CN106701692A - Recombinant adeno-associated virus for expressing HIV-1 (Human Immunodeficiency Virus-1) invasion inhibitor ECLD to inhibit HIV-1 infection - Google Patents
Recombinant adeno-associated virus for expressing HIV-1 (Human Immunodeficiency Virus-1) invasion inhibitor ECLD to inhibit HIV-1 infection Download PDFInfo
- Publication number
- CN106701692A CN106701692A CN201611272379.7A CN201611272379A CN106701692A CN 106701692 A CN106701692 A CN 106701692A CN 201611272379 A CN201611272379 A CN 201611272379A CN 106701692 A CN106701692 A CN 106701692A
- Authority
- CN
- China
- Prior art keywords
- ecld
- escld
- associated virus
- virus
- infection
- 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
- 239000003112 inhibitor Substances 0.000 title claims abstract description 61
- 241000702421 Dependoparvovirus Species 0.000 title claims abstract description 60
- 230000009545 invasion Effects 0.000 title claims abstract description 55
- 241000713772 Human immunodeficiency virus 1 Species 0.000 title claims abstract description 38
- 208000031886 HIV Infections Diseases 0.000 title abstract description 11
- 230000014509 gene expression Effects 0.000 claims abstract description 33
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 30
- 208000015181 infectious disease Diseases 0.000 claims abstract description 22
- 210000004027 cell Anatomy 0.000 claims description 63
- 102000037865 fusion proteins Human genes 0.000 claims description 36
- 108020001507 fusion proteins Proteins 0.000 claims description 36
- 241000700605 Viruses Species 0.000 claims description 25
- 239000007788 liquid Substances 0.000 claims description 22
- 239000006228 supernatant Substances 0.000 claims description 21
- 230000009385 viral infection Effects 0.000 claims description 14
- 239000013603 viral vector Substances 0.000 claims description 13
- 230000000694 effects Effects 0.000 claims description 12
- 210000004907 gland Anatomy 0.000 claims description 11
- 239000013612 plasmid Substances 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 8
- 102100030703 Interleukin-22 Human genes 0.000 claims description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 6
- 230000008859 change Effects 0.000 claims description 6
- 108010074109 interleukin-22 Proteins 0.000 claims description 6
- 210000004698 lymphocyte Anatomy 0.000 claims description 6
- 238000002360 preparation method Methods 0.000 claims description 6
- 238000010361 transduction Methods 0.000 claims description 6
- 230000026683 transduction Effects 0.000 claims description 6
- 239000013598 vector Substances 0.000 claims description 6
- 238000013461 design Methods 0.000 claims description 5
- 238000004806 packaging method and process Methods 0.000 claims description 5
- 238000000926 separation method Methods 0.000 claims description 5
- 230000009466 transformation Effects 0.000 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 4
- 108020004414 DNA Proteins 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 238000000197 pyrolysis Methods 0.000 claims description 3
- 238000001890 transfection Methods 0.000 claims description 3
- 239000000969 carrier Substances 0.000 claims description 2
- 238000005336 cracking Methods 0.000 claims description 2
- 238000000432 density-gradient centrifugation Methods 0.000 claims description 2
- 230000004069 differentiation Effects 0.000 claims description 2
- 239000006166 lysate Substances 0.000 claims description 2
- 210000005259 peripheral blood Anatomy 0.000 claims description 2
- 239000011886 peripheral blood Substances 0.000 claims description 2
- 230000002093 peripheral effect Effects 0.000 claims description 2
- 239000003726 plant lectin Substances 0.000 claims description 2
- 230000029812 viral genome replication Effects 0.000 claims description 2
- 210000002845 virion Anatomy 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims 4
- 125000003729 nucleotide group Chemical group 0.000 claims 4
- 108091026890 Coding region Proteins 0.000 claims 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 230000003248 secreting effect Effects 0.000 claims 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims 1
- 102000002322 Egg Proteins Human genes 0.000 claims 1
- 108010000912 Egg Proteins Proteins 0.000 claims 1
- 101710121417 Envelope glycoprotein Proteins 0.000 claims 1
- 108091081024 Start codon Proteins 0.000 claims 1
- 102100021696 Syncytin-1 Human genes 0.000 claims 1
- 230000002596 correlated effect Effects 0.000 claims 1
- 235000014103 egg white Nutrition 0.000 claims 1
- 210000000969 egg white Anatomy 0.000 claims 1
- 235000013601 eggs Nutrition 0.000 claims 1
- 230000004927 fusion Effects 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 11
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 230000002155 anti-virotic effect Effects 0.000 abstract 1
- 210000003527 eukaryotic cell Anatomy 0.000 abstract 1
- 239000001963 growth medium Substances 0.000 description 10
- 230000009465 prokaryotic expression Effects 0.000 description 10
- 239000000523 sample Substances 0.000 description 9
- 238000005406 washing Methods 0.000 description 8
- 208000002267 Anti-neutrophil cytoplasmic antibody-associated vasculitis Diseases 0.000 description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 description 7
- 150000001413 amino acids Chemical group 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 239000006210 lotion Substances 0.000 description 7
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 210000004369 blood Anatomy 0.000 description 5
- 230000005764 inhibitory process Effects 0.000 description 5
- 229960005486 vaccine Drugs 0.000 description 5
- 208000030507 AIDS Diseases 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 210000002751 lymph Anatomy 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 239000006180 TBST buffer Substances 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000005090 green fluorescent protein Substances 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000003472 neutralizing effect Effects 0.000 description 3
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241000701161 unidentified adenovirus Species 0.000 description 3
- 108010037897 DC-specific ICAM-3 grabbing nonintegrin Proteins 0.000 description 2
- -1 ECL nitrite ions Chemical class 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 208000036142 Viral infection Diseases 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000011225 antiretroviral therapy Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000031700 light absorption Effects 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 2
- 230000008782 phagocytosis Effects 0.000 description 2
- 239000013595 supernatant sample Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 208000029483 Acquired immunodeficiency Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 241000175212 Herpesvirales Species 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 239000005089 Luciferase Substances 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241000701945 Parvoviridae Species 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 102100025292 Stress-induced-phosphoprotein 1 Human genes 0.000 description 1
- 101710172711 Structural protein Proteins 0.000 description 1
- 108700005077 Viral Genes Proteins 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 229940124524 integrase inhibitor Drugs 0.000 description 1
- 239000002850 integrase inhibitor Substances 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000000527 lymphocytic effect Effects 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000014599 transmission of virus Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70514—CD4
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/7056—Lectin superfamily, e.g. CD23, CD72
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14121—Viruses as such, e.g. new isolates, mutants or their genomic sequences
-
- 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
- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Engineering & Computer Science (AREA)
- Toxicology (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses a recombinant adeno-associated virus for expressing an HIV-1 (Human Immunodeficiency Virus-1) invasion inhibitor ECLD to inhibit HIV-1 infection, and relates to genetic engineering and anti-virus techniques. ECLD and ESCLD genes of an invasion inhibitor are transferred into eukaryotic cells by using the recombinant adeno-associated virus carrying the ECLD and ESCLD genes of the invasion inhibitor, then the invasion inhibitor can be expressed, and infection of HIV-1 can be effectively inhibited by using the invasion inhibitor. The invasion inhibitor is packaged by using the recombinant adeno-associated virus, advantages of the invasion inhibitor and the recombinant adeno-associated virus are combined, and the invasion inhibitor is subjected to eukaryotic expression. A novel method for inhibiting HIV-1 infection is provided, and significant values can be made for developing methods for treating/preventing HIV-1.
Description
Technical field
Can express HIV-1 invasions and suppress the present invention relates to genetic engineering and viral infection resisting technology, more particularly to one kind
The recombinant adeno-associated virus of agent ECLD or ESCLD.Specifically, the present invention is using recombinant adeno-associated virus infection cell expression
Invasion inhibitor fusion proteins ECLD, ESCLD, target the HIV-1 major receptors CD4 of people's CD4+T lymphocytic cell surfaces, so as to press down
HIV-1 viruses infection processed.
Background technology
1981, U.S. CDC (Centers for Disease Control) reported acquired immunodeficiency for the first time
Syndrome, i.e. AIDS；Nineteen eighty-three, French scientist is isolated to human immunodeficiency virus type 1 (human first
Immunodeficiency virus-1, HIV-1), and confirm that it is to cause the cause of disease of acquired immunodeficiency syndrome.To mesh
Before untill, the whole world there are about 78,000,000 people's aids infections virus, and have 39,000,000 people to die from AIDS viral infection and its initiation
Complication.At the year end of cut-off 2013, still there are about 35,000,000 people and carry AIDS virus.
In view of HIV-1 severe popular situation, between past 35 years, substantial amounts of research has been carried out for HIV-1.But
Up to the present still there is not emerging for effective preventative vaccine or curative means, and in numerous healing means, the most
Important achievement is exactly HAART (highly active antiretroviraltherapy, HAART).Continuation
HAART so that the virus load in HIV-1 the infected's body maintains a low-level for a long time, significantly extends AIDS
In the life-span of patient, its quality of life is seted to greatly improve the drawbacks of still HAART also has it from ignoring, due to joint
Medication causes its high expense, and Long-term taking medicine is to the burden of liver kidney, and the resistance to the action of a drug is all caused caused by HIV-1 variations
HAART is not the long-term solution to anti-HIV-1.And in the research of preventative vaccine, although constantly proposing new think of
Road and direction, but still fail to find a kind of vaccine to most of crowds with protectiveness high.
Setback on vaccine so that increasing research focuses on remaining aspect, wherein, invasion/infection inhibitor
Research seems especially prominent.HIV-1 invasion cells are the first steps of HIV-1 infection, and the invasion cell processes of HIV-1 are very multiple
It is miscellaneous, cause that HIV-1 is more sensitive to the change in phagocytic process, the change of any condition is likely to make in phagocytic process
Into HIV-1 invasion cell failures, therefore also create the site that can much target or albumen and suppress the invasion of HIV-1.
Adeno-associated virus (Adenovirus-associated Virus, AAV) carrier system can transduction target gene enter
Enter cell and obtain expression steady in a long-term.The gland relevant viral vector derives from adeno-associated virus.Adeno-associated virus
(adenovirus-associated virus, AAV) is a kind of virus of the non-pathogenic for belonging to Parvoviridae, virus knot
Structure is regular dodecahedron, and diameter is about 20nm.Up to the present it is separated to have 100 various serotypes.Adeno-associated virus is most
Main the characteristics of is must just to be replicated in the presence of helper virus (adenovirus or herpesviral).Adeno-associated virus is
Single-stranded DNA viruses, the full-length genome of its wild type is about 4.7kb, and there is inverted terminal repeat (ITRs) to regulate and control at two ends
Two expression of gene of rep on its genome, cap.Rep expresses 4 albumen, and the duplication to AAV, transcription is integrated and packed
It is extremely important.Cap then expresses 3 structural proteins of AAV.After its invasion cell, small part is incorporated into No. 19 chromosomes of the mankind
A specific site on (AAVS), it is most of to form chromosomoids.The ITRs sequences of adeno-associated virus contain its replicate and
Element necessary to packaging.Therefore, the wild type gene group for encoding viral gene is replaced with into various target genes, borrows it and enter
The function of invading cell and be incorporated on chromosome transduces into cell target gene, here it is the origin of gland relevant viral vector.
AAV carriers are combined with invasion inhibitor, if it is possible to routinely express one kind in vivo and efficiently enter
Inhibitor is invaded, the need for can evading to traditional vaccine to a certain extent.In addition, the efficient anti-reverse transcription disease for being used now
What malicious therapy was used is generally a kind of RTI, a kind of integrase inhibitor and a kind of combination of protease inhibitors
Body, and drug resistance that the mutability high of HIV is brought is so that medicine is had too many difficulties to cope with using upper, therefore, it is badly in need of the new medicine of exploitation.
And inhibitor is invaded as an emerging aspect, and not yet there is good medicine to emerge, develop new efficient invasion and suppress
Agent can cause that the selection of antiretroviral therapy is more various, reduce the appearance of resistance strain.
Based on background above, this laboratory early stage is researched and developed and constructs invasion inhibitor fusion proteins CLD (by sCD4Molecule
Combine with sDC-SIGN molecules and form) (A of patent document CN 102617738), and albumen is obtained by Prokaryotic expression, purification,
Its biological function for being provided simultaneously with suppressing HIV-1 infection and propagating is demonstrated on cell, primary cell and mucous membrane tissue.
Protokaryon CLD shows excellent antiviral activity, but the fusion protein CLD and CLD of prokaryotic expression
Two functional domains for coming from people are distinct：Prokaryotes lack protein post-translational modification.Meanwhile, the expression of albumen,
Purifying can also greatly increase the cost for using.In order to promote the practical of CLD, it is necessary to further set up one kind can be true
The expression system of continuous expression CLD in nucleus, and verify its suppression situation to HIV-1.Therefore the present invention is on the basis of CLD
On transformed, and devise it is a kind of ECLD and ESCLD can be obtained with the system of eukaryotic expression CLD, the system and albumen are also
Can be used for the application of the medicine for developing anti-HIV-1 infection.
The content of the invention
It is related it is an object of the invention to provide a kind of restructuring gland that can express HIV-1 invasion inhibitor ECLD or ESCLD
Virus and its carrier and the viral preparation method；After the virus infected cell, cell expression invasion inhibitor ECLD or
ESCLD is so as to suppress HIV-1 infection.
What the object of the invention was realized in：
Using existing gland relevant viral vector system, a kind of new recombinant adeno-associated virus are built.The virus can transduce warp
Invasion inhibitor ESCLD or the ECLD gene for crossing transformation enters cell, the eukaryotic expression albumen, using the inhibitor ECLD of expression
Or ESCLD suppresses HIV-1 poisoning intrusions and infection.
The fusion protein ECLD or ESCLD is a kind of invasion inhibitor, and the composition of the fusion protein is a solubility
CD4(itself D1 and D2 area, that is to say CD to molecule4The functional areas of molecule, sCD4) with the Neck domains of DC-SIGN and CRD structures
The sDC-SIGN in domain (removing the Intracellular domain of DC-SIGN), by the 3-9 G for repeating between two albumen4S (4 glycine and one
Individual serine) it is connected, and transformed on this basis, obtain existing protein sequence, such as SEQ ID NO.1 to SEQ
Shown in ID NO.7, and construct the system that it can be allowed to be expressed in eukaryotic.
Cell is entered by the gene of recombinant adeno-associated virus transduction fusion protein ECLD or ESCLD, what expression was obtained melts
Hop protein ECLD or ESCLD compared to prokaryotic expression CLD, in the level of Protein secondary structure and higher structure
Different, and the ECLD of eukaryotic expression or ESCLD, its inhibition is more preferable.ICs of the ECLD or ESCLD to different virus50Value
Than CLD to the IC of different virus50Value can be low 3-5 times, such as Fig. 7；And compare the currently known preferable broad spectrum activity of some effects
The IC of neutralizing antibody, ECLD or ESCLD50Value is also decreased obviously, and its multiple for reducing can reach 10-1000 times, such as Fig. 8.
Therefore, the ESCLD or ECLD of eukaryotic expression have significant progressive compared to the CLD of prokaryotic expression.
More notably, the CLD of prokaryotic expression is not good to the inhibition of HIV-1 early stage Strain, but very
The ECLD or ESCLD of nuclear expression system expression have good inhibition, such as Fig. 5 to HIV-1 early stage Strain.And early stage disease
Strain is the decision sex factor in HIV-1 primary infections, and the neutralising capacity to these strains largely determines it
Suppress HIV-1 viral transmissions ability, can more react its suppress HIV-1 ability, and inhibitor present in prior art and
Antibody is not very good to the inhibition and broad spectrum activity of HIV-1 early stage Strain, can not especially obtain a kind of broad spectrum activity and
The inhibitor that inhibition is all significantly increased.And the ECLD or ESCLD transduceed by recombinant adeno-associated virus were deposited compared to before
Inhibitor and antibody, especially compared to the CLD of prokaryotic expression, its suppress HIV-1 early stage Strain broad spectrum activity and suppression
Effect is all significantly increased, and achieves unexpected technique effect.
Specifically, the technical scheme is that：
First, a kind of gland relevant viral vector for expressing invasion inhibitor fusion proteins ECLD or ESCLD, the carrier are designed：
1. inhibitor fusion proteins ECLD or ESCLD are invaded in expression；
2. expression vector skeleton for transformation come adeno-associated virus Helper-free systems carrier；；
3. expression vector characteristic profiles are as shown in Figure 1.
2nd, the preparation method this method for carrying the recombinant adeno-associated virus of invasion inhibitor ECLD or ESCLD gene includes
Following steps：
1. the gland relevant viral vector of expression invasion inhibitor fusion proteins ECLD and ESCLD builds
Design is entered with structure invasion inhibitor fusion proteins ECLD and ESCLD, the then vector plasmid to adeno-associated virus system
Row transformation, and ECLD or ESCLD elements are cloned into vector plasmid：
2. the packaging of adeno-associated virus and checking
By gland relevant viral vector system transfections to HEK293 cells, after transfecting 66-72 hours, cell, multigelation 4 are collected
Secondary releasing virus, and by 10000 revs/min of ten minutes supernatants of the collection containing virion of centrifugation.Utilize afterwards
The adeno-associated virus infection HEK293 cells for carrying inhibitor gene are packaged, supernatant is collected afterwards within 48 hours or cracking is thin
Whether born of the same parents, whether have in detection supernatant has the expression of ECLD in ESCLD or cell and verifies whether virus is correctly wrapped
Fill and with infectivity.
3rd, application of the recombinant adeno-associated virus of invasion inhibitor ECLD or ESCLD gene in HIV-1 infection is carried：
①CD4 +The separation of T lymphocytes and culture
Density gradient centrifugation is carried out by lymphocyte separation medium, from people's fresh peripheral blood, human peripheral lymphocyte is separated,
Again CD is obtained by adding IL-2 (interleukin-22) and PHA (phytohemagglutin phytolectin) cultures differentiation in 7 days4 +T lymphocytes：
2. adeno-associated virus transduction effect detection
The adeno-associated virus of preparation is added in the HEK293 cells of culture, infection monitors GFP expressions and egg after 48 hours
The efficiency of white expression；
3. invasion the inhibitor ECLD or ESCLD of adeno-associated virus transduction expression suppress HIV-1 virus infection
By carry ECLD or ESCLD adeno-associated virus infect HEK293 cells, infection 48 hours afterwards collect contain into
Invade the supernatant or the cell pyrolysis liquid containing ECLD of inhibitor ESCLD；Virus is incubated 1 hour with supernatant or lysate again
Afterwards, for infecting CD4 +T lymphocytes, detect the change of virus replication level.
Sequence table explanation
SEQ ID NO.1：The amino acid sequence of invasion inhibitor fusion proteins ECLD45
SEQ ID NO.2：The amino acid sequence of invasion inhibitor fusion proteins ECLD40
SEQ ID NO.3：The amino acid sequence of invasion inhibitor fusion proteins ECLD35
SEQ ID NO.4：The amino acid sequence of invasion inhibitor fusion proteins ECLD30
SEQ ID NO.5：The amino acid sequence of invasion inhibitor fusion proteins ECLD25
SEQ ID NO.6：The amino acid sequence of invasion inhibitor fusion proteins ECLD20
SEQ ID NO.7：The amino acid sequence of invasion inhibitor fusion proteins ECLD15
SEQ ID NO.8：The DNA sequence dna of coding invasion inhibitor fusion proteins ECLD45
SEQ ID NO.9：The DNA sequence dna of coding invasion inhibitor fusion proteins ECLD40
SEQ ID NO.10：The DNA sequence dna of coding invasion inhibitor fusion proteins ECLD35
SEQ ID NO.11：The DNA sequence dna of coding invasion inhibitor fusion proteins ECLD30
SEQ ID NO.12：The DNA sequence dna of coding invasion inhibitor fusion proteins ECLD25
SEQ ID NO.13：The DNA sequence dna of coding invasion inhibitor fusion proteins ECLD20
SEQ ID NO.14：The DNA sequence dna of coding invasion inhibitor fusion proteins ECLD15
SEQ ID NO.15：Forward primer used by the recombinant adeno-associated virus of the gene that structure is inserted with ESCLD
SEQ ID NO.16：Negative sense primer used by the recombinant adeno-associated virus of the gene that structure is inserted with ESCLD
SEQ ID NO.17：Forward primer sequence for extending ECLD connections (linker)
SEQ ID NO.18：Negative sense primer sequence for extending ECLD connections (linker)
Brief description of the drawings
Fig. 1 is the pAAV-IRES-hrGFP gland relevant viral vector collection of illustrative plates for carrying invasion inhibitor ESCLD genes；
Fig. 2 is adeno-associated virus infection 293T 48 hours infection figures afterwards under fluorescence microscope of cell of packaging；
Fig. 3 is the efficiency of the adeno-associated virus infection 293T of different virus titre；
Fig. 4 is cell express express target protein figure after Western Blot detect adeno-associated virus infection cell；
Fig. 5 is existed by the CLD35 of packaged adeno-associated virus eukaryotic expression ESCLD15 to ESCLD45 and prokaryotic expression
Suppress the effect of HIV-1 early stage Strain CH058.c/2960 infection in cell line；
Fig. 6 is the CLD35 by packaged adeno-associated virus eukaryotic expression ECLD15 to ECLD45 and prokaryotic expression in original
For the effect of the infection for suppressing the different early stage Strain of HIV-1 on cell.
Fig. 7 is ESCLD40 from the CLD35 of prokaryotic expression to the IC of different strain HIV-150The comparing of value
Fig. 8 is the IC of ESCLD40 and the preferable broad spectrum activity neutralizing antibody of some known effects to early stage virus strain HIV-150Value
Comparing (IC50, μ g/mL)
Specific embodiment
Below in conjunction with the accompanying drawings and example in detail：
Embodiment 1 carries the preparation method of the adeno-associated virus of invasion inhibitor ESCLD
The design of 1.ECLD
By the prokaryotic expression CLD having been built up, carry out transforming the DNA fragmentation for having obtained destination protein, such as SEQ on its basis
Shown in ID NO.8 to SEQ ID NO.14
2. the design of primer
With the ECLD as template, using software Design primers.The several to primer of highest scoring are chosen, and signal peptide is introduced in front end
Sequence and extension connection, the extension are connected as from 3 G4S extends to 9 always.
Primer sequence information is as shown in table 1：
Table 1：The ESCLD primer sequences of design
Table 2：Primer sequence for extending ECLD connections (linker)
The structure of 3.ESCLD vector plasmids
First expanded with PCR and obtain purpose fragment, then the linker of extension is inserted among ECLD by infusion
Expanded by PCR and obtain aim sequence, then aim sequence is connected with vector plasmid by the means of molecular cloning obtained
Our purpose carrier (carrying the aav plasmid of ESCLD genes)
4. packaging carries the adeno-associated virus of ESCLD genes
1) the 293T cells of culture are made after single cell suspension, according to 1 × 107Cell number add 10cm culture dish
Among, and supplemented medium is to 10mL.
2) treat to be transfected when cell number 70%-90% to culture dish long (usually 24 hours afterwards), transfect institute
Reagent is Lipo2000.The ratio between plasmid pAAV-RC, pHelper, pAAV-1RES-hrGFP used are 1: 2: 1, altogether
It is transferred to 16 μ g plasmids.The ratio of plasmid consumption and Lipo2000 is 1: 2.
3) carry out within 4-6 hours changing liquid after transfecting, collect virus within 72 hours after transfection.Culture medium is discarded first, and 1mL is used afterwards
New culture medium be put into the cell of whole culture dish is resuspended in the centrifuge tube of 1.5mL, be put into -80 DEG C of refrigerators, treat to freeze completely
After firmly (about 10-15 minutes), then it is transferred into dissolving (about 2-3 minutes) among 37 DEG C of water-baths.The process is repeatedly
Carry out 4 times.Finally, centrifuge tube is centrifuged ten minutes with 10000 revs/min of rotating speed, is divided in transfer supernatant to new centrifuge tube
Install, be stored in -80 DEG C it is standby.
5. the infectivity of the adeno-associated virus for carrying SCLD genes packed is verified
1) the 293T cells of culture are made after single cell suspension, according to 5 × 105Cell number add 12 orifice plates in, and
Per hole supplemented medium to 1mL, it is placed in cell culture incubator and cultivates 24 hours.
2) supernatant discarded, the AAV virus liquids required for adding experiment, and add fresh culture medium to 500 μ L.Left and right shake it is even it
After be put into incubator 15 minutes afterwards continue shake it is even, be repeated 3 times.Final being put into cell culture incubator carries out incubated overnight.
3) after incubated overnight, discard infection culture medium and add fresh culture to 1mL.Continue to put into cell culture incubator training
Support, 48 hours after culture to infection virus, collect supernatant standby.Cell in culture plate adds the PBS re-suspended cells in 1mL/ holes.
4) it is centrifuged 5 minutes with 1200 revs/min of rotating speed, supernatant discarded adds the PBS re-suspended cells of 1mL, is repeated 3 times.
5) paraformaldehyde of the 2% of 300 μ L of often pipe addition is fixed.
6) upper machine testing green fluorescent protein GFP after fixing, to determine the infection rate of AAV infection cells, as a result such as Fig. 2, Fig. 3
It is shown.
6. can the adeno-associated virus infection cell for carrying ESCLD genes packed of checking correct express express target protein
1) supernatant collected in 4 is carried out into PAGE gel electrophoresis, the sample required for electrophoresis added loading before loading
Buffer solution (5 ×), boiling water bath 10 minutes.Sample added by per hole is 20 μ L.In the electrophoretic buffer for preparing, 60V constant pressures first
Half an hour, treat to be changed to 120V constant pressures, electrophoresis 1 hour to one and a half hours again in the complete electrophoretic migration of sample to separation gel.
2) transferring film：The transferring film buffer solution for preparing in advance is put to 4 DEG C of precoolings, and the size according to institute's transferring film prepares filter paper and pvdf membrane,
Pvdf membrane needs pretreatment, that is, be put into 10 seconds preactivates in methyl alcohol, to place into and invade bubble in transferring film buffer solution.Transferring film system put to
Sequentially it is：Anode-filter paper (3 layers)-pvdf membrane-gel-filter paper (3 layers)-negative electrode.Condition is 200mA constant currents, according to albumen size
Turn the different time (about 1kDa turns 1 minute)
3) after transferring film is completed, take out film and put into confining liquid closing 1-2 hours.After closing is completed, 3 are washed with TBST washing lotions
It is secondary, 5 minutes every time.
4) film is put into the primary antibody solution for preparing again, is incubated at room temperature 1-2 hours, be incubated after completing, 3 are washed with TBST washing lotions
It is secondary, 5 minutes every time.
5) film is put into the two corresponding anti-solution of the HRP marks for preparing again, is incubated at room temperature 1 hour, be incubated after completing, washed with TBST
Liquid is washed 5 times, every time 5 minutes.
6) develop the color：Dripped after ECL nitrite ions are prepared on film, developed the color, as a result as shown in Figure 4.
Embodiment 2 carries application of the adeno-associated virus of invasion inhibitor ESCLD or ECLD in HIV-1 infection is suppressed
1. fusion protein ESCLD suppresses the experiment of HIV-1 infection in cell line
1) the TZM-b1 cells of culture are made after single cell suspension, according to 1 × 104/ hole cell number add 96 orifice plates it
In, final volume is 100 μ L
2) the packaged adeno-associated virus infection 293T cells containing ESCLD genes, collect supernatant after 48h, take 100 μ L of supernatant
And the CLD and 200TCID of prokaryotic expression50HIV-1 viruses are common to be incubated a hour in 37 DEG C of incubators.
3) culture medium that cell is completed in the orifice plate of the previous day 96 is discarded, then the virus after incubation is added jointly with supernatant, then general
Culture medium is mended to 200 μ L.And DMEM to final concentration of 40 μ g/ holes is added in the process.
4) 96 orifice plates are continued to put to 37 DEG C of incubator cultures 48 hours.Culture medium is discarded, with the PBS one time in 200 μ L/ holes
Afterwards, the NP40 cell lysis in 200 μ L/ holes are added 5 minutes.
5) last to measure LUC values according to the method for surveying luciferase, the LUC values according to its ESCLD sample subtract virus-free infection
The LUC values of the Supernatant samples received receive the LUC values of Supernatant samples divided by empty AAV infection again, calculate ESCLD and suppress HIV-1's
Infection rate, as shown in Figure 5.
2. fusion protein ECLD suppresses the experiment of HIV-1 infection on primary cell
1) new blood for being obtained from donor adds anti-coagulants, is centrifuged stand-by (time is no more than 8 hours).
2) often pipe adds 25mL new bloods to 50mL centrifuge tubes, adds isometric PBS, mixing of turning upside down.
3) 15mL centrifuge tubes are previously added 7mL lymph separating liquids, in the blood for adding 7mL to mix.When adding blood, action
It is slow and soft, keep the good layering between lymph separating liquid and blood.
4) lifting speed is set to room temperature centrifugation in 0,800g/ minutes 30 minutes
5) after centrifugation is completed, orlop is red blood cell, and centre is lymph separating liquid, and upper strata is serum.In serum and lymph point
There is the milky lymphocyte for a layer, as needing between chaotropic layer.Carefully draw, as far as possible remaining liquid of few suction.
6) lymphocyte is drawn in other 15mL centrifuge tubes, adds the D-HANKS buffer solution re-suspended cells of three times volume,
Room temperature is centrifuged ten minutes within 300g/ minutes.
7) repeat step 6,3-5 times.Finally give PBMC.
8) PBMC that will be obtained is with having added the RPMI-1640 culture mediums of FBS to carry out cell density adjustment so that its whole cell density
It is 5 × 106/mL。
9) every milliliter of cell liquid adds the μ L of PHA 1 (final concentration of 1 μ g/mL), and IL-220U is put into 37 degree of constant temperature cell culture incubators
In cultivated.
10) carried out changing liquid every three days, change the method for liquid half-and-half to change liquid.Cell liquid is all sucked into 15mL centrifuge tubes,
1200 revs/min are centrifuged 5 minutes, and the culture medium of cumulative volume half is siphoned away afterwards, add fresh culture medium, and add half
PHA and IL-2.From after separating cell, according to above-mentioned cultural method culture 7 days afterwards, the CD for being broken up4 +T cell liquid can be with
For testing.According to 1.5 × 104/ hole cell number is added among 96 orifice plates, and final volume is 100 μ L.
11) by the cell pyrolysis liquid containing fusion protein ECLD and 1 hour of virus incubation, it is incubated after completing and is added to CD4 +
Among T cell liquid.
12) infect 3 hours afterwards, 1200 revs/min are centrifuged 10 minutes, carefully draw supernatant, add the μ L of PBS 200 resuspended.
13) repeat the above steps 3 to 4 times, fully erased free virus.
14) the fresh μ L of 1640 culture medium 200 are added, and is proportionally added into IL-2.
15) three days are cultivated afterwards, 1200 revs/min are centrifuged 5 minutes, draw 100 μ L of supernatant, carry out the measure of p24, as a result as schemed
Shown in 6.
The ESCLD of embodiment 3 suppresses the measure of different HIV-1 strain IC50
The expression of fusion protein ESCLD in 1.ELISA quantitative determination supernatants
1) after the mice serum containing ESCLD antibody is diluted in 1: 2000 ratio with PBS in advance, by the amount of the μ L of every hole 50
Add elisa plate and seal, ambient temperature overnight storage.
2) coating buffer is removed, adds 400 μ L washing lotions, wash 3 times.
3) after washing, 300 μ L confining liquids are added, is placed on 37 DEG C of constant incubators and closes 2 hours.
4) confining liquid is discarded, 400 μ L washing lotions are added, is washed 3 times.
5) standard items (for drawing standard curve) and sample are added with 50 μ L/ holes, the initial dilution ratio of standard items is 1:
30, gradient dilution is carried out with 1: 3 ratio, altogether 8 gradients.Add be placed on after sample and standard items 37 DEG C it is incubated
1 hour of case.
6) standard items and sample are discarded, 400 μ L washing lotions are added, is washed 3 times.
7) antibody (1: 10000 dilution) of the rabbit anti-DC-SIGN in source is added with 50 μ L/ holes, 37 DEG C of constant incubators 1 is placed on small
When.
8) antibody is discarded, 400 μ L washing lotions are added, is washed 3 times.
9) goat anti-rabbit igg (1: 10000 dilution) is added with 50 μ L/ holes, is placed on 1 hour of 37 DEG C of constant incubators.
10) antibody liquid is discarded, 400 μ L washing lotions are added, is washed 5 times.
11) TMB nitrite ions are added with 50 μ L/ holes, room temperature lucifuge develops the color 5 minutes.
12) 50 μ L reaction terminating liquids are added after colour developing is completed per hole.
13) machine on (light absorption value of selection is 570nm and 450nm) reading, according to the numeric renderings standard curve for measuring, further according to
The protein concentration of the quantitative sample of the light absorption value of standard curve and testing sample.
2. fusion protein ESCLD IC50The measure of value
1) the TZM-b1 cells of culture are made after single cell suspension, according to 1 × 104/ hole cell number add 96 orifice plates it
In, final volume is 100 μ L
2) the ESCLD samples that will measure concentration carry out constant gradient dilution with gradient 1: 2.Last row is to be added without ESCLD
Control value.
3) strain of IC50 can not be measured for 1: 2 gradient dilution, is added according to the μ L of gradient 100,80,60,50,40,20,10,0
ESCLD samples.
4) subsequent step is with reference to embodiment 2.1.Finally carry out the measure of LUC.Calculated according to the relation between concentration and inhibiting rate
Go out IC50Value.
Claims (13)
1. a kind of recombinant adeno-associated virus, it is characterised in that：After infection cell so that cell expression invasion inhibitor fusion egg
White ECLD, its sequence is any one in amino acid sequence shown in SEQ ID NO.1 to SEQ ID NO.7.
2. a kind of recombinant adeno-associated virus, it is characterised in that：After infection cell so that cell is expressed and secretes invasion inhibitor
To extracellular, its described ESCLD is ECLD albumen n ends addition coding sequence of secretory signal peptide shape described in claim 1 to fusion protein ESCLD
Into.
3. a kind of recombined glandulae correlation viral vectors, it is characterised in that：Invade inhibitor fusion proteins ECLD's with that can express
Nucleotide sequence, the sequence is any one in the nucleotide sequence shown in SEQ ID NO.8 to SEQ ID NO.14.
4. recombined glandulae correlation viral vectors described in claim 3, it is characterised in that：The gland that expression vector skeleton comes for transformation
The pAAV-IRES-hrGFP carriers or pAAV-MCS of correlated virus Helper-free systems.
5. the recombined glandulae correlation viral vectors described in claim 3 or 4, are further characterized in that：In the nucleotide sequence of the ECLD
The nucleotide sequence of a segment signal peptide is added with after initiation codon.
6. the recombined glandulae correlation viral vectors described in claim 5, are further characterized in that：Its described signal peptide is HIV-1CN54
The signal peptide of envelope glycoprotein.
7. the preparation method of the recombinant adeno-associated virus described in claim 1 or 2, it is characterised in that：
1. the gland relevant viral vector of expression invasion inhibitor fusion proteins ECLD and ESCLD builds
Design is entered with structure invasion inhibitor fusion proteins ECLD and ESCLD, the then vector plasmid to adeno-associated virus system
Row transformation, and ECLD and ESCLD elements are cloned into vector plasmid；
2. the packaging of adeno-associated virus and checking
By gland relevant viral vector system transfections to HEK293 cells, after transfecting 66-72 hours, cell, multigelation 4 are collected
Secondary releasing virus, and by 10000 revs/min of ten minutes supernatants of the collection containing virion of centrifugation, utilize afterwards
The adeno-associated virus infection HEK293 cells for carrying inhibitor gene are packaged, supernatant is collected afterwards within 48 hours or cracking is thin
Whether born of the same parents, whether have in detection supernatant has the expression of ECLD in ESCLD or cell and verifies whether virus is correctly wrapped
Fill and with infectivity.
8. the application of recombinant adeno-associated virus described in claim 1 or 2, it is characterised in that：
①CD4 +The separation of T lymphocytes and culture
Density gradient centrifugation is carried out by lymphocyte separation medium, from people's fresh peripheral blood, human peripheral lymphocyte is separated,
Again CD is obtained by adding IL-2 (interleukin-22) and PHA (phytohemagglutin phytolectin) cultures differentiation in 7 days4 +T lymphocytes；
2. adeno-associated virus transduction effect detection
The adeno-associated virus of preparation is added in the HEK293 cells of culture, infection monitors GFP expressions and egg after 48 hours
The efficiency of white expression；
3. invasion the inhibitor ECLD or ESCLD of adeno-associated virus transduction expression suppress HIV-1 virus infection by carrying
The adeno-associated virus infection HEK293 cells of ECLD or ESCLD, infection is collected and contains invasion inhibitor ESCLD for 48 hours afterwards
Supernatant or the cell pyrolysis liquid containing ECLD；Virus is incubated after 1 hour with supernatant or lysate again, for feeling
Dye CD4 +T lymphocytes, detect the change of virus replication level.
It is 9. a kind of to invade inhibitor fusion proteins ECLD, it is characterised in that：Its sequence is SEQ ID NO.1 to SEQ ID NO.7
Any one in shown amino acid sequence.
It is 10. a kind of to invade inhibitor fusion proteins ESCLD, it is characterised in that：Albumen as claimed in claim 9, connects in its N-terminal
There is coding sequence of secretory signal peptide.
Invasion inhibitor fusion proteins described in 11. claims 9 or 10, it is characterised in that：Expressed by eukaryotic expression system
Arrive.
A kind of 12. DNA, it is characterised in that：The protein described in claim 9 or 10 can be encoded.
A kind of 13. cells, it is characterised in that：Can be by recombinant adeno-associated virus infection and the institute of eukaryotic expression claim 9 or 10
The protein stated.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611272379.7A CN106701692B (en) | 2016-12-29 | 2016-12-29 | Recombinant adeno-associated virus capable of expressing HIV-1 invasion inhibitor ECLD to inhibit HIV-1 infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611272379.7A CN106701692B (en) | 2016-12-29 | 2016-12-29 | Recombinant adeno-associated virus capable of expressing HIV-1 invasion inhibitor ECLD to inhibit HIV-1 infection |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106701692A true CN106701692A (en) | 2017-05-24 |
| CN106701692B CN106701692B (en) | 2023-05-23 |
Family
ID=58906717
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611272379.7A Active CN106701692B (en) | 2016-12-29 | 2016-12-29 | Recombinant adeno-associated virus capable of expressing HIV-1 invasion inhibitor ECLD to inhibit HIV-1 infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106701692B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023284889A1 (en) * | 2021-07-12 | 2023-01-19 | 成都维瑾柏鳌生物医药科技有限公司 | Cld protein mutant and use thereof |
| WO2024125271A1 (en) * | 2022-12-13 | 2024-06-20 | 成都维瑾柏鳌生物医药科技有限公司 | Recombinant protein against hiv-1 and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009118710A2 (en) * | 2008-03-27 | 2009-10-01 | Elevation Biotech | Cd4-related polypeptides and methods of use |
| CN101591379A (en) * | 2008-05-27 | 2009-12-02 | 中国疾病预防控制中心性病艾滋病预防控制中心 | Based on the amino acid mutation of EIAV attenuated live vaccine and the anti-HIV vaccine that makes up |
| CN102617738A (en) * | 2011-01-26 | 2012-08-01 | 中国科学院武汉病毒研究所 | Polypeptides, coding sequences, preparation method and use of recombinant fusion protein CD4-LINKER-DC-SIGN (CLD) |
| CN104694573A (en) * | 2015-03-26 | 2015-06-10 | 中国科学院武汉病毒研究所 | Method for inhibiting HIV-1 infectious agent from infecting primary lymphocyte by utilizing CRISPR/Cas9 |
-
2016
- 2016-12-29 CN CN201611272379.7A patent/CN106701692B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009118710A2 (en) * | 2008-03-27 | 2009-10-01 | Elevation Biotech | Cd4-related polypeptides and methods of use |
| CN101591379A (en) * | 2008-05-27 | 2009-12-02 | 中国疾病预防控制中心性病艾滋病预防控制中心 | Based on the amino acid mutation of EIAV attenuated live vaccine and the anti-HIV vaccine that makes up |
| CN102617738A (en) * | 2011-01-26 | 2012-08-01 | 中国科学院武汉病毒研究所 | Polypeptides, coding sequences, preparation method and use of recombinant fusion protein CD4-LINKER-DC-SIGN (CLD) |
| CN104694573A (en) * | 2015-03-26 | 2015-06-10 | 中国科学院武汉病毒研究所 | Method for inhibiting HIV-1 infectious agent from infecting primary lymphocyte by utilizing CRISPR/Cas9 |
Non-Patent Citations (2)
| Title |
|---|
| NICHOLE CERUTTI ET AL.: "Stabilization of HIV-1 gp120-CD4 Receptor Complex through Targeted Interchain Disulfide Exchange" * |
| TAO DU ET AL.: "Bifunctional CD4-DC-SIGN Fusion Proteins Demonstrate Enhanced Avidity to gp120 and Inhibit HIV-1 Infection and Dissemination" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023284889A1 (en) * | 2021-07-12 | 2023-01-19 | 成都维瑾柏鳌生物医药科技有限公司 | Cld protein mutant and use thereof |
| WO2024125271A1 (en) * | 2022-12-13 | 2024-06-20 | 成都维瑾柏鳌生物医药科技有限公司 | Recombinant protein against hiv-1 and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106701692B (en) | 2023-05-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shen et al. | Identification and functional analysis of novel nonstructural proteins of human bocavirus 1 | |
| Sun et al. | Molecular characterization of infectious clones of the minute virus of canines reveals unique features of bocaviruses | |
| Emerson et al. | ORF3 protein of hepatitis E virus is not required for replication, virion assembly, or infection of hepatoma cells in vitro | |
| Déctor et al. | Rotavirus gene silencing by small interfering RNAs | |
| Snyder et al. | Functional characterization of the alphavirus TF protein | |
| Kuo et al. | Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier | |
| Pudupakam et al. | Mutational analysis of the hypervariable region of hepatitis e virus reveals its involvement in the efficiency of viral RNA replication | |
| Corey et al. | Mutations in the stalk of the measles virus hemagglutinin protein decrease fusion but do not interfere with virus-specific interaction with the homologous fusion protein | |
| Stucker et al. | The role of evolutionary intermediates in the host adaptation of canine parvovirus | |
| Surjit et al. | The ORF2 protein of hepatitis E virus binds the 5′ region of viral RNA | |
| García-Escudero et al. | Inducible gene expression from African swine fever virus recombinants: analysis of the major capsid protein p72 | |
| Lou et al. | Human parvovirus B19 DNA replication induces a DNA damage response that is dispensable for cell cycle arrest at phase G2/M | |
| Meng et al. | Vaccinia virus A6 is essential for virion membrane biogenesis and localization of virion membrane proteins to sites of virion assembly | |
| Von Messling et al. | Amino-terminal precursor sequence modulates canine distemper virus fusion protein function | |
| Kuo et al. | Recognition of the murine coronavirus genomic RNA packaging signal depends on the second RNA-binding domain of the nucleocapsid protein | |
| Wang et al. | Parvovirus expresses a small noncoding RNA that plays an essential role in virus replication | |
| WO2023216826A1 (en) | Monoclonal antibody a38 against rift valley fever virus and use | |
| Padidam et al. | A phage single-stranded DNA (ssDNA) binding protein complements ssDNA accumulation of a geminivirus and interferes with viral movement | |
| Plattet et al. | Signal peptide and helical bundle domains of virulent canine distemper virus fusion protein restrict fusogenicity | |
| CN101748124B (en) | siRNA segment and application thereof used for curing and/or preventing Peste des petits ruminants | |
| CN106701692A (en) | Recombinant adeno-associated virus for expressing HIV-1 (Human Immunodeficiency Virus-1) invasion inhibitor ECLD to inhibit HIV-1 infection | |
| Zhang et al. | Primary virus envelope cross-reactivity of the broadening neutralizing antibody response during early chronic human immunodeficiency virus type 1 infection | |
| Wei et al. | Recovery of infectious virus by transfection of in vitro-generated RNA from tulane calicivirus cDNA | |
| Snyder et al. | Rescue of infectious particles from preassembled alphavirus nucleocapsid cores | |
| Komatsu et al. | Identification of a proline-kinked amphipathic α-helix downstream from the methyltransferase domain of a potexvirus replicase and its role in virus replication and perinuclear complex formation |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20220715 Address after: No. 202, floor 2, building 11, No. 88, Keyuan South Road, high tech Zone, Chengdu, Sichuan 610041 Applicant after: Chengdu Weijin Boao Biomedical Technology Co.,Ltd. Address before: 430071 No. 44, Middle District, little Hongshan, Wuchang District, Hubei, Wuhan Applicant before: Wuhan Institute of Virology, Chinese Academy of Sciences |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240613 Address after: 100089, 5th Floor, Building 7, Courtyard A2, West Third Ring North Road, Haidian District, Beijing Patentee after: Zhongguancun Technology Leasing Co.,Ltd. Country or region after: China Address before: No. 202, floor 2, building 11, No. 88, Keyuan South Road, high tech Zone, Chengdu, Sichuan 610041 Patentee before: Chengdu Weijin Boao Biomedical Technology Co.,Ltd. Country or region before: China |
|
| TR01 | Transfer of patent right |