CN105820257A - Human adenovirus antigen epitope chimeric protein as well as preparation and application thereof - Google Patents
Human adenovirus antigen epitope chimeric protein as well as preparation and application thereof Download PDFInfo
- Publication number
- CN105820257A CN105820257A CN201610269189.3A CN201610269189A CN105820257A CN 105820257 A CN105820257 A CN 105820257A CN 201610269189 A CN201610269189 A CN 201610269189A CN 105820257 A CN105820257 A CN 105820257A
- Authority
- CN
- China
- Prior art keywords
- amino acids
- adenovirus
- chimeric protein
- epitope
- protein
- 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.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/075—Adenoviridae
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Virology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Urology & Nephrology (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Hematology (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Gastroenterology & Hepatology (AREA)
- Plant Pathology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses human adenovirus antigen epitope chimeric protein as well as preparation and application thereof, and relates to fields of gene engineering techniques, vaccines and diagnostic reagents. The amino acid sequences of hexon protein of type 3, type 7, type 11, type 14 and type 55 of adenovirus are analyzed through computer analysis, protein fragments with good antigenicity can be screened respectively, the fragments are connected by using two glycine and one serine, then chimeric protein with multiple antigen fragments in serial connection can be formed, pronucleus preferred codons are selected and interpreted into corresponding nucleotide sequences, and full-length genes can be synthesized in a chemical manner. The chimeric protein can be obtained through expression purification by using a gene engineering technique, and the chimeric protein comprises 363 amino acids in whole length. The expressed chimeric protein can be applied to vaccine research, HAdV antibody or antigen detection and the like, and is related to fields such as gene engineering techniques, vaccines and diagnostic reagents.
Description
Technical field
The present inventor's Adenovirus Antigen epi-position chimeric protein and preparation thereof, application relate to technique for gene engineering, vaccine and diagnostic reagent field.The present invention is adenovirus hominis (HAdV) 3 type, 7 types, 11 types, 14 types and 55 type specific antigen neoepitope Western fragments to be connected, and utilizes technique for gene engineering, the preparation chimeric protein containing five kinds of type adenovirus strong antigen epi-positions.Analyzed by computer, from five kinds of type adenovirus hexons, filter out the protein fragments containing strong antigen epi-position, connect with two glycine and a serine between protein fragments, be connected into epitope chimeric protein more than.The gene order of this chimeric protein of chemosynthesis, and utilize technique for gene engineering to express this chimeric protein.The chimeric protein expressed can be used for the detection of HAdV antibody or antigen, and the development etc. of vaccine.
Background technology
Adenovirus hominis, i.e. HAdV(HumanAdenovirus), it is a kind of virus at the Dense crowd such as kindergarten, army with high hazardness.Since in December, 2011, successively there is a lot of epidemic that breaks out through respiratory infectious in China different regions, epidemic situation involves wide, and infectiousness is strong, identifies being respectively B group 55 type, 7 types and 14 type adenoviruss through laboratory Pathogen test.
Adenovirus mainly causes respiratory tract disease, but also can infect the positions such as digestive tract, urinary tract, eye, cardiac muscle and cause disease.It has been generally acknowledged that B1, C, E group adenovirus mainly causes respiratory tract disease, and B2 group mainly causes urinary system infection.The respiratory tract disease caused by adenovirus outbreak of epidemic in new recruit is repeatedly reported in the whole world.
Adenovirus is a kind of uncanned double-stranded DNA virus, belongs to Adenoviridae, full-length genome 34.7kb, and capsid is in 20 body structures of rule, diameter 80-100nm.Core is made up of distrand DNA and protein, has outward nucleocapsid, above has 252 granules, conjuncted is made up of 240 six adjacent bodies and 12 five.Epi-position on six adjacent bodies is the standard of difference different serotypes, is the viral position most sensitive to immunoselection pressure.
At present, the method for test in laboratory adenovirus infection typically has two kinds.One is that this method is more complicated by polymerase chain reaction specific detection adenoviral nucleic acid, is difficult to operation, and adenovirus typing is more difficult.Two is to utilize the specific IgM in enzyme-linked immunosorbent assay detection serum and IgG antibody.Generally, when virus infects, human immune system can excite humoral immunization and cell immune response and gradually control to infect, remove virus.When infection viremia occurs in early days, viral nucleic acid can be detected from patients serum and nose, pharyngeal secretion thing.Stronger immunoreation can be induced after adenovirus infection, produce specific antibody.After general morbidity 1 week, IgM in the patient started to produce, and within 7-10 days, IgG starts generation, gradually rises subsequently.Therefore may utilize the specific antibody in specific antigen detection serum.But current a kind of antigen is typically only capable to detect the adenovirus antibody of a kind of type, so needing to use multiple antigen to improve antibody detection rate when detecting HAdV antibody in serum, make a definite diagnosis infection type, improve testing cost, and still suffer from certain probability of false negative.Developing multiple epitopes recombinant antigen is the developing direction setting up adenovirus specific antibody quick detection reagent, is also the developing direction developed vaccine.
Summary of the invention
The present invention seeks to provide a kind of adenovirus hominis epitope chimeric protein for above-mentioned weak point, on the hexon of adenovirus hominis (HAdV) shell, there is multiple epi-position, it is the virion position most sensitive to immunoselection pressure, induction body produces many strain specific antibodieies, so this albumen is used as antigen and develops the ideal protein of adenovirus hominis (HAdV) antibody test reagent.
The aminoacid sequence of adenovirus hexon is analyzed by computer, filter out the protein fragments that five kinds of type adenoviruss contain strong antigen epi-position respectively, connect with two glycine and a serine between protein fragments, be connected into epitope chimeric protein more than.The gene order of this chimeric protein of chemosynthesis, and utilize technique for gene engineering to express this chimeric protein.The chimeric protein expressed can be used for the detection of HAdV antibody or antigen, and the development etc. of vaccine.
The present invention is the chemosynthesis adenovirus hominis (HAdV) gene order by the epitope chimeric protein of adenovirus type III, 7 types, 11 types, 14 types and 55 types, utilizes technique for gene engineering, the preparation chimeric protein containing five type Adenovirus Antigen epi-positions.Analyzed the aminoacid sequence of adenovirus hominis 3,7,11,14 and 55 type hexon by computer, filter out the protein fragments containing strong antigen epi-position respectively.Adenovirus type III filters out four protein fragments altogether, is the 136th～151 amino acids respectively, totally 16 aminoacid；164th～187 amino acids, totally 24 aminoacid；265th～284 amino acids, totally 20 aminoacid；422nd～437 amino acids, totally 16 aminoacid.Adenovirus type VII filters out four protein fragments altogether, is the 136th～148 amino acids respectively, totally 13 aminoacid；161st～181 amino acids, totally 21 aminoacid；258th～276 amino acids, totally 19 aminoacid；415th～426 amino acids, totally 12 aminoacid.Adenovirus type XI totally two protein fragments, are the 136th～160 amino acids respectively, totally 25 aminoacid；174th～186 amino acids, totally 13 aminoacid.Adenovirus 14 type totally four protein fragments, are the 136th～160 amino acids respectively, totally 25 aminoacid；174th～196 amino acids, totally 23 aminoacid；268th～286 amino acids, totally 19 aminoacid；415th～450 amino acids, totally 36 aminoacid.Adenovirus 55 type totally two protein fragments, are the 136th～158 amino acids respectively, totally 23 aminoacid；172nd～184 amino acids, totally 13 aminoacid.Connect with two glycine and a serine between protein fragments, be connected into epitope chimeric protein more than, 363 aminoacid of total length.Select the codon of prokaryote preference, chimeric protein is translated into corresponding nucleotide sequence, chemosynthesis full-length gene order.Utilize this chimeric protein of technique for gene engineering expression and purification.This chimeric protein can be used for vaccine and HAdV antibody or the detection of antigen, and prepares anti-HAdV monoclonal antibody and multi-resistance etc. for immunity.
Adenovirus hominis (HAdV) chimeric protein and preparation thereof, application take following steps to implement:
1. the screening of five type Adenovirus Antigen epi-positions and the chemosynthesis of chimeric protein genetic fragment:
Utilize software analysis adenovirus hominis 3 types such as ANTHEWIN, 7 types, 11 types, 14 types and the aminoacid sequence of 55 type hexons, filter out the protein fragments in each type adenovirus hexon with strong antigen respectively.Connect with two glycine and a serine between protein fragments, be connected into epitope chimeric protein more than.Select the codon of prokaryote preference, chimeric protein is translated into corresponding nucleotide sequence.5' end in chimeric protein genetic fragment adds NdeI restriction enzyme site (lower setting-out part), adds termination codon (TAA) and EcoRI restriction enzyme site (lower setting-out part) at 3' end.The genetic fragment making synthesis be prone to be cloned into plasmid pET-28a (+) in NdeI and EcoRI restriction enzyme site in.The full-length gene order of this chimeric protein of chemosynthesis.
The adenovirus type III epitope aminoacid sequence of screening:
136th～151 aa:
164th～187 aa:
265th～284 aa:
422nd～437 aa:
The adenovirus type VII epitope aminoacid sequence of screening:
136th～148 aa:
161st～181 aa:
258th～276 aa:
415th～426 aa:
The adenovirus type XI epitope aminoacid sequence of screening:
136th～160 aa:
174th～186 aa:
The adenovirus 14 type epitope aminoacid sequence of screening:
136th～160 aa:
174th～196 aa:
268th～286 aa:
415th～450 aa:
The adenovirus 55 type epitope aminoacid sequence of screening:
136th～158 aa:
172nd～184 aa:
Adenovirus type III epitope aminoacid sequence:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThrGlyGlySer
LysGluGlyLeuGlnIleGlyLysAspIleThrThrThrGluGlyGluGluLysPro
IleTyrAlaAspLysGlyGlySerAspGlyArgAspAlaValAlaGlyAlaLeuAla
ProGluIleValLeuTyrThrGluAsnGlyGlySerIleLysValLysThrAspAsp
ThrAsnGlyTrpGluLysAspAlaAsn
Adenovirus type VII epitope aminoacid sequence:
IleValThrThrGlyGluAspAsnAlaThrThrTyrThrGlyGlySerLysGluGly
LeuGluIleGlyLysAspIleThrAlaAspAsnLysProIleTyrAlaAspLysGly
GlySerAspGlyArgGluAlaAlaAspAlaPheSerProGluIleValLeuTyrThr
GluAsnGlyGlySerIleLysProArgAspThrAlaTrpGluLysAspThr
Adenovirus type XI epitope aminoacid sequence:
IleAlaGluGlyValLysAsnThrThrGlyGluGluHisValThrGluGluGluThr
AsnThrThrThrTyrThrGlyGlySerLysGluGlyLeuProValGlyLeuGluVal
SerAspGlu
Adenovirus 14 type epitope aminoacid sequence:
LeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAspGlyGluAsnAsp
GluLysAlaThrTyrThrGlyGlySerLysAspGlyLeuProIleGlyLeuGluVal
ProAlaGluGlyAspProLysProIleTyrAlaAsnLysGlyGlySerAspLeuArg
SerGlnLysGlnGlyLeuLysProLysIleValMetTyrAlaGluAsnGlyGlySer
IleGlyProArgThrAspSerTyrLysGluIleGlnLeuAsnGlyAspGlnAlaTrp
LysAspValAsnProAsnGlyIleSerGluLeuValLysGlyAsnProPhe
Adenovirus 55 type epitope aminoacid sequence:
IleAlaGluGlyValLysAsnGlyGluGluArgValThrGluGluGluAsnAsnThr
ThrThrTyrThrGlyGlySerLysGluGlyLeuProIleGlyLeuLysValSerAsp
Glu
Chimeric protein amino acid sequence:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThrGlyGlySer
LysGluGlyLeuGlnIleGlyLysAspIleThrThrThrGluGlyGluGluLysPro
IleTyrAlaAspLysGlyGlySerAspGlyArgAspAlaValAlaGlyAlaLeuAla
ProGluIleValLeuTyrThrGluAsnGlyGlySerIleLysValLysThrAspAsp
ThrAsnGlyTrpGluLysAspAlaAsnGlyGlySerIleValThrThrGlyGluAsp
AsnAlaThrThrTyrThrGlyGlySerLysGluGlyLeuGluIleGlyLysAspIle
ThrAlaAspAsnLysProIleTyrAlaAspLysGlyGlySerAspGlyArgGluAla
AlaAspAlaPheSerProGluIleValLeuTyrThrGluAsnGlyGlySerIleLys
ProArgAspThrAlaTrpGluLysAspThrGlyGlySerIleAlaGluGlyValLys
AsnThrThrGlyGluGluHisValThrGluGluGluThrAsnThrThrThrTyrThr
GlyGlySerLysGluGlyLeuProValGlyLeuGluValSerAspGluGlyGlySer
LeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAspGlyGluAsnAsp
GluLysAlaThrTyrThrGlyGlySerLysAspGlyLeuProIleGlyLeuGluVal
ProAlaGluGlyAspProLysProIleTyrAlaAsnLysGlyGlySerAspLeuArg
SerGlnLysGlnGlyLeuLysProLysIleValMetTyrAlaGluAsnGlyGlySer
IleGlyProArgThrAspSerTyrLysGluIleGlnLeuAsnGlyAspGlnAlaTrp
LysAspValAsnProAsnGlyIleSerGluLeuValLysGlyAsnProPheGlyGly
SerIleAlaGluGlyValLysAsnGlyGluGluArgValThrGluGluGluAsnAsn
ThrThrThrTyrThrGlyGlySerLysGluGlyLeuProIleGlyLeuLysValSer
AspGlu
The DNA sequence (1104bp) of the chimeric protein of chemosynthesis
2. the structure of expression HAdV epitope chimeric protein recombiant plasmid:
Extract plasmid pET-28a(+) (U.S.'s Novagen Products), with NdeI and EcoRI double digestion, reclaim the plasmid large fragment of enzyme action after electrophoresis, be dissolved in deionized water.By the HAdV epitope chimeric protein genetic fragment of NdeI and EcoRI double digestion chemosynthesis, after electrophoresis reclaims, it is dissolved in deionized water.Take DNA fragmentation after above-mentioned enzyme action, be inserted into carrier pET-28a(+) in NdeI and EcoRI site between, express containing the chimeric protein of five type Adenovirus Antigen epi-positions.
3. the screening of recombiant plasmid and qualification:
By recombinant plasmid transformed e. coli bl21 (DE3), coating, containing kanamycin (60 μ g/mL) LB flat board, puts 37 DEG C overnight.Next day, random picking converted bacterium colony and comparison bacterium (plasmid pET-28a transformed bacteria), extracts plasmid, verifies with NdeI and EcoRI double digestion, and the agarose gel electrophoresis result of 1.0% shows, cuts the genes of interest fragment of 1104bp.Meanwhile, the plasmid containing this genetic fragment carrying out DNA sequence analysis, sequence analysis confirms that recombiant plasmid contains the five type Adenovirus Antigen epitope gene fragments linked together, and sequence is the most correct:
ATTGTTACGACGAACGGTGATAATGCTGTTACGACGACGACGAATACGGGCGGCTCCAAAGAAGGTCTGCAAATCGGCAAAGACATCACCACGACCGAAGGCGAAGAAAAACCGATCTACGCAGATAAAGGCGGTAGTGATGGTCGCGATGCAGTTGCTGGTGCCCTGGCACCGGAAATTGTCCTGTATACCGAAAACGGCGGTAGCATCAAAGTTAAAACCGATGACACGAACGGCTGGGAAAAAGATGCGAATGGCGGTAGCATCGTCACGACCGGTGAAGACAACGCCACGACCTATACCGGCGGTTCTAAAGAAGGCCTGGAAATTGGTAAAGATATCACCGCAGACAATAAACCGATTTACGCTGATAAAGGCGGTAGCGACGGTCGTGAAGCGGCCGATGCGTTTTCTCCGGAAATTGTGCTGTATACCGAAAATGGCGGTTCAATCAAACCGCGCGATACCGCATGGGAAAAAGACACGGGCGGTTCGATTGCTGAAGGCGTGAAAAACACGACCGGTGAAGAACATGTTACCGAAGAAGAAACGAATACGACCACGTACACCGGCGGTTCTAAAGAAGGCTTACCGGTGGGTCTGGAAGTTAGTGATGAAGGCGGTTCCCTGGACAAAGGCGTTGAAACCACGGAAGAACGTCAGAACGAAGATGGTGAAAATGACGAAAAAGCCACCTATACGGGCGGTAGCAAAGATGGTCTGCCGATTGGCCTGGAAGTGCCGGCCGAAGGCGATCCGAAACCGATTTATGCCAACAAAGGCGGTAGCGATCTGCGTTCTCAGAAACAAGGTCTGAAACCGAAAATTGTTATGTATGCAGAAAACGGCGGTTCAATCGGCCCGCGCACCGATTCGTACAAAGAAATTCAGCTGAATGGTGATCAAGCTTGGAAAGACGTCAACCCGAATGGCATCAGTGAACTGGTGAAAGGTAACCCGTTCGGCGGTTCCATTGCCGAAGGCGTCAAAAATGGTGAAGAACGCGTGACCGAAGAAGAAAACAATACGACGACGTACACGGGCGGTAGCAAAGAAGGTCTGCCGATTGGTCTGAAAGTCTCCGATGAATAA
The expression of recombinant plasmid built contains the chimeric protein of five type Adenovirus Antigen epi-positions, 363 aminoacid of total length, it is adenovirus type III epitope protein fragment at its N end, it is sequentially connected in series 7 types, 11 types, 14 type epitope protein fragments, C end is adenovirus 55 type epitope protein fragment, each fragment length is followed successively by 85 aminoacid of 3 type length, 7 type length 74 aminoacid, 11 type length 41 aminoacid, 14 type length 112 aminoacid, 55 type length 39 aminoacid, are connected by two glycine and a serine between fragment, and its aminoacid sequence is as follows:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThrGlyGlySer
LysGluGlyLeuGlnIleGlyLysAspIleThrThrThrGluGlyGluGluLysPro
IleTyrAlaAspLysGlyGlySerAspGlyArgAspAlaValAlaGlyAlaLeuAla
ProGluIleValLeuTyrThrGluAsnGlyGlySerIleLysValLysThrAspAsp
ThrAsnGlyTrpGluLysAspAlaAsnGlyGlySerIleValThrThrGlyGluAsp
AsnAlaThrThrTyrThrGlyGlySerLysGluGlyLeuGluIleGlyLysAspIle
ThrAlaAspAsnLysProIleTyrAlaAspLysGlyGlySerAspGlyArgGluAla
AlaAspAlaPheSerProGluIleValLeuTyrThrGluAsnGlyGlySerIleLys
ProArgAspThrAlaTrpGluLysAspThrGlyGlySerIleAlaGluGlyValLys
AsnThrThrGlyGluGluHisValThrGluGluGluThrAsnThrThrThrTyrThr
GlyGlySerLysGluGlyLeuProValGlyLeuGluValSerAspGluGlyGlySer
LeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAspGlyGluAsnAsp
GluLysAlaThrTyrThrGlyGlySerLysAspGlyLeuProIleGlyLeuGluVal
ProAlaGluGlyAspProLysProIleTyrAlaAsnLysGlyGlySerAspLeuArg
SerGlnLysGlnGlyLeuLysProLysIleValMetTyrAlaGluAsnGlyGlySer
IleGlyProArgThrAspSerTyrLysGluIleGlnLeuAsnGlyAspGlnAlaTrp
LysAspValAsnProAsnGlyIleSerGluLeuValLysGlyAsnProPheGlyGly
SerIleAlaGluGlyValLysAsnGlyGluGluArgValThrGluGluGluAsnAsn
ThrThrThrTyrThrGlyGlySerLysGluGlyLeuProIleGlyLeuLysValSer
AspGlu
4. the Screening and Identification of expressed fusion protein engineering bacteria:
By the positive transformant containing recombiant plasmid, it is seeded in the test tube containing 3mLLB culture medium (containing kanamycin 60 μ g/mL), 37 DEG C of shaken cultivation 3h, add IPTG to final concentration 0.5mmol/L, continue shaken cultivation induction 6h, centrifugal thalline of collecting carries out SDS-PAGE detection, and recon expresses the HAdV chimeric protein that relative molecular weight is 55kD, and compares bacterium BL21 (DE3) without this protein band.
5. the purification of Adenovirus Antigen epi-position chimeric protein:
1) ultrasonic degradation of Adenovirus Antigen epi-position chimeric protein engineering bacteria
Centrifugal for the engineering bacteria of abduction delivering chimeric protein (8000rpm, 10min, 4 DEG C) is received bacterium, thalline is resuspended in the bacterial lysate (20mmol/LPBpH7.4,5% glycerol) of original fluid 1/10 volume, ice-bath ultrasonic breaks bacterium, centrifugal collection supernatant.
2) the amine sulfate fractional precipitation of Adenovirus Antigen epi-position chimeric protein
Accurately measure supernatant volume, in supernatant, add appropriate saturated amine sulfate solution, stirring while adding, make final concentration of the 25% of amine sulfate, put in ice bath overnight.Centrifugal collect supernatant, add appropriate solid sulfur acid amide powder, stirring while adding, make final concentration of the 50% of amine sulfate, put in ice bath overnight.Next day centrifugal collecting precipitation.Suspend with 200mL balance liquid (50mMTris-HCl, pH7.5) and precipitate, load in bag filter, to 1000mL balance liquid dialysed overnight, change liquid 4 times.Centrifugal collection supernatant, directly goes up DEAE-SeparoseFF anion column purification.
3) DEAE-SeparoseFF anion column purification
Balance DEAE-SeparoseFF anion column, the direct upper prop of supernatant then upper step dialysed, flow velocity about 100mL/h is rinsed with balance liquid (50MmTris-HCl, pH7.5).Fully wash post with balance liquid after end of the sample, the most successively with containing 100,200,300,400,500, the balance liquid eluted protein of 1000mmol/LNaCl, collect each eluting peak albumen, with 12%SDS-PAGE electrophoresis detection each peak albumen, determine which eluting peak is containing the Adenovirus Antigen epi-position chimeric protein expressed.300mmol/LNaCl eluted protein peak is Adenovirus Antigen epi-position chimeric protein.
6. the Adenovirus Antigen epi-position chimeric protein will expressed, prepares anti-HAdV monoclonal antibody and multi-resistance etc. for vaccine, HAdV antibody or the detection of antigen and for immunity.
7. the strong antigen protein fragments of five kinds of type adenovirus hexons is connected, carry out expressing, preparing with the form of fusion protein.
8. by gene recombination technology, utilize yeast cells, insect cell, mammalian cell and genetically modified animals and plants carry out recombinant expressed, prepare Adenovirus Antigen epi-position chimeric protein.
English abbreviation illustrates: HAdV (Humanadenovirus): adenovirus hominis;EDTA: tetramethylethylenediamine；IPTG: isopropylthiogalactoside；DTT: dithiothreitol, DTT；SDS: dodecyl sodium sulfonate is received；PAGE: polyacrylamide gel electrophoresis；PB: phosphate buffer；KD: kilodalton.
The present invention compared with prior art has the advantage that
The Adenovirus Antigen epi-position chimeric protein that we express, has a more advantages:
1. being expressed by five kinds of type Adenovirus Antigen neoepitope Western segment compositions, ratio uses single protein fragments more convenient, economical respectively.When making Detection of antigen anti-HAdV antibody with chimeric protein, it is no longer necessary to prepare these protein fragments respectively, it is not required that adjusting the use ratio of these antigens, therefore application is convenient and cost is relatively low.
The most presently used adenovirus vaccine is mainly inactivated vaccine and attenuated live vaccine, but adenovirus has carcinogenecity to animal, dangerous big, therefore develops the novel nucleocapsid Component Vaccines without DNA and seems extremely urgent.We utilize technique for gene engineering to express the preparation chimeric protein containing the adjacent isoantigen epi-position of multiple adenovirus six, lay the foundation for developing recombinant vaccine.The advantage that recombinant vaccine has safety, low cost.
3. the chimeric protein containing five type Adenovirus Antigen epi-positions expressed exists with soluble form, it is easy to purification, and need not renaturation process.
Accompanying drawing explanation
Below with reference to accompanying drawing, the invention will be further described:
Fig. 1 is the result that adenovirus type III hexon epitope is analyzed by molecular biology software.Result shows, contains 4 strong hydrophilic epitopes at adenovirus type III hexon N end: 136～151aa, 164～187aa, 265～284aa, 422～437aa, i.e. the position of arrows in figure.
Fig. 2 is the result that adenovirus type VII hexon epitope is analyzed by molecular biology software.Result shows, contains 4 strong hydrophilic epitopes at adenovirus type VII hexon N end: 136～148aa, 161～181aa, 258～276aa, 415～426aa, i.e. the position of arrows in figure.
Fig. 3 is the result that adenovirus type XI hexon epitope is analyzed by molecular biology software.Result shows, contains 2 strong hydrophilic epitopes at adenovirus type XI hexon N end: 136～160aa, 174～186aa, i.e. the position of arrows in figure.
Fig. 4 is the result that adenovirus 14 type hexon epitope is analyzed by molecular biology software.Result shows, contains 4 strong hydrophilic epitopes at adenovirus 14 type hexon N end: 136～160aa, 174～196aa, 268～286aa, 415～450aa, i.e. the position of arrows in figure.
Fig. 5 is the result that adenovirus 55 type hexon epitope is analyzed by molecular biology software.Result shows, contains 2 strong hydrophilic epitopes at adenovirus type III hexon N end: 136～158aa, 172～184aa, i.e. the position of arrows in figure.
Fig. 6 is the construction of recombinant plasmid flow chart expressing five kinds of type Adenovirus Antigen epi-position chimeric proteins.
Fig. 7 is the SDS-PAGE analysis result expressing Adenovirus Antigen epi-position chimeric protein recombinant bacterium.By in the recombinant plasmid transformed of structure to escherichia coli, after choosing single bacterium colony, screen the SDS-PAGE of superior strain.M: albumen marker；Cloudy: containing pET-28a(+) the negative control bacterium of plasmid；1-5: Adenovirus Antigen epi-position chimeric protein recombinant bacterium, five recons all express the chimeric protein that relative molecular weight is 55kDa, the i.e. position of arrows in figure.
Fig. 8 is to express Adenovirus Antigen epi-position chimeric protein SDS-PAGE analysis result after purification.M: albumen Marker；1: Adenovirus Antigen epi-position chimeric protein after purification, concentration is 0.5mg/mL, relative molecular weight 55kDa；2: concentration is the BSA of 0.5mg/mL.
Detailed description of the invention
The detailed description of embodiment of the present invention:
Analysis, gene chemical synthesis and the expression of adenovirus hexon epitope
Analyze adenovirus type III, 7 types, 11 types, 14 types and the aminoacid sequence of 55 type hexons by computer, filter out the protein fragments wherein with strong antigen respectively.Adenovirus type III filters out four protein fragments altogether, and adenovirus type VII filters out four protein fragments altogether, adenovirus type XI totally two protein fragments, adenovirus 14 type totally four protein fragments, adenovirus 55 type totally two protein fragments.Connect with two glycine and a serine between protein fragments, be connected into epitope chimeric protein more than.Select the codon of prokaryote preference, chimeric protein is translated into corresponding nucleotide sequence.The full-length gene order of this chimeric protein of chemosynthesis, and be cloned into plasmid pET-28a (+) in NdeI/EcoRI site, express Adenovirus Antigen epi-position chimeric protein.By recombinant plasmid transformed e. coli bl21 (DE3), screening obtains the engineering bacteria of high efficient expression Adenovirus Antigen epi-position chimeric protein, and the HAdV chimeric protein of expression accounts for the 60% of tropina total amount.
Materials and methods
1. strain and plasmid: Host Strains BL21 (DE3) and expression vector pET-28a (+) it is U.S.'s Novagen Products.
2. molecular biology reagents: restricted enzyme NdeI, EcoRI and T4DNA ligase is TaKaRa Products.Plasmid purification kit and the test kit reclaiming DNA fragmentation in agarose gel are TaKaRa Products.DTT and IPTG is TaKaRa Products.Other reagent is import or domestic analytical reagent.
3. the synthesis of genetic fragment: helped synthesis by Dalian TaKaRa company.
4. the enzyme action of gene clone method: DNA, connection, electrophoresis；The extraction of plasmid, conversion；The general molecular cloning method such as the SDS-PAGE analysis of albumen is carried out according to a conventional method.Other test kit by specification operates.
5.DNA sequence analysis: with TaKaRa company plasmid purification kit plasmid purification, with the full-automatic sequencer of DNA.
Result
The screening of 1.HAdV hexon epitope and the chemosynthesis of genetic fragment thereof:
Utilize the softwares such as ANTHEWIN, analyze adenovirus type III, 7 types, 11 types, 14 types and the aminoacid sequence of 55 type hexons by computer, find all have multiple fragment to contain stronger antigenic determinant.Adenovirus type III filters out four protein fragments (Fig. 1) altogether, is the 136th～151 aa, totally 16 aminoacid respectively；164th～187 amino acids, totally 24 aminoacid；265th～284 amino acids, totally 20 aminoacid；422nd～437 amino acids, totally 16 aminoacid.Adenovirus type VII filters out four protein fragments (Fig. 2) altogether, is the 136th～148 amino acids respectively, totally 13 aminoacid；161st～181 amino acids, totally 21 aminoacid；258th～276 amino acids, totally 19 aminoacid；415th～426 amino acids, totally 12 aminoacid.Adenovirus type XI totally two protein fragments (Fig. 3), are the 136th～160 amino acids respectively, totally 25 aminoacid；174th～186 amino acids, totally 13 aminoacid.Adenovirus 14 type totally four protein fragments (Fig. 4), are the 136th～160 amino acids respectively, totally 25 aminoacid；174th～196 amino acids, totally 23 aminoacid；268th～286 amino acids, totally 19 aminoacid；415th～450 amino acids, totally 36 aminoacid.Adenovirus 55 type totally two protein fragments (Fig. 5), are the 136th～158 amino acids respectively, totally 23 aminoacid；172nd～184 amino acids, totally 13 aminoacid.Connect with two glycine and a serine between protein fragments, be connected into epitope chimeric protein more than.Select the codon of prokaryote preference, chimeric protein is translated into corresponding nucleotide sequence.5' end in chimeric protein gene order adds NdeI restriction enzyme site, adds termination codon (TAA) and EcoRI restriction enzyme site at 3' end.The full-length gene order of this chimeric protein of chemosynthesis.
The adenovirus type III epitope aminoacid sequence of screening:
136th～151 aa:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThr
164th～187 aa:
LysGluGlyLeuGlnIleGlyLysAspIleThrThrThrGluGlyGluGluLysPro
IleTyrAlaAspLys
265th～284 aa:
AspGlyArgAspAlaValAlaGlyAlaLeuAlaProGluIleValLeuTyrThrGlu
Asn
422nd～437 aa:
IleLysValLysThrAspAspThrAsnGlyTrpGluLysAspAlaAsn
The adenovirus type VII epitope aminoacid sequence of screening:
136th～148 aa:
IleValThrThrGlyGluAspAsnAlaThrThrTyrThr
161st～181 aa:
LysGluGlyLeuGluIleGlyLysAspIleThrAlaAspAsnLysProIleTyrAlaAspLys
258th～276 aa:
AspGlyArgGluAlaAlaAspAlaPheSerProGluIleValLeuTyrThrGluAsn
415th～426 aa:
IleLysProArgAspThrAlaTrpGluLysAspThr
The adenovirus type XI epitope aminoacid sequence of screening:
136th～160 aa:
IleAlaGluGlyValLysAsnThrThrGlyGluGluHisValThrGluGluGluThr
AsnThrThrThrTyrThr
174th～186 aa:
LysGluGlyLeuProValGlyLeuGluValSerAspGlu
The adenovirus 14 type epitope aminoacid sequence of screening:
136th～160 aa:
LeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAspGlyGluAsnAsp
GluLysAlaThrTyrThr
174th～196 aa:
LysAspGlyLeuProIleGlyLeuGluValProAlaGluGlyAspProLysProIle
TyrAlaAsnLys
268th～286 aa:
AspLeuArgSerGlnLysGlnGlyLeuLysProLysIleValMetTyrAlaGluAsn
415th～450 aa:
IleGlyProArgThrAspSerTyrLysGluIleGlnLeuAsnGlyAspGlnAlaTrp
LysAspValAsnProAsnGlyIleSerGluLeuValLysGlyAsnProPhe
The adenovirus 55 type epitope aminoacid sequence of screening:
136th～158 aa:
IleAlaGluGlyValLysAsnGlyGluGluArgValThrGluGluGluAsnAsnThr
ThrThrTyrThr
172nd～184 aa:
LysGluGlyLeuProIleGlyLeuLysValSerAspGlu
Adenovirus type III epitope aminoacid sequence:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThrGlyGlySer
LysGluGlyLeuGlnIleGlyLysAspIleThrThrThrGluGlyGluGluLysPro
IleTyrAlaAspLysGlyGlySerAspGlyArgAspAlaValAlaGlyAlaLeuAla
ProGluIleValLeuTyrThrGluAsnGlyGlySerIleLysValLysThrAspAsp
ThrAsnGlyTrpGluLysAspAlaAsn
Adenovirus type VII epitope aminoacid sequence:
IleValThrThrGlyGluAspAsnAlaThrThrTyrThrGlyGlySerLysGluGly
LeuGluIleGlyLysAspIleThrAlaAspAsnLysProIleTyrAlaAspLysGly
GlySerAspGlyArgGluAlaAlaAspAlaPheSerProGluIleValLeuTyrThr
GluAsnGlyGlySerIleLysProArgAspThrAlaTrpGluLysAspThr
Adenovirus type XI epitope aminoacid sequence:
IleAlaGluGlyValLysAsnThrThrGlyGluGluHisValThrGluGluGluThr
AsnThrThrThrTyrThrGlyGlySerLysGluGlyLeuProValGlyLeuGluVal
SerAspGlu
Adenovirus 14 type epitope aminoacid sequence:
LeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAspGlyGluAsnAsp
GluLysAlaThrTyrThrGlyGlySerLysAspGlyLeuProIleGlyLeuGluVal
ProAlaGluGlyAspProLysProIleTyrAlaAsnLysGlyGlySerAspLeuArg
SerGlnLysGlnGlyLeuLysProLysIleValMetTyrAlaGluAsnGlyGlySer
IleGlyProArgThrAspSerTyrLysGluIleGlnLeuAsnGlyAspGlnAlaTrp
LysAspValAsnProAsnGlyIleSerGluLeuValLysGlyAsnProPhe
Adenovirus 55 type epitope aminoacid sequence:
IleAlaGluGlyValLysAsnGlyGluGluArgValThrGluGluGluAsnAsnThr
ThrThrTyrThrGlyGlySerLysGluGlyLeuProIleGlyLeuLysValSerAsp
Glu
Chimeric protein amino acid sequence:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThrGlyGlySer
LysGluGlyLeuGlnIleGlyLysAspIleThrThrThrGluGlyGluGluLysPro
IleTyrAlaAspLysGlyGlySerAspGlyArgAspAlaValAlaGlyAlaLeuAla
ProGluIleValLeuTyrThrGluAsnGlyGlySerIleLysValLysThrAspAsp
ThrAsnGlyTrpGluLysAspAlaAsnGlyGlySerIleValThrThrGlyGluAsp
AsnAlaThrThrTyrThrGlyGlySerLysGluGlyLeuGluIleGlyLysAspIle
ThrAlaAspAsnLysProIleTyrAlaAspLysGlyGlySerAspGlyArgGluAla
AlaAspAlaPheSerProGluIleValLeuTyrThrGluAsnGlyGlySerIleLys
ProArgAspThrAlaTrpGluLysAspThrGlyGlySerIleAlaGluGlyValLys
AsnThrThrGlyGluGluHisValThrGluGluGluThrAsnThrThrThrTyrThr
GlyGlySerLysGluGlyLeuProValGlyLeuGluValSerAspGluGlyGlySer
LeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAspGlyGluAsnAsp
GluLysAlaThrTyrThrGlyGlySerLysAspGlyLeuProIleGlyLeuGluVal
ProAlaGluGlyAspProLysProIleTyrAlaAsnLysGlyGlySerAspLeuArg
SerGlnLysGlnGlyLeuLysProLysIleValMetTyrAlaGluAsnGlyGlySer
IleGlyProArgThrAspSerTyrLysGluIleGlnLeuAsnGlyAspGlnAlaTrp
LysAspValAsnProAsnGlyIleSerGluLeuValLysGlyAsnProPheGlyGly
SerIleAlaGluGlyValLysAsnGlyGluGluArgValThrGluGluGluAsnAsn
ThrThrThrTyrThrGlyGlySerLysGluGlyLeuProIleGlyLeuLysValSer
AspGlu
The DNA sequence (1104bp) of the chimeric protein of chemosynthesis
The structure of 2.HAdV epitope chimeric protein recombiant plasmid:
Extract plasmid pET28a(+) (U.S.'s Novagen Products), with NdeI and EcoRI double digestion, reclaim the plasmid large fragment of enzyme action after electrophoresis, be dissolved in deionized water.By the HAdV epitope chimeric protein genetic fragment of NdeI and EcoRI double digestion chemosynthesis, after electrophoresis reclaims, it is dissolved in deionized water.Take DNA fragmentation after above-mentioned enzyme action, be inserted into carrier pET28a(+) in NdeI and EcoRI site between, express containing the chimeric protein of five kinds of type Adenovirus Antigen epi-positions.Build flow process and see Fig. 6.
3. the screening of recombiant plasmid and qualification:
The recombinant plasmid transformed e. coli bl21 (DE3) upper step connected, by converted product coating containing kanamycin (60 μ g/mL) LB flat board, puts 37 DEG C overnight.5 transformant bacterium colonies of random choose, were inoculated into respectively in the test tube containing 4mL LB liquid medium (containing kanamycin 60 μ g/mL), put 37 DEG C of shaken cultivation 6h, take bacterium solution 1mL next day, centrifugal receipts bacterium.Respectively with 50 μ L deionized water suspension thalline, boiling water boiling 5min, centrifugal (4 DEG C, 12000rpm) 5min, take supernatant (inside having plasmid) 1 μ L and be used as pcr template, the primer held by chimeric protein genetic fragment 5 ' (5 '-CATATGATTGTTACGACGAACGGTGATAAT-3 ') and 3 ' primer (the 5 '-GAATTCTTATT held
CATCGGAGACTTT-3 ') composition primer pair, the PCR amplification positive recombiant plasmid containing chimeric protein genetic fragment, the genetic fragment of long 1104bp should be amplified.PCR reaction density is: each 1 μ L of plasmid template 1 μ L, upstream and downstream primer, 10xBuffer5.0 μ L, 2.5mmol/LdNTP4.0 μ L, Taq DNA polymerase 0.5 μ L (2.5U), deionized water 37.5 μ L, cumulative volume 50 μ L.Amplification condition is: 94 DEG C 30 seconds, 60 DEG C 30 seconds, 72 DEG C 60 seconds, 30 circulations；Last 72 DEG C extend 7 minutes.Taking pcr amplification product 5 μ L, Agarose gel detection with 1.2% is as a result, 8 transformants all amplify the genes of interest fragment of 1104bp.Confirm, all contain the tandem gene of five kinds of type Adenovirus Antigen epitope gene fragments.
Extracting the plasmid of transformant, the tandem gene sequence of five kinds of type Adenovirus Antigen epitope gene fragments in mensuration plasmid, DNA sequence analysis confirms, recombiant plasmid contains five kinds of type Adenovirus Antigen epitope gene fragments, and sequence is the most correct:
The expression of recombinant plasmid built contains the chimeric protein of five kinds of type Adenovirus Antigen epi-positions, 363 aminoacid of total length, it is adenovirus type III epitope protein fragment at its N end, it is sequentially connected in series 7 types, 11 types, 14 type epitope protein fragments, C end is adenovirus 55 type epitope protein fragment, each fragment length is followed successively by 85 aminoacid of 3 type length, 7 type length 74 aminoacid, 11 type length 41 aminoacid, 14 type length 112 aminoacid, 55 type length 39 aminoacid, are connected by two glycine and a serine between fragment, and its aminoacid sequence is as follows:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThrGlyGlySer
LysGluGlyLeuGlnIleGlyLysAspIleThrThrThrGluGlyGluGluLysPro
IleTyrAlaAspLysGlyGlySerAspGlyArgAspAlaValAlaGlyAlaLeuAla
ProGluIleValLeuTyrThrGluAsnGlyGlySerIleLysValLysThrAspAsp
ThrAsnGlyTrpGluLysAspAlaAsnGlyGlySerIleValThrThrGlyGluAsp
AsnAlaThrThrTyrThrGlyGlySerLysGluGlyLeuGluIleGlyLysAspIle
ThrAlaAspAsnLysProIleTyrAlaAspLysGlyGlySerAspGlyArgGluAla
AlaAspAlaPheSerProGluIleValLeuTyrThrGluAsnGlyGlySerIleLys
ProArgAspThrAlaTrpGluLysAspThrGlyGlySerIleAlaGluGlyValLys
AsnThrThrGlyGluGluHisValThrGluGluGluThrAsnThrThrThrTyrThr
GlyGlySerLysGluGlyLeuProValGlyLeuGluValSerAspGluGlyGlySer
LeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAspGlyGluAsnAsp
GluLysAlaThrTyrThrGlyGlySerLysAspGlyLeuProIleGlyLeuGluVal
ProAlaGluGlyAspProLysProIleTyrAlaAsnLysGlyGlySerAspLeuArg
SerGlnLysGlnGlyLeuLysProLysIleValMetTyrAlaGluAsnGlyGlySer
IleGlyProArgThrAspSerTyrLysGluIleGlnLeuAsnGlyAspGlnAlaTrp
LysAspValAsnProAsnGlyIleSerGluLeuValLysGlyAsnProPheGlyGly
SerIleAlaGluGlyValLysAsnGlyGluGluArgValThrGluGluGluAsnAsn
ThrThrThrTyrThrGlyGlySerLysGluGlyLeuProIleGlyLeuLysValSer
AspGlu
4. the Screening and Identification of expressed fusion protein engineering bacteria:
Above-mentioned transformant is seeded in the test tube containing 3mLLB culture medium (containing kanamycin 60 μ g/mL), 37 DEG C of shaken cultivation 3h, add IPTG to final concentration 0.5mmol/L, continue shaken cultivation induction 6h, centrifugal thalline of collecting carries out SDS-PAGE detection, and 5 transformants express Adenovirus Antigen epi-position chimeric protein (Fig. 7) that relative molecular weight is 55kD.
The purification of Adenovirus Antigen epi-position chimeric protein
According to the aminoacid sequence of Adenovirus Antigen epi-position chimeric protein, analyze its physicochemical property, determine suitable purification process.Through computer analysis, the isoelectric point, IP of Adenovirus Antigen epi-position chimeric protein is pH4.365, and therefore we determine in the 50MmTris-HCl buffer that pH is 7.5, purify with DEAE-SeparoseFF anion column.Specifically comprise the following steps that
Material and method
1. main agents:
DEAE-SeparoseFF polyanionic gel is Pharmacia Products, and IPTG, DTT are TaTaRa Products.Other reagent is domestic or Import Analysis pure reagent.
2. the abduction delivering of Adenovirus Antigen epi-position chimeric protein engineering bacteria and ultrasonic degradation
From the LB flat board being inoculated with No. 1 engineering bacteria, choose single bacterium colony with toothpick, be inoculated in the conical flask containing 200mLLB fluid medium, add kanamycin to final concentration 60 μ g/mL, put overnight incubation in 37 DEG C of shaking tables.Next day, bacterium solution is inoculated into 4 respectively containing the conical flasks of 200mLLB fluid medium, every bottle graft kind bacterium solution 50mL, puts shaken cultivation 1 hour in 37 DEG C of shaking tables, then add IPTG to final concentration 0.1mmol/L, abduction delivering 4 hours.
Centrifugal for the 1000mL engineering bacteria of abduction delivering chimeric protein (8000rpm, 30min, 4 DEG C) is received bacterium, thalline is resuspended in the bacterial lysate (20mmol/LPBpH7.4,10mmol/LEDTA, 1mmol/LDTT, 5% glycerol) of 100mL, ice-bath ultrasonic breaks bacterium 10min, centrifugal (8000rpm, 30min, 4 DEG C) collects supernatant.
3. the amine sulfate fractional precipitation of Adenovirus Antigen epi-position chimeric protein
Accurately measure supernatant volume, in supernatant, add appropriate saturated amine sulfate solution, stirring while adding, make final concentration of the 25% of amine sulfate, put in ice bath overnight.Next day, centrifugal (12000rpm, 20min, 4 DEG C) collected supernatant, added appropriate solid sulfur acid amide powder, stirring while adding, made final concentration of the 50% of amine sulfate, put in ice bath overnight.Next day, centrifugal (12000rpm, 20min, 4 DEG C) collected precipitation.Suspend with 200mL balance liquid (50mMTris-HCl, pH7.5) and precipitate, load in bag filter, to 1000mL balance liquid dialysed overnight, change liquid 4 times.Centrifugal (12000rpm, 20min, 4 DEG C) collects supernatant, directly goes up DEAE-SeparoseFF anion column purification.
4.DEAE-SeparoseFF anion column purification
Balance DEAE-SeparoseFF anion column, the direct upper prop of supernatant then upper step dialysed, loading flow velocity 100mL/h is rinsed with balance liquid (50mMTris-HCl, pH7.5).Fully wash post with balance liquid after end of the sample, the most successively with containing 100,200,300,400,500, the eluant solution albumen of 1000mmol/LNaCl, collect each eluting peak albumen, with 12%SDS-PAGE electrophoresis detection each peak albumen, determine which eluting peak is containing the Adenovirus Antigen epi-position chimeric protein expressed.
Result
Variable concentrations NaCl albumen of eluting from DEAE-SeparoseFF post is carried out SDS-PAGE analysis, and from electrophoresis result, Adenovirus Antigen epi-position chimeric protein is present in 300mmol/LNaCl eluted protein peak.
By 300mmol/LNaCl eluted protein peak by dialysis concentration, SDS-PAGE measures chimeric protein concentration, it is known that the chimeric protein concentration of purification gained is 0.5mg/mL(Fig. 8).
The qualification of Adenovirus Antigen epi-position chimeric protein and application
The Adenovirus Antigen epi-position chimeric protein of purification is used as immunogen, immunity BALB/c mouse, prepares antiserum.By indirect ELISA experimental technique, five kinds of type Adenovirus Antigen neoepitope Western are utilized chimeric protein antiserum to be detected, to identify the immunogenicity of Adenovirus Antigen epi-position chimeric protein.Experimental result shows, this chimeric protein has good immunogenicity, can be used as the research of adenovirus vaccine.
Material and method
1. adenovirus type III, 7 types, 11 types, 14 types and the epitope protein of 55 types: preserved by the preparation of this laboratory.
2. integrated enzyme reaction material: elisa plate is that 96 orifice plates are produced in Shenzhen, the sheep anti-mouse igg of horseradish peroxidase (HRP) labelling is purchased from Sigma company.Other material is the conventional material of integrated enzyme reaction.
3. prepared by chimeric protein antiserum: after chimeric protein and Freund's complete adjuvant equal-volume are sufficiently mixed emulsifying, lumbar injection immune mouse.Afterwards every two weeks, after chimeric protein and incomplete Freund's adjuvant equal-volume mixing and emulsifying, carry out booster immunization.Immunity 4 times altogether.The 4th immunity two weeks after, eye socket takes blood, prepares antiserum.
4. chimeric protein immunogenicity detection: use indirect elisa method that chimeric protein immunogenicity is detected.Basic step is: dilutes five kinds of type Adenovirus Antigen neoepitope Western extremely final concentration of 2 μ g/mL respectively with CBS buffer (pH9.6), is coated 96 hole ELISA Plate, every hole 100 μ L, 4 DEG C of overnight incubation；Fully washing plate 5 times with 1 ‰ PBST afterwards, the calf serum (FBS) of 10% is as confining liquid, and every hole adds 150 μ L, closes 2h for 37 DEG C；1 ‰ PBST fully wash plate 5 times, chimeric protein antiserum is in following ratio gradient dilution (diluent is the calf serum of 10%) 1:10000,1:20000,1:40000,1:80000,1:160000,1:320000,1:640000,1:1280000,100 μ L/ holes, hatch 2h for 37 DEG C, and negative serum presses 1:10000 dilution as comparison；1 ‰ PBST fully wash plate 5 times；Add the sheep anti-mouse igg of 1:5000 dilution (diluent is the calf serum of 10%), 100 μ L/ holes, hatch 1h for 37 DEG C；1 ‰ PBST fully wash plate 5 times, add TMB nitrite ion, 50 μ L/ holes, and 37 DEG C, lucifuge reacts 10min；Add the HCl of 1mol/L, 50 μ L/ holes, terminate reaction；Microplate reader measures each hole A450Value, if P/N > 2.1, can the result of determination positive.
Result
1. indirect elisa method detection Adenovirus Antigen epi-position chimeric protein immunogenicity
The adenovirus type III of purification, 7 types, 11 types, 14 types and 55 type epitope proteins CBS buffer is diluted coated elisa plate, the antiserum that detection Adenovirus Antigen epi-position chimeric protein immune mouse obtains.Result shows, adenovirus type III Protein Detection chimeric protein serum antibody titer is 1:320000；Adenovirus type VII Protein Detection chimeric protein serum antibody titer is 1:640000；Adenovirus type XI Protein Detection chimeric protein serum antibody titer is 1:640000；Adenovirus 14 type Protein Detection chimeric protein serum antibody titer is 1:1280000；Adenovirus 55 type Protein Detection chimeric protein serum antibody titer is 1:640000.After Adenovirus Antigen epi-position chimeric protein immunity body is described, it is possible to effective stimulation body produces the antibody for each type Adenovirus Antigen epi-position, has preferable immunogenicity, lays the foundation for developing adenovirus vaccine further.
Adenovirus hominis (HAdV) epitope chimeric protein sequence table sees appendix document: nucleotide or the readable carrier of aminoacid sequence list machine.
Adenovirus hominis (HAdV) epitope chimeric protein sequence table
<110>Li Yuexi
<120>adenovirus hominis epitope chimeric protein and preparation, application
<160>2
<210>1
<211>363
<212>PRT
<213>adenovirus hominis
<220>
<223>containing polytype adenovirus hominis epitope.
<400>1
<210>2
<211>1092
<212>DNA
<213>artificial sequence
<220>
<221>CDS
<222>(1)...(1092)
<223>genetic fragment of synthetic, the coding chimeric protein containing Adenovirus Antigen epi-position.
<220>
<221>mis-feature
<222>(1090)...(1092)
<223>termination codon increased during synthetic gene.
<400>2
Claims (11)
1. an adenovirus hominis epitope chimeric protein, it is formed by connecting by adenovirus type III, 7 types, 11 types, 14 types and 55 type epitope protein fragments, connecting with glycine and serine between protein fragments, 363 aminoacid of chimeric protein total length, aminoacid sequence is as follows:
。
Adenovirus hominis epitope chimeric protein the most according to claim 1, it is characterised in that: its gene order is as follows:
ATTGTTACGACGAACGGTGATAATGCTGTTACGACGACGACGAATACG
GGCGGCTCCAAAGAAGGTCTGCAAATCGGCAAAGACATCACCACGACC
GAAGGCGAAGAAAAACCGATCTACGCAGATAAAGGCGGTAGTGATGGT
CGCGATGCAGTTGCTGGTGCCCTGGCACCGGAAATTGTCCTGTATACC
GAAAACGGCGGTAGCATCAAAGTTAAAACCGATGACACGAACGGCTGG
GAAAAAGATGCGAATGGCGGTAGCATCGTCACGACCGGTGAAGACAAC
GCCACGACCTATACCGGCGGTTCTAAAGAAGGCCTGGAAATTGGTAAA
GATATCACCGCAGACAATAAACCGATTTACGCTGATAAAGGCGGTAGC
GACGGTCGTGAAGCGGCCGATGCGTTTTCTCCGGAAATTGTGCTGTAT
ACCGAAAATGGCGGTTCAATCAAACCGCGCGATACCGCATGGGAAAAA
GACACGGGCGGTTCGATTGCTGAAGGCGTGAAAAACACGACCGGTGAA
GAACATGTTACCGAAGAAGAAACGAATACGACCACGTACACCGGCGGT
TCTAAAGAAGGCTTACCGGTGGGTCTGGAAGTTAGTGATGAAGGCGGT
TCCCTGGACAAAGGCGTTGAAACCACGGAAGAACGTCAGAACGAAGAT
GGTGAAAATGACGAAAAAGCCACCTATACGGGCGGTAGCAAAGATGGT
CTGCCGATTGGCCTGGAAGTGCCGGCCGAAGGCGATCCGAAACCGATT
TATGCCAACAAAGGCGGTAGCGATCTGCGTTCTCAGAAACAAGGTCTG
AAACCGAAAATTGTTATGTATGCAGAAAACGGCGGTTCAATCGGCCCG
CGCACCGATTCGTACAAAGAAATTCAGCTGAATGGTGATCAAGCTTGG
AAAGACGTCAACCCGAATGGCATCAGTGAACTGGTGAAAGGTAACCCG
TTCGGCGGTTCCATTGCCGAAGGCGTCAAAAATGGTGAAGAACGCGTG
ACCGAAGAAGAAAACAATACGACGACGTACACGGGCGGTAGCAAAGAA
GGTCTGCCGATTGGTCTGAAAGTCTCCGATGAA。
Adenovirus hominis epitope chimeric protein the most according to claim 1, it is characterized in that: derive from the epitope protein fragment totally 4 of adenovirus type III, specially the 136th amino acids is to the 151st amino acids, the 164th amino acids to the 187th amino acids, the 265th amino acids to the 284th amino acids, the 422nd amino acids to the 437th amino acids, and each protein fragments aminoacid sequence is as follows:
136th～151 aa:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThr
164th～187 aa:
LysGluGlyLeuGlnIleGlyLysAspIleThrThrThrGluGlyGlu
GluLysProIleTyrAlaAspLys
265th～284 aa:
AspGlyArgAspAlaValAlaGlyAlaLeuAlaProGluIleValLeu
TyrThrGluAsn
422nd～437 aa:
IleLysValLysThrAspAspThrAsnGlyTrpGluLysAspAlaAsn
Connecting with glycine and serine between 4 protein fragments, connect into new recombiant protein fragment, aminoacid sequence is as follows:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThr
GlyGlySerLysGluGlyLeuGlnIleGlyLysAspIleThrThrThr
GluGlyGluGluLysProIleTyrAlaAspLysGlyGlySerAspGly
ArgAspAlaValAlaGlyAlaLeuAlaProGluIleValLeuTyrThr
GluAsnGlyGlySerIleLysValLysThrAspAspThrAsnGlyTrp
GluLysAspAlaAsn。
Adenovirus hominis epitope chimeric protein the most according to claim 1, it is characterized in that: derive from the epitope protein fragment totally 4 of adenovirus type VII, specially the 136th amino acids is to the 148th amino acids, the 161st amino acids to the 181st amino acids, the 258th amino acids to the 276th amino acids, the 415th amino acids to the 426th amino acids, and each protein fragments aminoacid sequence is as follows:
136th～148 aa:
IleValThrThrGlyGluAspAsnAlaThrThrTyrThr
161st～181 aa:
LysGluGlyLeuGluIleGlyLysAspIleThrAlaAspAsnLysPro
IleTyrAlaAspLys
258th～276 aa:
AspGlyArgGluAlaAlaAspAlaPheSerProGluIleValLeuTyr
ThrGluAsn
415th～426 aa:
IleLysProArgAspThrAlaTrpGluLysAspThr
Connecting with glycine and serine between 4 protein fragments, connect into new recombiant protein fragment, aminoacid sequence is as follows:
IleValThrThrGlyGluAspAsnAlaThrThrTyrThrGlyGlySer
LysGluGlyLeuGluIleGlyLysAspIleThrAlaAspAsnLysPro
IleTyrAlaAspLysGlyGlySerAspGlyArgGluAlaAlaAspAla
PheSerProGluIleValLeuTyrThrGluAsnGlyGlySerIleLys
ProArgAspThrAlaTrpGluLysAspThr。
Adenovirus hominis epitope chimeric protein the most according to claim 1, derive from the epitope protein fragment totally 2 sections of adenovirus type XI, specially the 136th amino acids is to the 160th amino acids, the 174th amino acids to the 186th amino acids, and each protein fragments aminoacid sequence is as follows:
136th～160 aa:
IleAlaGluGlyValLysAsnThrThrGlyGluGluHisValThrGlu
GluGluThrAsnThrThrThrTyrThr
174th～186 aa:
LysGluGlyLeuProValGlyLeuGluValSerAspGlu
Connecting with glycine and serine between 2 protein fragments, connect into new recombiant protein fragment, aminoacid sequence is as follows:
IleAlaGluGlyValLysAsnThrThrGlyGluGluHisValThrGlu
GluGluThrAsnThrThrThrTyrThrGlyGlySerLysGluGlyLeu
ProValGlyLeuGluValSerAspGlu。
6. the adenovirus hominis epitope chimeric protein described in claim 1, it is characterized in that: derive from the epitope protein fragment totally 4 of adenovirus 14 type, specially the 136th amino acids is to the 160th amino acids, the 174th amino acids to the 196th amino acids, the 268th amino acids to the 286th amino acids, the 415th amino acids to the 450th amino acids, and each protein fragments aminoacid sequence is as follows:
136th～160 aa:
LeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAspGly
GluAsnAspGluLysAlaThrTyrThr
174th～196 aa:
LysAspGlyLeuProIleGlyLeuGluValProAlaGluGlyAspPro
LysProIleTyrAlaAsnLys
268th～286 aa:
AspLeuArgSerGlnLysGlnGlyLeuLysProLysIleValMetTyr
AlaGluAsn
415th～450 aa:
IleGlyProArgThrAspSerTyrLysGluIleGlnLeuAsnGlyAsp
GlnAlaTrpLysAspValAsnProAsnGlyIleSerGluLeuValLys
GlyAsnProPhe
Connecting with glycine and serine between 4 protein fragments, connect into new recombiant protein fragment, aminoacid sequence is as follows:
LeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAspGly
GluAsnAspGluLysAlaThrTyrThrGlyGlySerLysAspGlyLeu
ProIleGlyLeuGluValProAlaGluGlyAspProLysProIleTyr
AlaAsnLysGlyGlySerAspLeuArgSerGlnLysGlnGlyLeuLys
ProLysIleValMetTyrAlaGluAsnGlyGlySerIleGlyProArg
ThrAspSerTyrLysGluIleGlnLeuAsnGlyAspGlnAlaTrpLys
AspValAsnProAsnGlyIleSerGluLeuValLysGlyAsnProPhe。
Adenovirus hominis epitope chimeric protein the most according to claim 1, it is characterized in that: derive from the epitope protein fragment totally 2 of adenovirus 55 type, specially the 136th amino acids is to the 158th amino acids, the 172nd amino acids to the 184th amino acids, and each protein fragments aminoacid sequence is as follows:
136th～158 aa:
IleAlaGluGlyValLysAsnGlyGluGluArgValThrGluGluGlu
AsnAsnThrThrThrTyrThr
172nd～184 aa:
LysGluGlyLeuProIleGlyLeuLysValSerAspGlu
Connecting with glycine and serine between 2 protein fragments, connect into new recombiant protein fragment, aminoacid sequence is as follows:
IleAlaGluGlyValLysAsnGlyGluGluArgValThrGluGluGlu
AsnAsnThrThrThrTyrThrGlyGlySerLysGluGlyLeuProIle
GlyLeuLysValSerAspGlu。
8. the adenovirus hominis epitope chimeric protein described in claim 1, utilizes technique for gene engineering to express, prepare this albumen, and concrete grammar is as follows:
The screening of Adenovirus Antigen neoepitope Western fragment:
Utilize the softwares such as ANTHEWIN, adenovirus type III, 7 types, 11 types, 14 types and the aminoacid sequence of 55 type hexons is analyzed by computer, find all have multiple fragment to contain stronger antigenic determinant, adenovirus type III filters out four protein fragments altogether, be respectively the 136th amino acids to the 151st amino acids, totally 16 aminoacid；164th amino acids to the 187th amino acids, totally 24 aminoacid；265th amino acids to the 284th amino acids, totally 20 aminoacid；422nd amino acids to the 437th amino acids, totally 16 aminoacid, adenovirus type VII filters out four protein fragments altogether, be respectively the 136th amino acids to the 148th amino acids, totally 13 aminoacid；161st amino acids to the 181st amino acids, totally 21 aminoacid；258th amino acids to the 276th amino acids, totally 19 aminoacid；415th amino acids to the 426th amino acids, totally 12 aminoacid, adenovirus type XI totally two protein fragments, be respectively the 136th amino acids to the 160th amino acids, totally 25 aminoacid；174th amino acids to the 186th amino acids, totally 13 aminoacid, adenovirus 14 type totally four protein fragments, be respectively the 136th amino acids to the 160th amino acids, totally 25 aminoacid；174th amino acids to the 196th amino acids, totally 23 aminoacid；268th amino acids to the 286th amino acids, totally 19 aminoacid；415th amino acids to the 450th amino acids, totally 36 aminoacid, adenovirus 55 type totally two protein fragments, be respectively the 136th amino acids to the 158th amino acids, totally 23 aminoacid；172nd amino acids is to the 184th amino acids, totally 13 aminoacid, connect with two glycine and a serine between protein fragments, it is connected into epitope chimeric protein more than, selecting the codon of prokaryote preference, chimeric protein is translated into corresponding nucleotide sequence, the 5' end in chimeric protein gene order adds NdeI restriction enzyme site, termination codon (TAA) and EcoRI restriction enzyme site, the full-length gene order of this chimeric protein of chemosynthesis is added at 3' end；
The adenovirus type III epitope aminoacid sequence of screening:
136th～151 aa:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThr
164th～187 aa:
LysGluGlyLeuGlnIleGlyLysAspIleThrThrThrGluGlyGlu
GluLysProIleTyrAlaAspLys
265th～284 aa:
AspGlyArgAspAlaValAlaGlyAlaLeuAlaProGluIleValLeu
TyrThrGluAsn
422nd～437 aa:
IleLysValLysThrAspAspThrAsnGlyTrpGluLysAspAlaAsn
The adenovirus type VII epitope aminoacid sequence of screening:
136th～148 aa:
IleValThrThrGlyGluAspAsnAlaThrThrTyrThr
161st～181 aa:
LysGluGlyLeuGluIleGlyLysAspIleThrAlaAspAsnLysPro
IleTyrAlaAspLys
258th～276 aa:
AspGlyArgGluAlaAlaAspAlaPheSerProGluIleValLeuTyr
ThrGluAsn
415th～426 aa:
IleLysProArgAspThrAlaTrpGluLysAspThr
The adenovirus type XI epitope aminoacid sequence of screening:
136th～160 aa:
IleAlaGluGlyValLysAsnThrThrGlyGluGluHisValThrGlu
GluGluThrAsnThrThrThrTyrThr
174th～186 aa:
LysGluGlyLeuProValGlyLeuGluValSerAspGlu
The adenovirus 14 type epitope aminoacid sequence of screening:
136th～160 aa:
LeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAspGly
GluAsnAspGluLysAlaThrTyrThr
174th～196 aa:
LysAspGlyLeuProIleGlyLeuGluValProAlaGluGlyAspPro
LysProIleTyrAlaAsnLys
268th～286 aa:
AspLeuArgSerGlnLysGlnGlyLeuLysProLysIleValMetTyr
AlaGluAsn
415th～450 aa:
IleGlyProArgThrAspSerTyrLysGluIleGlnLeuAsnGlyAsp
GlnAlaTrpLysAspValAsnProAsnGlyIleSerGluLeuValLys
GlyAsnProPhe
The adenovirus 55 type epitope aminoacid sequence of screening:
136th～158 aa:
IleAlaGluGlyValLysAsnGlyGluGluArgValThrGluGluGlu
AsnAsnThrThrThrTyrThr
172nd～184 aa:
LysGluGlyLeuProIleGlyLeuLysValSerAspGlu
Adenovirus type III epitope aminoacid sequence:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThr
GlyGlySerLysGluGlyLeuGlnIleGlyLysAspIleThrThrThr
GluGlyGluGluLysProIleTyrAlaAspLysGlyGlySerAspGly
ArgAspAlaValAlaGlyAlaLeuAlaProGluIleValLeuTyrThr
GluAsnGlyGlySerIleLysValLysThrAspAspThrAsnGlyTrp
GluLysAspAlaAsn
Adenovirus type VII epitope aminoacid sequence:
IleValThrThrGlyGluAspAsnAlaThrThrTyrThrGlyGlySer
LysGluGlyLeuGluIleGlyLysAspIleThrAlaAspAsnLysPro
IleTyrAlaAspLysGlyGlySerAspGlyArgGluAlaAlaAspAla
PheSerProGluIleValLeuTyrThrGluAsnGlyGlySerIleLys
ProArgAspThrAlaTrpGluLysAspThr
Adenovirus type XI epitope aminoacid sequence:
IleAlaGluGlyValLysAsnThrThrGlyGluGluHisValThrGlu
GluGluThrAsnThrThrThrTyrThrGlyGlySerLysGluGlyLeu
ProValGlyLeuGluValSerAspGlu
Adenovirus 14 type epitope aminoacid sequence:
LeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAspGly
GluAsnAspGluLysAlaThrTyrThrGlyGlySerLysAspGlyLeu
ProIleGlyLeuGluValProAlaGluGlyAspProLysProIleTyr
AlaAsnLysGlyGlySerAspLeuArgSerGlnLysGlnGlyLeuLys
ProLysIleValMetTyrAlaGluAsnGlyGlySerIleGlyProArg
ThrAspSerTyrLysGluIleGlnLeuAsnGlyAspGlnAlaTrpLys
AspValAsnProAsnGlyIleSerGluLeuValLysGlyAsnProPhe
Adenovirus 55 type epitope aminoacid sequence:
IleAlaGluGlyValLysAsnGlyGluGluArgValThrGluGluGlu
AsnAsnThrThrThrTyrThrGlyGlySerLysGluGlyLeuProIle
GlyLeuLysValSerAspGlu
Chimeric protein amino acid sequence:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThr
GlyGlySerLysGluGlyLeuGlnIleGlyLysAspIleThrThrThr
GluGlyGluGluLysProIleTyrAlaAspLysGlyGlySerAspGly
ArgAspAlaValAlaGlyAlaLeuAlaProGluIleValLeuTyrThr
GluAsnGlyGlySerIleLysValLysThrAspAspThrAsnGlyTrp
GluLysAspAlaAsnGlyGlySerIleValThrThrGlyGluAspAsn
AlaThrThrTyrThrGlyGlySerLysGluGlyLeuGluIleGlyLys
AspIleThrAlaAspAsnLysProIleTyrAlaAspLysGlyGlySer
AspGlyArgGluAlaAlaAspAlaPheSerProGluIleValLeuTyr
ThrGluAsnGlyGlySerIleLysProArgAspThrAlaTrpGluLys
AspThrGlyGlySerIleAlaGluGlyValLysAsnThrThrGlyGlu
GluHisValThrGluGluGluThrAsnThrThrThrTyrThrGlyGly
SerLysGluGlyLeuProValGlyLeuGluValSerAspGluGlyGly
SerLeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAsp
GlyGluAsnAspGluLysAlaThrTyrThrGlyGlySerLysAspGly
LeuProIleGlyLeuGluValProAlaGluGlyAspProLysProIle
TyrAlaAsnLysGlyGlySerAspLeuArgSerGlnLysGlnGlyLeu
LysProLysIleValMetTyrAlaGluAsnGlyGlySerIleGlyPro
ArgThrAspSerTyrLysGluIleGlnLeuAsnGlyAspGlnAlaTrp
LysAspValAsnProAsnGlyIleSerGluLeuValLysGlyAsnPro
PheGlyGlySerIleAlaGluGlyValLysAsnGlyGluGluArgVal
ThrGluGluGluAsnAsnThrThrThrTyrThrGlyGlySerLysGlu
GlyLeuProIleGlyLeuLysValSerAspGlu
The DNA sequence (1104bp) of chemosynthesis chimeric protein
The structure of HAdV epitope chimeric protein recombiant plasmid:
Extract plasmid pET-28a(+) (U.S.'s Novagen Products), with NdeI and EcoRI double digestion, the plasmid large fragment of enzyme action is reclaimed after electrophoresis, it is dissolved in deionized water, by the HAdV epitope chimeric protein genetic fragment of NdeI and EcoRI double digestion chemosynthesis, after electrophoresis reclaims, it is dissolved in deionized water, take DNA fragmentation after above-mentioned enzyme action, be inserted into carrier pET28a(+) in NdeI and EcoRI site between, express containing the chimeric protein of five kinds of type Adenovirus Antigen epi-positions；
The screening of recombiant plasmid and qualification:
By recombinant plasmid transformed e. coli bl21 (DE3), coating, containing kanamycin (60 μ g/mL) LB flat board, puts 37 DEG C overnight；Next day, random picking converted bacterium colony and comparison bacterium (plasmid pET28a transformed bacteria), extracts plasmid, verifies with NdeI and EcoRI double digestion, and the agarose gel electrophoresis result of 1.0% shows, cuts the genes of interest fragment of 1104bp；Meanwhile, the plasmid containing this genetic fragment carrying out DNA sequence analysis, sequence analysis confirms that recombiant plasmid contains the five kinds of type Adenovirus Antigen epitope gene fragments linked together, and sequence is the most correct:
ATTGTTACGACGAACGGTGATAATGCTGTTACGACGACGACGAATACGGGCGGCTCCAAAGAAGGTCTGCAAATCGGCAAAGACATCACCACGACCGAAGGCGAAGAAAAACCGATCTACGCAGATAAAGGCGGTAGTGATGGTCGCGATGCAGTTGCTGGTGCCCTGGCACCGGAAATTGTCCTGTATACCGAAAACGGCGGTAGCATCAAAGTTAAAACCGATGACACGAACGGCTGGGAAAAAGATGCGAATGGCGGTAGCATCGTCACGACCGGTGAAGACAACGCCACGACCTATACCGGCGGTTCTAAAGAAGGCCTGGAAATTGGTAAAGATATCACCGCAGACAATAAACCGATTTACGCTGATAAAGGCGGTAGCGACGGTCGTGAAGCGGCCGATGCGTTTTCTCCGGAAATTGTGCTGTATACCGAAAATGGCGGTTCAATCAAACCGCGCGATACCGCATGGGAAAAAGACACGGGCGGTTCGATTGCTGAAGGCGTGAAAAACACGACCGGTGAAGAACATGTTACCGAAGAAGAAACGAATACGACCACGTACACCGGCGGTTCTAAAGAAGGCTTACCGGTGGGTCTGGAAGTTAGTGATGAAGGCGGTTCCCTGGACAAAGGCGTTGAAACCACGGAAGAACGTCAGAACGAAGATGGTGAAAATGACGAAAAAGCCACCTATACGGGCGGTAGCAAAGATGGTCTGCCGATTGGCCTGGAAGTGCCGGCCGAAGGCGATCCGAAACCGATTTATGCCAACAAAGGCGGTAGCGATCTGCGTTCTCAGAAACAAGGTCTGAAACCGAAAATTGTTATGTATGCAGAAAACGGCGGTTCAATCGGCCCGCGCACCGATTCGTACAAAGAAATTCAGCTGAATGGTGATCAAGCTTGGAAAGACGTCAACCCGAATGGCATCAGTGAACTGGTGAAAGGTAACCCGTTCGGCGGTTCCATTGCCGAAGGCGTCAAAAATGGTGAAGAACGCGTGACCGAAGAAGAAAACAATACGACGACGTACACGGGCGGTAGCAAAGAAGGTCTGCCGATTGGTCTGAAAGTCTCCGATGAATAA
The expression of recombinant plasmid built contains the chimeric protein of five kinds of type Adenovirus Antigen epi-positions, 363 aminoacid of total length, it is adenovirus type III epitope protein fragment at its N end, it is sequentially connected in series 7 types, 11 types, 14 type epitope protein fragments, C end is adenovirus 55 type epitope protein fragment, each fragment length is followed successively by 85 aminoacid of 3 type length, 7 type length 74 aminoacid, 11 type length 41 aminoacid, 14 type length 112 aminoacid, 55 type length 39 aminoacid, are connected by two glycine and a serine between protein fragments, and its aminoacid sequence is as follows:
IleValThrThrAsnGlyAspAsnAlaValThrThrThrThrAsnThr
GlyGlySerLysGluGlyLeuGlnIleGlyLysAspIleThrThrThr
GluGlyGluGluLysProIleTyrAlaAspLysGlyGlySerAspGly
ArgAspAlaValAlaGlyAlaLeuAlaProGluIleValLeuTyrThr
GluAsnGlyGlySerIleLysValLysThrAspAspThrAsnGlyTrp
GluLysAspAlaAsnGlyGlySerIleValThrThrGlyGluAspAsn
AlaThrThrTyrThrGlyGlySerLysGluGlyLeuGluIleGlyLys
AspIleThrAlaAspAsnLysProIleTyrAlaAspLysGlyGlySer
AspGlyArgGluAlaAlaAspAlaPheSerProGluIleValLeuTyr
ThrGluAsnGlyGlySerIleLysProArgAspThrAlaTrpGluLys
AspThrGlyGlySerIleAlaGluGlyValLysAsnThrThrGlyGlu
GluHisValThrGluGluGluThrAsnThrThrThrTyrThrGlyGly
SerLysGluGlyLeuProValGlyLeuGluValSerAspGluGlyGly
SerLeuAspLysGlyValGluThrThrGluGluArgGlnAsnGluAsp
GlyGluAsnAspGluLysAlaThrTyrThrGlyGlySerLysAspGly
LeuProIleGlyLeuGluValProAlaGluGlyAspProLysProIle
TyrAlaAsnLysGlyGlySerAspLeuArgSerGlnLysGlnGlyLeu
LysProLysIleValMetTyrAlaGluAsnGlyGlySerIleGlyPro
ArgThrAspSerTyrLysGluIleGlnLeuAsnGlyAspGlnAlaTrp
LysAspValAsnProAsnGlyIleSerGluLeuValLysGlyAsnPro
PheGlyGlySerIleAlaGluGlyValLysAsnGlyGluGluArgVal
ThrGluGluGluAsnAsnThrThrThrTyrThrGlyGlySerLysGlu
GlyLeuProIleGlyLeuLysValSerAspGlu
The Screening and Identification of expression chimeric protein engineering bacteria:
By the positive transformant containing recombiant plasmid, it is seeded in the test tube containing 3mLLB culture medium (containing kanamycin 60 μ g/mL), 37 DEG C of shaken cultivation 3h, add IPTG to final concentration 0.5mmol/L, continue shaken cultivation induction 6h, centrifugal thalline of collecting carries out SDS-PAGE detection, and it is 55kD's that recon expresses relative molecular weight
HAdV chimeric protein, and compare bacterium BL21 (DE3) without this protein band；
The purification of adenovirus hominis epitope chimeric protein:
1) ultrasonic degradation of Adenovirus Antigen epi-position chimeric protein engineering bacteria is expressed
Centrifugal for the engineering bacteria of abduction delivering chimeric protein (8000rpm, 10min, 4 DEG C) is received bacterium, thalline is resuspended in the bacterial lysate (20mmol/LPBpH7.4,5% glycerol) of original fluid 1/10 volume, ice-bath ultrasonic breaks bacterium, centrifugal collection supernatant；
2) the amine sulfate fractional precipitation of Adenovirus Antigen epi-position chimeric protein is expressed
Accurately measure supernatant volume, in supernatant, add appropriate saturated amine sulfate solution, stirring while adding, make final concentration of the 25% of amine sulfate, put in ice bath overnight；
Centrifugal collect supernatant, add appropriate solid sulfur acid amide powder, stirring while adding, make final concentration of the 50% of amine sulfate, put in ice bath overnight；Next day centrifugal collecting precipitation；Suspend with 200mL balance liquid (50mMTris-HCl, pH7.5) and precipitate, load in bag filter, to 1000mL balance liquid dialysed overnight, change liquid 4 times；Centrifugal collection supernatant, directly goes up DEAE-SeparoseFF anion column purification；
3) DEAE-SeparoseFF anion column purification
Rinsing balance DEAE-SeparoseFF anion column with balance liquid (50mMTris-HCl, pH7.5), the direct upper prop of supernatant then upper step dialysed, flow velocity is 100mL/h；Fully wash post with balance liquid after end of the sample, the most successively with containing 100,200,300,400,500, the eluant solution albumen of 1000mmol/LNaCl, collect each eluting peak albumen, with 12%SDS-PAGE electrophoresis detection each peak albumen, determine which eluting peak is containing the Adenovirus Antigen epi-position chimeric protein expressed；300mmol/LNaCl eluted protein peak is Adenovirus Antigen epi-position chimeric protein.
9. the adenovirus hominis epitope chimeric protein described in claim 1 is used for developing adenovirus vaccine, antibody or antigen detecting agent, and prepares anti-adenovirus monoclonal antibody and multi-resistance for immunity.
10. the adenovirus hominis epitope chimeric protein described in claim 1, it is possible to utilize yeast cells, insect cell, mammalian cell and genetically modified animals and plants to carry out recombinant expressed, preparation.
Adenovirus hominis epitope chimeric protein described in 11. claim 1, in chimeric protein, each type Adenovirus Antigen neoepitope Western fragment arbitrarily connects, and carries out expressing, preparing with the form of chimeric protein.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610269189.3A CN105820257B (en) | 2016-04-27 | 2016-04-27 | Adenovirus hominis epitope chimeric protein and its preparation, application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610269189.3A CN105820257B (en) | 2016-04-27 | 2016-04-27 | Adenovirus hominis epitope chimeric protein and its preparation, application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105820257A true CN105820257A (en) | 2016-08-03 |
| CN105820257B CN105820257B (en) | 2019-02-22 |
Family
ID=56527606
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610269189.3A Active CN105820257B (en) | 2016-04-27 | 2016-04-27 | Adenovirus hominis epitope chimeric protein and its preparation, application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105820257B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107090047A (en) * | 2017-03-10 | 2017-08-25 | 兰州理工大学 | The series connection epitope peptide of five kinds of strawberry viruses of detection simultaneously and its preparation method of chicken yolk antibody |
| CN107365365A (en) * | 2017-08-30 | 2017-11-21 | 广州医科大学附属第医院 | The adenovirus cilia protein peptide of recombination expression, adenovirus subunit vaccine and preparation method thereof |
| CN107602672A (en) * | 2017-08-30 | 2018-01-19 | 广州医科大学附属第医院 | The adenovirus cilia protein peptide of recombination expression, adenovirus subunit vaccine and preparation method thereof |
| CN108456245A (en) * | 2017-12-28 | 2018-08-28 | 肇庆大华农生物药品有限公司 | A kind of Hexon albumen and preparation method thereof, monoclonal antibody and kit |
| CN111044728A (en) * | 2019-09-27 | 2020-04-21 | 广州医科大学附属第一医院 | IgM antibody colloidal gold test strip for rapidly detecting adenovirus and preparation method thereof |
| CN111537711A (en) * | 2020-04-28 | 2020-08-14 | 北京贝尔生物工程股份有限公司 | Kit for rapidly and accurately detecting adenovirus type 3 and preparation method thereof |
| CN111999494A (en) * | 2020-08-18 | 2020-11-27 | 北京贝尔生物工程股份有限公司 | Kit for detecting adenovirus IgM antibody by magnetic particle chemiluminescence method and preparation method thereof |
| CN113045674A (en) * | 2021-03-31 | 2021-06-29 | 李巍 | Human adenovirus five serotype universal antigen epitope fusion protein and preparation and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2217699A2 (en) * | 2007-10-22 | 2010-08-18 | University of Rochester | Respiratory syncytial virus vaccine based on chimeric papillomavirus virus-like particles or capsomeres |
| CN105348391A (en) * | 2015-11-03 | 2016-02-24 | 李越希 | Enteric cytopathogenic human orphan virus 6-type VP1 protein specific epitope, fusion protein of enteric cytopathogenic human orphan virus 6-type VP1 protein specific epitope and preparation method and use of fusion protein |
| CN105671212A (en) * | 2016-04-14 | 2016-06-15 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation and application of adenovirus parting gene chip |
-
2016
- 2016-04-27 CN CN201610269189.3A patent/CN105820257B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2217699A2 (en) * | 2007-10-22 | 2010-08-18 | University of Rochester | Respiratory syncytial virus vaccine based on chimeric papillomavirus virus-like particles or capsomeres |
| CN105348391A (en) * | 2015-11-03 | 2016-02-24 | 李越希 | Enteric cytopathogenic human orphan virus 6-type VP1 protein specific epitope, fusion protein of enteric cytopathogenic human orphan virus 6-type VP1 protein specific epitope and preparation method and use of fusion protein |
| CN105671212A (en) * | 2016-04-14 | 2016-06-15 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Preparation and application of adenovirus parting gene chip |
Cited By (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107090047A (en) * | 2017-03-10 | 2017-08-25 | 兰州理工大学 | The series connection epitope peptide of five kinds of strawberry viruses of detection simultaneously and its preparation method of chicken yolk antibody |
| CN107090047B (en) * | 2017-03-10 | 2021-05-07 | 兰州理工大学 | Tandem epitope peptide for simultaneously detecting five strawberry viruses and preparation method of chicken egg yolk antibody thereof |
| CN107365365A (en) * | 2017-08-30 | 2017-11-21 | 广州医科大学附属第医院 | The adenovirus cilia protein peptide of recombination expression, adenovirus subunit vaccine and preparation method thereof |
| CN107602672A (en) * | 2017-08-30 | 2018-01-19 | 广州医科大学附属第医院 | The adenovirus cilia protein peptide of recombination expression, adenovirus subunit vaccine and preparation method thereof |
| CN107365365B (en) * | 2017-08-30 | 2021-01-01 | 广州医科大学附属第一医院 | Recombinant expression adenovirus cilia protein peptide, adenovirus subunit vaccine and preparation method thereof |
| CN107602672B (en) * | 2017-08-30 | 2021-06-18 | 广州医科大学附属第一医院 | Recombinant expression adenovirus cilia protein peptide, adenovirus subunit vaccine and preparation method thereof |
| CN108456245A (en) * | 2017-12-28 | 2018-08-28 | 肇庆大华农生物药品有限公司 | A kind of Hexon albumen and preparation method thereof, monoclonal antibody and kit |
| CN111044728A (en) * | 2019-09-27 | 2020-04-21 | 广州医科大学附属第一医院 | IgM antibody colloidal gold test strip for rapidly detecting adenovirus and preparation method thereof |
| CN111537711A (en) * | 2020-04-28 | 2020-08-14 | 北京贝尔生物工程股份有限公司 | Kit for rapidly and accurately detecting adenovirus type 3 and preparation method thereof |
| CN111537711B (en) * | 2020-04-28 | 2021-02-05 | 北京贝尔生物工程股份有限公司 | Kit for rapidly and accurately detecting adenovirus type 3 and preparation method thereof |
| CN111999494A (en) * | 2020-08-18 | 2020-11-27 | 北京贝尔生物工程股份有限公司 | Kit for detecting adenovirus IgM antibody by magnetic particle chemiluminescence method and preparation method thereof |
| CN113045674A (en) * | 2021-03-31 | 2021-06-29 | 李巍 | Human adenovirus five serotype universal antigen epitope fusion protein and preparation and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105820257B (en) | 2019-02-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105820257A (en) | Human adenovirus antigen epitope chimeric protein as well as preparation and application thereof | |
| Liu et al. | Identification and characterization of a cross-neutralization epitope of Enterovirus 71 | |
| CN110759973B (en) | Cell strain for expressing African swine fever virus CD2v protein and application thereof | |
| CN110698543A (en) | Double-antigen indirect ELISA kit for African swine fever virus antibody detection | |
| CN113956362B (en) | Recombinant feline parvovirus VP2 protein antigen and application thereof in antibody diagnosis and vaccine preparation | |
| CN112175086A (en) | Monoclonal antibody of anti-porcine epidemic diarrhea virus nsp13 protein and application | |
| CN112574319A (en) | African swine fever virus P12 protein nanoparticle and preparation method and application thereof | |
| CN105348391A (en) | Enteric cytopathogenic human orphan virus 6-type VP1 protein specific epitope, fusion protein of enteric cytopathogenic human orphan virus 6-type VP1 protein specific epitope and preparation method and use of fusion protein | |
| CN109402069A (en) | A kind of pseudovirion and its preparation method and application | |
| CN106589082A (en) | Screening and application of active tuberculosis diagnosis molecules | |
| CN110845624A (en) | SUMO-CP fusion protein, preparation method thereof and preparation method of polyclonal antibody thereof | |
| CN113203854B (en) | Indirect ELISA kit for detecting cat coronavirus antibody | |
| CN112500494B (en) | Antigen for detecting novel coronavirus and preparation method thereof | |
| CN101152570B (en) | Epitope vaccine of hog cholera virus and its preparing process | |
| Li et al. | Mapping B-cell linear epitopes of NS3 protein of bovine viral diarrhea virus | |
| CN108680741B (en) | Infectious bronchitis virus 5b ELISA antibody detection kit and application thereof | |
| CN116023506A (en) | ASFV nonstructural protein dominant antigen epitope fusion protein, kit and application thereof | |
| HEYDARI et al. | Expression and purification of a novel computationally designed antigen for simultaneously detection of htlv-1 and hbv antibodies | |
| CN102621305B (en) | Pig cytomegalovirus antibody indirect blocking enzyme-linked immuno sorbent assay (ELISA) detection kit and detection method thereof | |
| CN105949320A (en) | Preparation and application of echovirus type 1 VP1 protein specific antigen epitope and fusion protein thereof | |
| CN105906716B (en) | Preparation and application of echovirus 9 type VP1 protein specific epitope and fusion protein thereof | |
| CN113512098A (en) | Indirect ELISA (enzyme-Linked immuno sorbent assay) method for identifying swine fever virus and bovine viral diarrhea virus serum antibodies and application thereof | |
| CN115521942A (en) | BVDV epitope gene vaccine construction method and application thereof | |
| Yi et al. | Identification of a novel Aleutian mink disease virus B-cell epitope using a monoclonal antibody against VP2 protein | |
| CN106399347A (en) | Preparation method of GyV5 novel annular virus VP3 protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20230912 Address after: 210002 No. 293 East Zhongshan Road, Jiangsu, Nanjing Patentee after: EASTERN THEATER DISEASE PREVENTION AND CONTROL CENTER OF PLA Address before: 210002 No. 293 East Zhongshan Road, Jiangsu, Nanjing Patentee before: Li Yuexi |