaat acgatttcag caagagaacc 900
tctgccctgg tgagcgccgg atggctgcag gaaggaaaag gcgagaacaa gttcgtggcc 960
accgccggag gagtgggact gagacataag ttctga 996
<210> 15
<211> 331
<212> PRT
<213> Artificial Sequence
<400> 15
Met Lys Lys Ser Leu Ile Ala Leu Thr Leu Ala Ala Leu Pro Val Ala
1 5 10 15
Ala Met Ala Asp Val Thr Leu Tyr Gly Thr Ile Lys Ala Gly Val Glu
20 25 30
Thr Ser Arg Ser Val Phe His Gln Asn Gly Gln Val Thr Glu Val Thr
35 40 45
Thr Ala Thr Gly Ile Val Asp Leu Gly Ser Lys Ile Gly Phe Lys Gly
50 55 60
Gln Glu Asp Leu Gly Asn Gly Leu Lys Ala Ile Trp Gln Val Glu Gln
65 70 75 80
Lys Ala Ser Ile Ala Gly Thr Asp Ser Gly Trp Gly Asn Arg Gln Ser
85 90 95
Phe Ile Gly Leu Lys Gly Gly Phe Gly Lys Leu Arg Val Gly Arg Leu
100 105 110
Asn Ser Val Leu Lys Asp Thr Gly Asp Ile Asn Pro Trp Asp Ser Lys
115 120 125
Ser Asp Tyr Leu Gly Val Asn Lys Ile Ala Glu Pro Glu Ala Arg Leu
130 135 140
Ile Ser Val Arg Tyr Asp Ser Pro Glu Phe Ala Gly Leu Ser Gly Ser
145 150 155 160
Val Gln Tyr Ala Leu Asn Asp Asn Ala Gly Arg His Asn Ser Glu Ser
165 170 175
Tyr His Ala Gly Phe Asn Tyr Lys Asn Gly Gly Phe Phe Val Gln Tyr
180 185 190
Gly Gly Ala Tyr Lys Arg His His Gln Val Gln Glu Gly Leu Asn Ile
195 200 205
Glu Lys Tyr Gln Ile His Arg Leu Val Ser Gly Tyr Asp Asn Asp Ala
210 215 220
Leu Tyr Ala Ser Val Ala Val Gln Gln Gln Asp Ala Lys Leu Thr Asp
225 230 235 240
Ala Ser Asn Ser His Asn Ser Gln Thr Glu Val Ala Ala Thr Leu Ala
245 250 255
Tyr Arg Phe Gly Asn Val Thr Pro Arg Val Ser Tyr Ala His Gly Phe
260 265 270
Lys Gly Leu Val Asp Asp Ala Asp Ile Gly Asn Glu Tyr Asp Gln Val
275 280 285
Val Val Gly Ala Glu Tyr Asp Phe Ser Lys Arg Thr Ser Ala Leu Val
290 295 300
Ser Ala Gly Trp Leu Gln Glu Gly Lys Gly Glu Asn Lys Phe Val Ala
305 310 315 320
Thr Ala Gly Gly Val Gly Leu Arg His Lys Phe
325 330
<210> 16
<211> 234
<212> DNA
<213> Artificial Sequence
<400> 16
ggtaccaagc ttaagtttaa accgctgatc agcctcgact gtgccttcta gttgccagcc 60
atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt 120
cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct 180
ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcag 234
<210> 17
<211> 906
<212> DNA
<213> Artificial Sequence
<400> 17
atggccagca gcgacgggga cagactgtgc agaagcaacg ccgtgaggag gaagaccacc 60
cccagctaca gcgggcagta cagaaccgcc agaagaagcg tggtggtggg gccccccgac 120
gactcagacg acagcctggg ctacatcacc acagtgggag ccgacagccc cagccccgtg 180
tacgctgacc tgtacttcga gcacaagaac acaacaccta gagtgcatca gcccaacgac 240
agcagcggca gcgaagacga cttcgaagac attgacgagg tggtggccgc cttcagagag 300
gccagactga gacacgagct ggtggaagac gccgtgtacg agaaccccct gagcgtggag 360
aagcctagca gatcctttac caagaacgcc gccgtgaagc ctaagctgga ggacagcccc 420
aagagagccc cccccggagc tggagctatt gcttccggca gacccatctc atttagcacc 480
gcccccaaaa ccgccacaag cagctggtgc ggccccaccc caagctataa caagagagtg 540
ttctgtgaag ccgtgagaag agtggccgcc atgcaggccc agaaggctgc cgaagccgcc 600
tggaactcca acccccccag aaacaacgct gaactggaca gactgctgac aggagccgtg 660
atcagaatta ccgtgcacga aggactgaac ctgatccagg ccgccaatga ggccgacctg 720
ggcgagggag ccagcgtgtc aaagagagga cacaacagaa agaccggcga cctgcagggc 780
ggcatgggca atgagccaat gtacgcccag gtgagaaaac ccaagagcag aaccgacaca 840
cagaccaccg gaagaatcac caacagaagc agagccagaa gcgcttccag aacagacgcc 900
agaaaa 906
<210> 18
<211> 302
<212> PRT
<213> Artificial Sequence
<400> 18
Met Ala Ser Ser Asp Gly Asp Arg Leu Cys Arg Ser Asn Ala Val Arg
1 5 10 15
Arg Lys Thr Thr Pro Ser Tyr Ser Gly Gln Tyr Arg Thr Ala Arg Arg
20 25 30
Ser Val Val Val Gly Pro Pro Asp Asp Ser Asp Asp Ser Leu Gly Tyr
35 40 45
Ile Thr Thr Val Gly Ala Asp Ser Pro Ser Pro Val Tyr Ala Asp Leu
50 55 60
Tyr Phe Glu His Lys Asn Thr Thr Pro Arg Val His Gln Pro Asn Asp
65 70 75 80
Ser Ser Gly Ser Glu Asp Asp Phe Glu Asp Ile Asp Glu Val Val Ala
85 90 95
Ala Phe Arg Glu Ala Arg Leu Arg His Glu Leu Val Glu Asp Ala Val
100 105 110
Tyr Glu Asn Pro Leu Ser Val Glu Lys Pro Ser Arg Ser Phe Thr Lys
115 120 125
Asn Ala Ala Val Lys Pro Lys Leu Glu Asp Ser Pro Lys Arg Ala Pro
130 135 140
Pro Gly Ala Gly Ala Ile Ala Ser Gly Arg Pro Ile Ser Phe Ser Thr
145 150 155 160
Ala Pro Lys Thr Ala Thr Ser Ser Trp Cys Gly Pro Thr Pro Ser Tyr
165 170 175
Asn Lys Arg Val Phe Cys Glu Ala Val Arg Arg Val Ala Ala Met Gln
180 185 190
Ala Gln Lys Ala Ala Glu Ala Ala Trp Asn Ser Asn Pro Pro Arg Asn
195 200 205
Asn Ala Glu Leu Asp Arg Leu Leu Thr Gly Ala Val Ile Arg Ile Thr
210 215 220
Val His Glu Gly Leu Asn Leu Ile Gln Ala Ala Asn Glu Ala Asp Leu
225 230 235 240
Gly Glu Gly Ala Ser Val Ser Lys Arg Gly His Asn Arg Lys Thr Gly
245 250 255
Asp Leu Gln Gly Gly Met Gly Asn Lys Pro Met Tyr Ala Gln Val Arg
260 265 270
Lys Pro Lys Ser Arg Thr Asp Thr Gln Thr Thr Gly Arg Ile Thr Asn
275 280 285
Arg Ser Arg Ala Arg Ser Ala Ser Arg Thr Asp Ala Arg Lys
290 295 300
<210> 19
<211> 96
<212> DNA
<213> Artificial Sequence
<400> 19
cgaaaaagaa gatcaggttc gggtgcgcca gtaaagcaga cattaaactt tgatttgctg 60
aaacttgcag gtgatgtaga gtcaaatcca ggtcca 96
<210> 20
<211> 837
<212> DNA
<213> Artificial Sequence
<400> 20
atgttctgca ccagccccgc caccagaggc gacagcagcg agagcaagcc cggagccagc 60
gtggacgtga acggcaagat ggagtacggc agcgcccccg gccccctgaa tggaagagac 120
accagcagag gacccggagc cttctgtacc cccggatggg agatccaccc cgccagactg 180
gtggaggaca tcaacagagt gttcctgtgc atcgcccagt cctccggcag agtgaccaga 240
gacagcagga gactgagaag gatttgcctg gacttttacc tgatgggcag aacaagacag 300
agacccaccc tggcctgctg ggaagagctg ctgcagctgc agcccacaca gacccagtgc 360
ctgagagcca ccctgatgga ggtgagccac cggcccccca gaggagaaga cggattcatc 420
gaagccccca atgtgcctct gcaccggagc gccctggaat gcgacgtgag cgacgacggc 480
ggcgaggacg acagcgacga tgacggcagc acccccagcg acgtgattga gttcagggac 540
tccgacgccg agtcctccga cggcgaagac tttatcgtgg aagaggagag cgaggagagc 600
acagatagct gcgagccaga cggcgtgccc ggcgattgtt accgggacgg cgacggctgt 660
aataccccaa gcccaaagag accccagaga gccattgaga gatacgccgg ggccgagacc 720
gccgaataca ccgctgccaa ggccctgaca gccctgggcg aaggcggagt ggactggaag 780
agaagaagac acgaggcccc cagaagacac gacatccccc ccccccacgg agtgtga 837
<210> 21
<211> 278
<212> PRT
<213> Artificial Sequence
<400> 21
Met Phe Cys Thr Ser Pro Ala Thr Arg Gly Asp Ser Ser Glu Ser Lys
1 5 10 15
Pro Gly Ala Ser Val Asp Val Asn Gly Lys Met Glu Tyr Gly Ser Ala
20 25 30
Pro Gly Pro Leu Asn Gly Arg Asp Thr Ser Arg Gly Pro Gly Ala Phe
35 40 45
Cys Thr Pro Gly Trp Glu Ile His Pro Ala Arg Leu Val Glu Asp Ile
50 55 60
Asn Arg Val Phe Leu Cys Ile Ala Gln Ser Ser Gly Arg Val Thr Arg
65 70 75 80
Asp Ser Arg Arg Leu Arg Arg Val Cys Leu Asp Phe Tyr Leu Met Gly
85 90 95
Arg Thr Arg Gln Arg Pro Thr Leu Ala Cys Trp Glu Glu Leu Leu Gln
100 105 110
Leu Gln Pro Thr Gln Thr Gln Cys Leu Arg Ala Thr Leu Met Glu Val
115 120 125
Ser His Arg Pro Pro Arg Gly Glu Asp Gly Phe Ile Glu Ala Pro Asn
130 135 140
Val Pro Leu His Arg Ser Ala Leu Glu Cys Asp Val Ser Asp Asp Gly
145 150 155 160
Gly Glu Asp Asp Ser Asp Asp Asp Gly Ser Thr Pro Ser Asp Val Ile
165 170 175
Glu Phe Arg Asp Ser Asp Ala Glu Ser Ser Asp Gly Glu Asp Phe Ile
180 185 190
Val Glu Glu Glu Ser Glu Glu Ser Thr Asp Ser Cys Glu Pro Asp Gly
195 200 205
Val Pro Gly Asp Cys Tyr Arg Asp Gly Asp Gly Cys Asn Thr Pro Ser
210 215 220
Pro Lys Arg Pro Gln Arg Ala Ile Glu Arg Tyr Ala Gly Ala Glu Thr
225 230 235 240
Ala Glu Tyr Thr Ala Ala Lys Ala Leu Thr Ala Leu Gly Glu Gly Gly
245 250 255
Val Asp Trp Lys Arg Arg Arg His Glu Ala Pro Arg Arg His Asp Ile
260 265 270
Pro Pro Pro His Gly Val
275
<210> 22
<211> 2526
<212> DNA
<213> Artificial Sequence
<400> 22
atgttcgccc tggtgctggc cgtggtgatc ctgcccctgt ggaccaccgc caacaagagc 60
tacgtgaccc ccacccccgc caccagaagc atcggccaca tgagcgccct gctgagagag 120
tacagcgaca gaaacatgag cctgaagctg gaagccttct accccaccgg cttcgacgag 180
gaactgatca aatccctgca ctggggcaac gacagaaagc acgtgttcct ggtgattgtg 240
aaagtgaacc ccaccaccca cgaaggcgac gtgggcctgg tgatcttccc caagtacctg 300
ctgagcccct accacttcaa ggccgagcac cgggccccct tccccgctgg aagattcgga 360
tttctgagcc accccgtgac acccgacgtg agtttctttg actcctcctt cgccccctac 420
ctgaccaccc agcacctggt ggccttcacc accttccccc ccaaccccct ggtgtggcac 480
ctggagagag ccgagaccgc cgccaccgct gagaggcctt tcggcgtgtc cctgctgccc 540
gccagaccca cagtgcctaa gaacaccatc ctggaacaca aggcccactt cgccacctgg 600
gacgccctgg ctagacacac cttctttagt gccgaggcca tcattaccaa ctccaccctg 660
agaatccacg tgcccctgtt cggcagcgtg tggcccatca gatactgggc taccggcagc 720
gtgctgctga cctccgactc tggcagagtg gaggtgaaca tcggcgtggg cttcatgagc 780
agcctgatta gcctgagcag cggactgccc atcgagctga tcgtggtgcc ccacaccgtg 840
aaactgaacg ccgtgaccag cgacaccacc tggttccagc tgaatccccc cggccccgac 900
cccggacctt cttatagagt gtacctgctg ggaagaggcc tggatatgaa cttctctaag 960
cacgccaccg tggacatctg cgcctacccc gaggaatccc tggactacag gtatcatctg 1020
agcatggccc acacagaggc cctgagaatg accacaaaag ccgaccagca cgacatcaac 1080
gaagagagct attaccacat cgccgccaga atcgccacct ccatcttcgc cctgagcgaa 1140
atgggaagaa caacagaata cttcctgctg gacgaaattg tggacgtgca gtaccagctg 1200
aagttcctga actacatcct gatgagaatc ggggccggcg ctcaccccaa cacaatcagc 1260
gggacctcag acctgatctt cgccgacccc agccagctgc acgatgagct gagcctgctg 1320
ttcggccagg tgaagcctgc caacgtggac tacttcatct cctacgacga ggccagagat 1380
cagctgaaga ccgcctacgc cctgagcaga ggacaggacc acgtgaacgc cctgagcctg 1440
gctagaaggg tgattatgag catttacaag ggcctgctgg tgaagcagaa cctgaacgcc 1500
accgagagac aggccctgtt cttcgccagc atgattctgc tgaactttag agagggcctg 1560
gagaactcta gcagagtgct ggacggccgg accaccctgc tgctgatgac cagcatgtgc 1620
accgccgctc acgccaccca ggccgctctg aacatccagg agggcctggc ctatctgaat 1680
cccagcaaac acatgttcac aatccccaac gtgtacagcc catgcatggg aagcctgaga 1740
acagatctga ccgaagaaat ccacgtgatg aacctgctga gtgctatccc cacccgcccc 1800
ggcctgaacg aggtgctgca cacccagctg gacgagagcg agatctttga cgccgccttc 1860
aagaccatga tgatcttcac cacctggacc gccaaagacc tgcacatcct gcacacacac 1920
gtgcccgagg tgttcacctg ccaggacgcc gccgccagaa acggcgagta tgtgctgatc 1980
ctgcccgccg tgcagggcca cagctacgtg atcaccagaa acaagcccca gagaggcctg 2040
gtgtacagcc tggccgacgt ggacgtgtat aatcccatca gcgtggtgta tctgagcaaa 2100
gacacctgcg tgtctgaaca cggcgtgatc gagaccgtgg ccctgcccca ccccgacaat 2160
ctgaaagagt gtctgtactg cggcagcgtg ttcctgagat acctgaccac tggagccatc 2220
atggacatta ttatcatcga ttctaaggac accgagagac agctggccgc catgggcaac 2280
agcacaatcc ccccctttaa ccccgatatg cacggcgacg acagcaaagc cgtgctgctg 2340
ttccccaacg gcaccgtggt gacactgctg ggattcgaga gaagacaggc catcagaatg 2400
agcggccagt acctgggcgc cagcctggga ggagccttcc tggctgtggt gggcttcggg 2460
atcatcggct ggatgctgtg cggcaacagt aggctgagag agtataacaa gatccccctg 2520
acatga 2526
<210> 23
<211> 841
<212> PRT
<213> Artificial Sequence
<400> 23
Met Phe Ala Leu Val Leu Ala Val Val Ile Leu Pro Leu Trp Thr Thr
1 5 10 15
Ala Asn Lys Ser Tyr Val Thr Pro Thr Pro Ala Thr Arg Ser Ile Gly
20 25 30
His Met Ser Ala Leu Leu Arg Glu Tyr Ser Asp Arg Asn Met Ser Leu
35 40 45
Lys Leu Glu Ala Phe Tyr Pro Thr Gly Phe Asp Glu Glu Leu Ile Lys
50 55 60
Ser Leu His Trp Gly Asn Asp Lys Lys His Val Phe Leu Val Ile Val
65 70 75 80
Lys Val Asn Pro Thr Thr His Glu Gly Asp Val Gly Leu Val Ile Phe
85 90 95
Pro Lys Tyr Leu Leu Ser Pro Tyr His Phe Lys Ala Glu His Arg Ala
100 105 110
Pro Phe Pro Ala Gly Arg Phe Gly Phe Leu Ser His Pro Val Thr Pro
115 120 125
Asp Val Ser Phe Phe Asp Ser Ser Phe Ala Pro Tyr Leu Thr Thr Gln
130 135 140
His Leu Val Ala Phe Thr Thr Phe Pro Pro Asn Pro Leu Val Trp His
145 150 155 160
Leu Glu Arg Ala Glu Thr Ala Ala Thr Ala Glu Arg Pro Phe Gly Val
165 170 175
Ser Leu Leu Pro Ala Arg Pro Thr Val Pro Lys Asn Thr Ile Leu Glu
180 185 190
His Lys Ala His Phe Ala Thr Trp Asp Ala Leu Ala Arg His Thr Phe
195 200 205
Phe Ser Ala Glu Ala Ile Ile Thr Asn Ser Thr Leu Arg Ile His Val
210 215 220
Pro Leu Phe Gly Ser Val Trp Pro Ile Arg Tyr Trp Ala Thr Gly Ser
225 230 235 240
Val Leu Leu Thr Ser Asp Ser Gly Arg Val Glu Val Asn Ile Gly Val
245 250 255
Gly Phe Met Ser Ser Leu Ile Ser Leu Ser Ser Gly Leu Pro Ile Glu
260 265 270
Leu Ile Val Val Pro His Thr Val Lys Leu Asn Ala Val Thr Ser Asp
275 280 285
Thr Thr Trp Phe Gln Leu Asn Pro Pro Gly Pro Asp Pro Gly Pro Ser
290 295 300
Tyr Arg Val Tyr Leu Leu Gly Arg Gly Leu Asp Met Asn Phe Ala Lys
305 310 315 320
His Ala Thr Val Asp Ile Cys Ala Tyr Pro Glu Glu Ser Leu Asp Tyr
325 330 335
Arg Tyr His Leu Ser Met Ala His Thr Glu Ala Leu Arg Met Thr Thr
340 345 350
Lys Ala Asp Gln His Asp Ile Asn Glu Glu Ser Tyr Tyr His Ile Ala
355 360 365
Ala Arg Ile Ala Thr Ser Ile Phe Ala Leu Ser Glu Met Gly Arg Thr
370 375 380
Thr Glu Tyr Phe Leu Leu Asp Glu Ile Val Asp Val Gln Tyr Gln Leu
385 390 395 400
Lys Phe Leu Asn Tyr Ile Leu Met Arg Ile Gly Ala Gly Ala His Pro
405 410 415
Asn Thr Ile Ser Gly Thr Ser Asp Leu Ile Phe Ala Asp Pro Ser Gln
420 425 430
Leu His Asp Glu Leu Ser Leu Leu Phe Gly Gln Val Lys Pro Ala Asn
435 440 445
Val Asp Tyr Phe Ile Ser Tyr Asp Glu Ala Arg Asp Gln Leu Lys Thr
450 455 460
Ala Tyr Ala Leu Ser Arg Gly Gln Asp His Val Asn Ala Leu Ser Leu
465 470 475 480
Ala Arg Arg Val Ile Met Ser Ile Tyr Lys Gly Leu Leu Val Lys Gln
485 490 495
Asn Leu Asn Ala Thr Glu Arg Gln Ala Leu Phe Phe Ala Ser Met Ile
500 505 510
Leu Leu Asn Phe Arg Glu Gly Leu Glu Asn Ser Ser Arg Val Leu Asp
515 520 525
Gly Arg Thr Thr Leu Leu Leu Met Thr Ser Met Cys Thr Ala Ala His
530 535 540
Ala Thr Gln Ala Ala Leu Asn Ile Gln Glu Gly Leu Ala Tyr Leu Asn
545 550 555 560
Pro Ser Lys His Met Phe Thr Ile Pro Asn Val Tyr Ser Pro Cys Met
565 570 575
Gly Ser Leu Arg Thr Asp Leu Thr Glu Glu Ile His Val Met Asn Leu
580 585 590
Leu Ser Ala Ile Pro Thr Arg Pro Gly Leu Asn Glu Val Leu His Thr
595 600 605
Gln Leu Asp Glu Ser Glu Ile Phe Asp Ala Ala Phe Lys Thr Met Met
610 615 620
Ile Phe Thr Thr Trp Thr Ala Lys Asp Leu His Ile Leu His Thr His
625 630 635 640
Val Pro Glu Val Phe Thr Cys Gln Asp Ala Ala Ala Arg Asn Gly Glu
645 650 655
Tyr Val Leu Ile Leu Pro Ala Val Gln Gly His Ser Tyr Val Ile Thr
660 665 670
Arg Asn Lys Pro Gln Arg Gly Leu Val Tyr Ser Leu Ala Asp Val Asp
675 680 685
Val Tyr Asn Pro Ile Ser Val Val Tyr Leu Ser Lys Asp Thr Cys Val
690 695 700
Ser Glu His Gly Val Ile Glu Thr Val Ala Leu Pro His Pro Asp Asn
705 710 715 720
Leu Lys Glu Cys Leu Tyr Cys Gly Ser Val Phe Leu Arg Tyr Leu Thr
725 730 735
Thr Gly Ala Ile Met Asp Ile Ile Ile Ile Asp Ser Lys Asp Thr Glu
740 745 750
Arg Gln Leu Ala Ala Met Gly Asn Ser Thr Ile Pro Pro Phe Asn Pro
755 760 765
Asp Met His Gly Asp Asp Ser Lys Ala Val Leu Leu Phe Pro Asn Gly
770 775 780
Thr Val Val Thr Leu Leu Gly Phe Glu Arg Arg Gln Ala Ile Arg Met
785 790 795 800
Ser Gly Gln Tyr Leu Gly Ala Ser Leu Gly Gly Ala Phe Leu Ala Val
805 810 815
Val Gly Phe Gly Ile Ile Gly Trp Met Leu Cys Gly Asn Ser Arg Leu
820 825 830
Arg Glu Tyr Asn Lys Ile Pro Leu Thr
835 840
<210> 24
<211> 2607
<212> DNA
<213> Artificial Sequence
<400> 24
atgttcgtga ccgccgtggt gtccgtgtcc cccagcagct tctacgagtc cctgcaggtg 60
gagcccaccc agtccgagga catcaccaga tccgcccacc tgggcgacgg cgacgagatc 120
agggaggcca ttcataagag ccaggacgcc gagaccaagc ccaccttcta cgtgtgcccc 180
cccccaaccg gcagcaccat cgtgagactg gaaccaccca gaacctgtcc cgactaccac 240
ctgggaaaga acttcaccga gggcattgcc gtggtgtaca aagagaacat cgccgcctac 300
aaattcaagg ccaccgtgta ctacaaagac gtgatcgtga gcaccgcctg ggccggctct 360
agctacaccc agatcacaaa cagatacgcc gacagagtgc ccatccccgt gtccgagatc 420
acagacacca tcgacaagtt cggaaaatgc agcagcaagg ccacctacgt gagaaacaac 480
cacaaagtgg aggccttcaa cgaggacaag aacccacagg acatgccact gatcgccagc 540
aagtacaaca gcgtgggcag caaggcctgg cacaccacca acgacaccta catggtggcc 600
ggcacccccg gcacctacag aaccggaacc tccgtgaact gcatcatcga ggaagtggag 660
gccagaagca tcttccccta cgacagcttc ggcctgagca caggcgacat catctacatg 720
agcccatttt tcggcctgag agacggcgcc tatcgcgagc acagcaacta cgccatggat 780
cggttccacc agttcgaggg atacaggcag agagacctgg acaccagagc cctgctggag 840
cccgctgcca gaaacttcct ggtgaccccc cacctgacag tgggctggaa ctggaagccc 900
aagagaaccg aggtgtgcag cctggtgaag tggagagaag tggaagatgt ggtgagagat 960
gaatacgccc acaactttag attcaccatg aaaaccctga gcaccacatt catcagcgag 1020
acaaacgagt tcaacctgaa ccagatccac ctgagccagt gcgtgaagga ggaggccaga 1080
gccattatca accggatcta caccaccaga tacaacagca gccacgtgag aaccggagac 1140
atccagacct acctggccag aggcggcttc gtggtggtgt tccagcccct gctgagcaac 1200
agcctggcca gactgtacct gcaggaactg gtgagggaaa acaccaacca cagcccccag 1260
aaacacccca ccagaaacac aagaagcaga agatcagtgc ccgtggaact gagagccaac 1320
agaacaatca ccaccaccag cagcgtggaa ttcgccatgc tgcagttcac ctacgaccac 1380
atccaggagc acgtgaacga aatgctggcc aggatcagca gtagctggtg ccagctgcag 1440
aacagagaaa gagccctgtg gagtgggctg ttccccatca accccagcgc cctggcctcc 1500
accattctgg atcagagagt gaaagccaga atcctgggcg acgtgatcag cgtgagcaac 1560
tgccccgaac tgggcagcga cacaagaatc atcctgcaga acagcatgag agtgagcggc 1620
tctactacca gatgctatag ccggcctctg atctctatcg tgagcctgaa tggaagcggg 1680
accgtggaag gacagctggg cacagacaac gaactgatca tgagcagaga tctgctggag 1740
ccatgcgtgg ctaaccacaa gcgctacttc ctgttcgggc accactacgt gtactatgag 1800
gactacagat acgtgagaga gatcgccgtg cacgacgtgg ggatgatttc tacatacgtg 1860
gacctgaatc tgacactgct gaaggaccgg gagttcatgc ccctgcaggt gtacacaaga 1920
gacgagctga gagacacagg cctgctggac tattctgaaa tccagaggag aaaccagatg 1980
cacagcctga gattctatga catcgacaag gtggtgcagt acgacagcgg gacagccatc 2040
atgcagggaa tggcccagtt cttccagggc ctggggaccg ccggccaggc tgtgggacat 2100
gtggtgctgg gggctaccgg cgccctgctg agtaccgtgc acgggtttac caccttcctg 2160
agcaacccct ttggcgccct ggccgtgggc ctgctggtgc tggctggact ggtggccgct 2220
ttttttgctt atcgctatgt gctgaagctg aagacctccc ccatgaaggc cctgtaccca 2280
ctgaccacca agggactgaa acagctgccc gaaggcatgg atcccttcgc cgagaagccc 2340
aacgccaccg acacccccat cgaagaaatc ggcgactccc agaacaccga acccagcgtg 2400
aacagcggct tcgacccaga caaattcaga gaagcccagg aaatgattaa gtacatgacc 2460
ctggtgagcg ccgccgagag acaggagagc aaggccagaa aaaagaacaa gaccagcgcc 2520
ctgctgacca gcagactgac cggcctggcc ctgagaaaca gaagaggcta cagcagagtg 2580
agaaccgaga acgtgaccgg cgtgtga 2607
<210> 25
<211> 868
<212> PRT
<213> Artificial Sequence
<400> 25
Met Phe Val Thr Ala Val Val Ser Val Ser Pro Ser Ser Phe Tyr Glu
1 5 10 15
Ser Leu Gln Val Glu Pro Thr Gln Ser Glu Asp Ile Thr Arg Ser Ala
20 25 30
His Leu Gly Asp Gly Asp Glu Ile Arg Glu Ala Ile His Lys Ser Gln
35 40 45
Asp Ala Glu Thr Lys Pro Thr Phe Tyr Val Cys Pro Pro Pro Thr Gly
50 55 60
Ser Thr Ile Val Arg Leu Glu Pro Pro Arg Thr Cys Pro Asp Tyr His
65 70 75 80
Leu Gly Lys Asn Phe Thr Glu Gly Ile Ala Val Val Tyr Lys Glu Asn
85 90 95
Ile Ala Ala Tyr Lys Phe Lys Ala Thr Val Tyr Tyr Lys Asp Val Ile
100 105 110
Val Ser Thr Ala Trp Ala Gly Ser Ser Tyr Thr Gln Ile Thr Asn Arg
115 120 125
Tyr Ala Asp Arg Val Pro Ile Pro Val Ser Glu Ile Thr Asp Thr Ile
130 135 140
Asp Lys Phe Gly Lys Cys Ser Ser Lys Ala Thr Tyr Val Arg Asn Asn
145 150 155 160
His Lys Val Glu Ala Phe Asn Glu Asp Lys Asn Pro Gln Asp Met Pro
165 170 175
Leu Ile Ala Ser Lys Tyr Asn Ser Val Gly Ser Lys Ala Trp His Thr
180 185 190
Thr Asn Asp Thr Tyr Met Val Ala Gly Thr Pro Gly Thr Tyr Arg Thr
195 200 205
Gly Thr Ser Val Asn Cys Ile Ile Glu Glu Val Glu Ala Arg Ser Ile
210 215 220
Phe Pro Tyr Asp Ser Phe Gly Leu Ser Thr Gly Asp Ile Ile Tyr Met
225 230 235 240
Ser Pro Phe Phe Gly Leu Arg Asp Gly Ala Tyr Arg Glu His Ser Asn
245 250 255
Tyr Ala Met Asp Arg Phe His Gln Phe Glu Gly Tyr Arg Gln Arg Asp
260 265 270
Leu Asp Thr Arg Ala Leu Leu Glu Pro Ala Ala Arg Asn Phe Leu Val
275 280 285
Thr Pro His Leu Thr Val Gly Trp Asn Trp Lys Pro Lys Arg Thr Glu
290 295 300
Val Cys Ser Leu Val Lys Trp Arg Glu Val Glu Asp Val Val Arg Asp
305 310 315 320
Glu Tyr Ala His Asn Phe Arg Phe Thr Met Lys Thr Leu Ser Thr Thr
325 330 335
Phe Ile Ser Glu Thr Asn Glu Phe Asn Leu Asn Gln Ile His Leu Ser
340 345 350
Gln Cys Val Lys Glu Glu Ala Arg Ala Ile Ile Asn Arg Ile Tyr Thr
355 360 365
Thr Arg Tyr Asn Ser Ser His Val Arg Thr Gly Asp Ile Gln Thr Tyr
370 375 380
Leu Ala Arg Gly Gly Phe Val Val Val Phe Gln Pro Leu Leu Ser Asn
385 390 395 400
Ser Leu Ala Arg Leu Tyr Leu Gln Glu Leu Val Arg Glu Asn Thr Asn
405 410 415
His Ser Pro Gln Lys His Pro Thr Arg Asn Thr Arg Ser Arg Arg Ser
420 425 430
Val Pro Val Glu Leu Arg Ala Asn Arg Thr Ile Thr Thr Thr Ser Ser
435 440 445
Val Glu Phe Ala Met Leu Gln Phe Thr Tyr Asp His Ile Gln Glu His
450 455 460
Val Asn Glu Met Leu Ala Arg Ile Ser Ser Ser Trp Cys Gln Leu Gln
465 470 475 480
Asn Arg Glu Arg Ala Leu Trp Ser Gly Leu Phe Pro Ile Asn Pro Ser
485 490 495
Ala Leu Ala Ser Thr Ile Leu Asp Gln Arg Val Lys Ala Arg Ile Leu
500 505 510
Gly Asp Val Ile Ser Val Ser Asn Cys Pro Glu Leu Gly Ser Asp Thr
515 520 525
Arg Ile Ile Leu Gln Asn Ser Met Arg Val Ser Gly Ser Thr Thr Arg
530 535 540
Cys Tyr Ser Arg Pro Leu Ile Ser Ile Val Ser Leu Asn Gly Ser Gly
545 550 555 560
Thr Val Glu Gly Gln Leu Gly Thr Asp Asn Glu Leu Ile Met Ser Arg
565 570 575
Asp Leu Leu Glu Pro Cys Val Ala Asn His Lys Arg Tyr Phe Leu Phe
580 585 590
Gly His His Tyr Val Tyr Tyr Glu Asp Tyr Arg Tyr Val Arg Glu Ile
595 600 605
Ala Val His Asp Val Gly Met Ile Ser Thr Tyr Val Asp Leu Asn Leu
610 615 620
Thr Leu Leu Lys Asp Arg Glu Phe Met Pro Leu Arg Val Tyr Thr Arg
625 630 635 640
Asp Glu Leu Arg Asp Thr Gly Leu Leu Asp Tyr Ser Glu Ile Gln Arg
645 650 655
Arg Asn Gln Met His Ser Leu Arg Phe Tyr Asp Ile Asp Lys Val Val
660 665 670
Gln Tyr Asp Ser Gly Thr Ala Ile Met Gln Gly Met Ala Gln Phe Phe
675 680 685
Gln Gly Leu Gly Thr Ala Gly Gln Ala Val Gly His Val Val Leu Gly
690 695 700
Ala Thr Gly Ala Leu Leu Ser Thr Val His Gly Phe Thr Thr Phe Leu
705 710 715 720
Ser Asn Pro Phe Gly Ala Leu Ala Val Gly Leu Leu Val Leu Ala Gly
725 730 735
Leu Val Ala Ala Phe Phe Ala Tyr Arg Tyr Val Leu Lys Leu Lys Thr
740 745 750
Ser Pro Met Lys Ala Leu Tyr Pro Leu Thr Thr Lys Gly Leu Lys Gln
755 760 765
Leu Pro Glu Gly Met Asp Pro Phe Ala Glu Lys Pro Asn Ala Thr Asp
770 775 780
Thr Pro Ile Glu Glu Ile Gly Asp Ser Gln Asn Thr Glu Pro Ser Val
785 790 795 800
Asn Ser Gly Phe Asp Pro Asp Lys Phe Arg Glu Ala Gln Glu Met Ile
805 810 815
Lys Tyr Met Thr Leu Val Ser Ala Ala Glu Arg Gln Glu Ser Lys Ala
820 825 830
Arg Lys Lys Asn Lys Thr Ser Ala Leu Leu Thr Ser Arg Leu Thr Gly
835 840 845
Leu Ala Leu Arg Asn Arg Arg Gly Tyr Ser Arg Val Arg Thr Glu Asn
850 855 860
Val Thr Gly Val
865

Claims (10)

1. A recombinant virus, which is any one of the following A1) to A8):
a1 A recombinant adenovirus expressing varicella zoster virus gE protein obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene into the genomic DNA of an AdC68XY3-empty virus;
a2 A recombinant adenovirus expressing varicella zoster virus gE protein obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene and CPG sequence into the genomic DNA of an AdC68XY3-empty virus;
a3 A recombinant adenovirus expressing varicella zoster virus gE protein and cytokine IL21 obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene and cytokine IL21 coding gene into the genome DNA of an AdC68XY3-empty virus;
a4 A recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein encoding gene and a T cell costimulatory molecule CD40L encoding gene into the genome DNA of an AdC68XY3-empty virus, and expressing the varicella zoster virus gE protein and the T cell costimulatory molecule CD 40L;
A5 A recombinant adenovirus obtained by inserting a DNA molecule containing a gene encoding the gE protein of varicella zoster virus and a DNA molecule containing a gene encoding the ProB protein of trimeric porin into the genomic DNA of AdC68XY3-empty virus, expressing the gE protein of varicella zoster virus and the ProB protein of trimeric porin;
a6 A recombinant adenovirus obtained by inserting a DNA molecule comprising a varicella zoster virus gE protein encoding gene, a varicella zoster virus Orf9 protein encoding gene and a varicella zoster virus Orf63 protein encoding gene into genomic DNA of an AdC68XY3-empty virus, expressing varicella zoster virus gE protein, varicella zoster virus Orf9 protein and varicella zoster virus Orf63 protein;
a7 A recombinant adenovirus expressing varicella zoster virus gE protein and varicella zoster virus gH protein obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene and varicella zoster virus gH protein coding gene into the genome DNA of an AdC68XY3-empty virus;
a8 A recombinant adenovirus expressing varicella zoster virus gE protein and varicella zoster virus gB protein obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene and varicella zoster virus gB protein coding gene into the genome DNA of an AdC68XY3-empty virus;
The nucleotide sequence of the genomic DNA of the AdC68XY3-empty virus is shown as a sequence 1.
2. The recombinant virus of claim 1, wherein: the CPG sequence is shown in SEQ ID NO. 6.
3. The recombinant virus according to claim 1 or 2, characterized in that:
in the A1), the DNA molecule containing the varicella zoster virus gE protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene and a BGH polyA signal a;
in the A2), the DNA molecule containing the varicella zoster virus gE protein coding gene and the CPG sequence sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a DNA molecule containing 4 continuous repeated CPG sequences and a BGH polyA signal a;
in the A3), the DNA molecule containing the varicella zoster virus gE protein coding gene and the cytokine IL21 coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a connector p2a sequence, the cytokine IL21 coding gene and a BGH polyA signal a;
in the A4), the DNA molecule containing the varicella zoster virus gE protein coding gene and the T cell costimulatory molecule CD40L coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a linker p2a sequence, the T cell costimulatory molecule CD40L coding gene and a BGH polyA signal a;
In the A5), the DNA molecule containing the varicella zoster virus gE protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene and a BGH polyA signal a; the DNA molecule containing the trimeric porin ProB coding gene sequentially comprises a CMV promoter b, the trimeric porin ProB coding gene and a BGH polyA signal b;
in the A6), the DNA molecule containing varicella zoster virus gE protein encoding gene, varicella zoster virus Orf9 protein encoding gene and varicella zoster virus Orf63 protein encoding gene sequentially comprises CMV promoter a, varicella zoster virus gE protein encoding gene, linker p2a sequence, varicella zoster virus Orf9 protein encoding gene, linker f2a sequence, varicella zoster virus Orf63 protein encoding gene and BGH polyA signal a;
in the A7), the DNA molecule containing the varicella zoster virus gE protein coding gene and the varicella zoster virus gH protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a linker p2a sequence, the varicella zoster virus gH protein coding gene and a BGH polyA signal a;
In the A8), the DNA molecule containing the varicella zoster virus gE protein coding gene and the varicella zoster virus gB protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a linker p2a sequence, the varicella zoster virus gB protein coding gene and a BGH polyA signal a;
the nucleotide sequence of the CMV promoter a is shown in a sequence 2;
the nucleotide sequence of the BGH polyA signal a is shown as a sequence 5;
the nucleotide sequence of the DNA molecule containing 4 continuous repeated CPG sequences is shown as a sequence 7;
the sequence of the linker p2a is shown as sequence 8;
the linker f2a sequence is shown as sequence 19;
the nucleotide sequence of the CMV promoter b is shown in a sequence 13;
the nucleotide sequence of the BGH polyA signal b is shown as sequence 16.
4. A recombinant virus according to any one of claims 1-3, wherein:
in the A1), the DNA molecule containing the varicella zoster virus gE protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene and a BGH polyA signal a;
in the A2), the DNA molecule containing the varicella zoster virus gE protein coding gene and the CPG sequence sequentially consists of a CMV promoter a, the varicella zoster virus gE protein coding gene, a DNA molecule containing 4 continuous repeated CPG sequences and a BGH polyA signal a;
In the A3), the DNA molecule containing the varicella zoster virus gE protein coding gene and the cytokine IL21 coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a connector p2a sequence, the cytokine IL21 coding gene and a BGH polyA signal a;
in the A4), the DNA molecule containing varicella zoster virus gE protein coding gene and T cell costimulatory molecule CD40L coding gene sequentially comprises CMV promoter a, varicella zoster virus gE protein coding gene, linker p2a sequence, T cell costimulatory molecule CD40L coding gene and BGH polyA signal a;
in the A5), the DNA molecule containing the varicella zoster virus gE protein coding gene sequentially consists of a CMV promoter a, the varicella zoster virus gE protein coding gene and a BGH polyA signal a; the DNA molecule containing the trimeric porin ProB coding gene sequentially comprises a CMV promoter b, the trimeric porin ProB coding gene and a BGH polyA signal b;
in the A6), the DNA molecule containing varicella zoster virus gE protein coding gene, varicella zoster virus Orf9 protein coding gene and varicella zoster virus Orf63 protein coding gene is sequentially composed of CMV promoter a, varicella zoster virus gE protein coding gene, linker p2a sequence, varicella zoster virus Orf9 protein coding gene, linker f2a sequence, varicella zoster virus Orf63 protein coding gene and BGH polyA signal a;
In the A7), the DNA molecule containing the varicella zoster virus gE protein coding gene and the varicella zoster virus gH protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a linker p2a sequence, the varicella zoster virus gH protein coding gene and a BGH polyA signal a;
in the A8), the DNA molecule containing the varicella zoster virus gE protein coding gene and the varicella zoster virus gB protein coding gene sequentially comprises a CMV promoter a, the varicella zoster virus gE protein coding gene, a linker p2a sequence, the varicella zoster virus gB protein coding gene and a BGH polyA signal a.
5. The recombinant virus of any one of claims 1-4, wherein:
in the A1), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene into an E1 deletion region of an AdC68XY3-empty virus;
in the A2), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene and CPG sequence into an E1 deletion region of an AdC68XY3-empty vector virus;
In the A3), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene and a cytokine IL21 coding gene into an E1 deletion region of an AdC68XY3-empty virus;
in the A4), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene and a T cell costimulatory molecule CD40L coding gene into an E1 deletion region of an AdC68XY3-empty virus;
in the A5), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene into an E1 deletion region of an AdC68XY3-empty virus, and inserting a DNA molecule containing a trimeric porin ProB coding gene into an E3 deletion region of the AdC68XY3-empty virus;
in the A6), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing varicella zoster virus gE protein coding gene, varicella zoster virus Orf9 protein coding gene and varicella zoster virus Orf63 protein coding gene into an E1 deletion region of an AdC68XY3-empty virus;
in the A7), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene and a varicella zoster virus gH protein coding gene into an E1 deletion region of an AdC68XY3-empty virus;
In the A8), the recombinant virus is a recombinant adenovirus obtained by inserting a DNA molecule containing a varicella zoster virus gE protein coding gene and a varicella zoster virus gB protein coding gene into an E1 deletion region of an AdC68XY3-empty virus.
6. The recombinant virus of any one of claims 1-5, wherein:
in the A1), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th site of an AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene;
in the A2), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th site of an AdC68XY3-empty virus with a DNA molecule containing varicella zoster virus gE protein coding gene and CPG sequence;
in the A3), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th site of an AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene and a cytokine IL21 coding gene;
in the A4), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th site of an AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene and a T cell costimulatory molecule CD40L coding gene;
In the A5), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th site of the AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene, and inserting a DNA molecule containing a trimeric porin ProB coding gene between 25317-25318 th site of the AdC68XY3-empty virus;
in the A6), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th position of an AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene, a varicella zoster virus Orf9 protein coding gene and a varicella zoster virus Orf63 protein coding gene;
in the A7), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th position of an AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene and a varicella zoster virus gH protein coding gene;
in the A8), the recombinant virus is a recombinant adenovirus obtained by replacing a DNA molecule shown in 518-624 th position of an AdC68XY3-empty virus with a DNA molecule containing a varicella zoster virus gE protein coding gene and a varicella zoster virus gB protein coding gene.
7. Use of the recombinant virus of any one of claims 1-6 in any one of the following X1) -X5):
x1) as varicella zoster vaccine;
x2) as a varicella zoster virus drug;
x3) preparing varicella zoster vaccine;
x4) preparing a varicella zoster virus drug;
x5) preparing a product for preventing and/or treating varicella or zoster.
8. A product comprising the recombinant virus of any one of claims 1-5 as an active ingredient; the use of the product is as follows Z1) or Z2):
z1) varicella zoster virus;
z2) preventing and/or treating varicella or zoster.
9. The use according to claim 6 or the product according to claim 7, characterized in that: the product is a vaccine or a medicament.
10. The use or product according to claim 8, wherein:
the vaccine has an immunizing dose of (0.1-10) ×10 10 vp；
Or, the number of immunization times of the vaccine is one time or two or more times;
alternatively, the vaccine may be administered by intramuscular injection, subcutaneous injection or nasal spray.
CN202210480448.2A 2022-05-05 2022-05-05 Varicella zoster virus vaccine and application thereof Pending CN117165538A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210480448.2A CN117165538A (en) 2022-05-05 2022-05-05 Varicella zoster virus vaccine and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210480448.2A CN117165538A (en) 2022-05-05 2022-05-05 Varicella zoster virus vaccine and application thereof

Publications (1)

Publication Number Publication Date
CN117165538A true CN117165538A (en) 2023-12-05

Family

ID=88939955

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210480448.2A Pending CN117165538A (en) 2022-05-05 2022-05-05 Varicella zoster virus vaccine and application thereof

Country Status (1)

Country Link
CN (1) CN117165538A (en)

Similar Documents

Publication Publication Date Title
CN113185613B (en) Novel coronavirus S protein and subunit vaccine thereof
AU2019271972B2 (en) Adenovirus polynucleotides and polypeptides
BE1023916A9 (en) NEW ADENOVIRUS
RU2731342C1 (en) Pharmaceutical agent and method for use thereof for inducing specific immunity to virus of severe acute respiratory syndrome sars-cov-2 (embodiments)
DK2753364T3 (en) CONDITIONING REPLYING CYTOMEGALOVIRUS AS VACCINE AGAINST CMV
KR20220016137A (en) modified adenovirus
CA2568838A1 (en) Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with sars corona virus spike protein
KR20220152248A (en) Compositions and methods for inducing an immune response
KR20210013589A (en) Immune checkpoint inhibitor co-expression vector
AU2017305176A1 (en) Compositions and methods of replication deficient adenoviral vectors for vaccine applications
KR20200066349A (en) Replicable adenovirus vector
KR20210090650A (en) Alphavirus neoantigen vectors and interferon inhibitors
KR20220041844A (en) HIV antigen and MHC complex
CN115867349A (en) Vaccines against coronaviruses and methods of use
CN111148528A (en) Influenza vaccine
US20070190065A1 (en) Nucleic acids, polypeptides, methods of expression, and immunogenic compositions associated with SARS corona virus spike protein
CN117165538A (en) Varicella zoster virus vaccine and application thereof
CN114891830B (en) Recombinant expression vector based on varicella-zoster virus, recombinant virus and application thereof
Dolter et al. Incorporation of CD4 into virions by a recombinant herpes simplex virus
KR20230006825A (en) Infectious disease antigens and vaccines
KR20230008707A (en) Vaccine composition for treatment of coronavirus
US20210299243A1 (en) Il-10 inhibition for vaccines and immunotherapy
CN115287271B (en) Method for detecting neutralizing activity of SARS-CoV-2 antibody
RU2761904C1 (en) Drug application for induction of specific immunity against severe acute respiratory syndrome virus sars-cov-2 in children
WO2010009465A2 (en) Htlv-ii vector and methods of use

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination