CN104001155B - A kind of Tat albumen and its preparation method and application - Google Patents
A kind of Tat albumen and its preparation method and application Download PDFInfo
- Publication number
- CN104001155B CN104001155B CN201410259996.8A CN201410259996A CN104001155B CN 104001155 B CN104001155 B CN 104001155B CN 201410259996 A CN201410259996 A CN 201410259996A CN 104001155 B CN104001155 B CN 104001155B
- Authority
- CN
- China
- Prior art keywords
- tat
- albumen
- hiv
- cell
- attenuation
- 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.)
- Active
Links
- 238000002360 preparation method Methods 0.000 title claims description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- 101710149951 Protein Tat Proteins 0.000 claims description 38
- 230000000694 effects Effects 0.000 claims description 21
- 230000006907 apoptotic process Effects 0.000 claims description 8
- 238000002703 mutagenesis Methods 0.000 claims description 8
- 231100000350 mutagenesis Toxicity 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 230000023603 positive regulation of transcription initiation, DNA-dependent Effects 0.000 claims description 7
- 230000002238 attenuated effect Effects 0.000 claims description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 2
- 230000000717 retained effect Effects 0.000 claims description 2
- 241000713772 Human immunodeficiency virus 1 Species 0.000 abstract description 18
- 208000032420 Latent Infection Diseases 0.000 abstract description 16
- 208000037771 disease arising from reactivation of latent virus Diseases 0.000 abstract description 16
- 230000003013 cytotoxicity Effects 0.000 abstract description 4
- 231100000135 cytotoxicity Toxicity 0.000 abstract description 4
- 230000005847 immunogenicity Effects 0.000 abstract description 4
- 238000001727 in vivo Methods 0.000 abstract description 2
- 239000003814 drug Substances 0.000 abstract 1
- 229940079593 drug Drugs 0.000 abstract 1
- 230000001483 mobilizing effect Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 21
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 17
- 235000004279 alanine Nutrition 0.000 description 17
- 238000002474 experimental method Methods 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 6
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 6
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 239000004474 valine Substances 0.000 description 6
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 230000009466 transformation Effects 0.000 description 5
- 108010002350 Interleukin-2 Proteins 0.000 description 4
- 102000000588 Interleukin-2 Human genes 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 108090000695 Cytokines Proteins 0.000 description 3
- 102000004127 Cytokines Human genes 0.000 description 3
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 3
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 3
- 239000012190 activator Substances 0.000 description 3
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 210000003462 vein Anatomy 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000029812 viral genome replication Effects 0.000 description 2
- WAEXFXRVDQXREF-UHFFFAOYSA-N vorinostat Chemical compound ONC(=O)CCCCCCC(=O)NC1=CC=CC=C1 WAEXFXRVDQXREF-UHFFFAOYSA-N 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 1
- 108010022579 ATP dependent 26S protease Proteins 0.000 description 1
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 1
- 108010082126 Alanine transaminase Proteins 0.000 description 1
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 1
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 1
- 108010040476 FITC-annexin A5 Proteins 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 102100030703 Interleukin-22 Human genes 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- 241001112090 Pseudovirus Species 0.000 description 1
- 244000062793 Sorghum vulgare Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000004159 blood analysis Methods 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 229940109239 creatinine Drugs 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 229940121372 histone deacetylase inhibitor Drugs 0.000 description 1
- 239000003276 histone deacetylase inhibitor Substances 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 108010074109 interleukin-22 Proteins 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 235000019713 millet Nutrition 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 229960000237 vorinostat Drugs 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
A kind of Tat albumen, is the aminoacid sequence of described Tat albumen as SEQ? NO:1, SEQ? NO:2, SEQ? NO:3, SEQ? shown in NO:4.Tat albumen of the present invention not only has good latent infection mobilizing function, and reduces cytotoxicity and immunogenicity in vivo.The potential drug that the albumen comprising said structure function can activate as HIV-1 latent infection is researched and developed.
Description
Technical field
The present invention relates to a kind of antiviral compound, more specifically, Tat albumen relating to a kind of improvement and its preparation method and application.
Background technology
HAART (HighlyActiveAntiretroviralTherapy, HAART) treatment effectively the viral load in patient body can be controlled to the degree that can't detect, but HIV-1(HIV (human immunodeficiency virus) one type of latent infection) provirus be integrated in the genome of host formed storage vault be later difficult to remove.Patient must long-term prescription to suppress virus replication, once withdrawal will cause the resilience of virus replication.The latent infection how removing HIV-1 has become the bottleneck problem of thorough treatment of AIDS.
The cytokines such as interleukin-22 (Interleukin2, IL-2) and anti-CD3 were once used to the latent infection activating HIV-1, and these cytokines make cell in integral level, there occurs activation, cause great toxic and side effects to body.Multiple acetylation of histone enzyme inhibitor (HistoneDeacetylaseinhibitor; HDACi) be also the activator of hiding that research is many at present; on the one hand because HDACi is also broad effect spectrum to the activation of gene; easily cause the unconventionality expression of other genes; the HDACi that effectiveness comparison is good in vitro on the other hand; as valprocacid (VPA) and Vorinostat (i.e. suberanilohydroxamicacid; SAHA) etc.; performance is clinically not good, can not drop into practical application.Therefore, new high specificity, efficient and the activator of hiding of safety is the task of top priority is found.
HIV-1Tat is the specific trans-activating factor of HIV-1, and this albumen is attached on the TAR of HIV-15 ' LTR specifically, and transcribing of HIV-1mRNA is raised hundreds of times.Tat albumen is also the crucial factor in the latent infection of HIV-1.This albumen, with wearing film peptide, is proved to be and has the efficient function through cell membrane; Once the vaccine being designed as HIV-1 enters clinical experiment and uses, comparatively safe to human body.But Tat albumen is proved to be has apoptosis-induced function, may impact immune cell function.
Summary of the invention
An object of the present invention is exactly find a kind of new AntiHIV1 RT activity approach,
First provide a kind of Tat albumen preparing the application in inverase, what the present invention adopted is that TAT-86 carrys out proving effect.
A kind of attenuation Tat albumen is further provided to prepare the application in inverase, shown in described attenuation Tat protein amino acid sequence SEQNO:1, SEQNO:2, SEQNO:3, SEQNO:4.
Further provide a kind of attenuation Tat albumen, described attenuation Tat protein amino acid sequence SEQNO:1(R4M4), SEQNO:2(R4M5), SEQNO:3(R4M7), SEQNO:4(R5M4) shown in.
A kind of preparation method of above-mentioned attenuation Tat albumen is provided further, it is characterized in that, first the site of rite-directed mutagenesis is selected, on the gene of Tat, rite-directed mutagenesis is carried out to these sites, finally retained the trans-activation function of Tat in a large number, and removed the attenuated proteins of wherein apoptosis and other activity.
The present invention has the following advantages:
1. the object of the present invention is to provide the HIV-1Tat albumen of some attenuations for activating HIV-1 latent infection.
2. the invention provides a kind of thinking of trans-activator Tat as activating dormant infection instrument utilizing HIV-1 self, and by accumulative sudden change, Tat albumen is transformed, make it more reliable in safety.
3. the invention provides the Tat albumen of four kinds of attenuations, can specific activation HIV-1 latent infection on a cellular level.
4. the invention provides a kind of building mode of the external latent infection model of HIV-1 of improvement.
5. the invention provides a kind of attenuation Tat albumen activates HIV-1 latent infection new method with SAHA coupling.
6. the function of attenuation Tat albumen provided by the invention to immunocyte does not impact.
7. what the present invention found Tat albumen possesses good transactivation activity, can effectively raise transcribing of HIV-1mRNA.
8.Tat albumen possesses good wears film activity, effectively can enter cell and each tissue performance function.
Accompanying drawing explanation
Fig. 1: HIV1Tat albumen transformation and screening principle.
The ideograph of Fig. 2: four kinds of attenuation Tat.
Fig. 3: four kinds of attenuation Tat can activate the promoter of HIV-1 in Tzm-b1.
The transformation of Fig. 4: Tat-R5M4 reduces cytotoxicity and apoptosis activity.
Fig. 5: Tat-R5M4 can wear film effectively enters cell and each tissue.
The structure of Fig. 6: HIV-1 external latent infection model.
Fig. 7: Tat-R5M4 effectively can activate the external model of hiding of HIV-1.
Fig. 8: the Tat-R5M4 CD4 that effectively can activate the latent infection state from clinical patient peripheral blood
+t cell.
Fig. 9: acute toxicity testing and immunogenicity detect.
Detailed description of the invention
The present invention is further described below in conjunction with the drawings and specific embodiments.Unless stated otherwise, the present invention adopts reagent, equipment and method are conventional commercial reagent, equipment and the conventional method used of the art.
Embodiment one: to transformation and the Activity determination of Tat albumen
In the past few decades, the 26S Proteasome Structure and Function of HIV-1Tat has had very detailed research.In the present invention, we go out except apoptosis activity by accumulation sudden change transformation, retain the Tat albumen of most of transactivation activity simultaneously.Because the research of first three domain of Tat albumen (amino acid/11-59) is very detailed, so we aminoacid 60-72 that first Selecting research is less carries out point mutation, the gene of sudden change is connected on carrier for expression of eukaryon and detects uciferase activity after transfection Tzm-b1 cell, wherein only have M36, M39, M51, M66, M67, M68, M69, M77 remain >=transactivation activity of 80%.Then we carry out combination superposition these sudden changes, have carried out six taking turns sudden change altogether, and finally obtain 4 candidate albumens: R4M4, R4M5, R4M7, R5M4, wherein R5M4 have accumulated maximum 5 point mutation.
Wherein R4M4 is in rite-directed mutagenesis, is replaced by the valine alanine of the 36th, and the glutamine alanine of the 66th replaces, and the valine alanine of the 67th replaces, and the serine alanine of the 68th replaces.
Wherein R4M5 is in rite-directed mutagenesis, is replaced by the isoleucine alanine of the 39th, and the glutamine alanine of the 66th replaces, and the valine alanine of the 67th replaces, and the serine alanine of the 68th replaces.
Wherein R4M7 is in rite-directed mutagenesis, and the glutamine alanine of the 66th replaces, and the valine alanine of the 67th replaces, and the serine alanine of the 68th replaces, and the serine alanine of the 77th replaces.
Wherein R5M4 is in rite-directed mutagenesis, replaced by the valine alanine of the 36th, the glutamine alanine of the 66th replaces, and the valine alanine of the 67th replaces, the serine alanine of the 68th replaces, and the serine alanine of the 77th replaces.
R4M4, R4M5, R4M7, R5M4 are cloned in prokaryotic expression carrier and carry out Expression and purification, the albumen of acquisition processes Tzm-b1 cell, detects the activity of FireflyLuciferase after 48 hours.
This experiment proves the R4M4 of transformation, and R4M5, R4M7, R5M4 have good transactivation activity in Tzm-b1.
Embodiment two: cytotoxicity and apoptosis activity detect
Respectively the Tat-86 of 10nM, 50nM, 100nM, 500nM, 1uM, 2uM, 3uM, 4uM or Tat-R5M4 and Tzm-b1 cell are hatched jointly, after 48 hours, detect cell viability by MTS method.Use 0.1ug respectively, 0.5ug, 1ug, the Tat-86(of 2ug carries out prokaryotic expression according to the technology of routine, citing document: ExpressionoffulllengthTatinE.colianditspurification) and Tat-R5M4 and Jurkat cell hatch altogether, with the two dyeing of Annexin-V-FITC and PE after 48 hours, by Flow cytometry apoptosis ratio.
This experiment proves that Tat-R5M4 significantly reduces cytotoxicity and apoptosis-induced ability.
Embodiment three: that detects Tat-R5M4 wears film activity
Film activity is worn in order to verify whether Tat-R5M4 affects after sudden change, Tat-R5M4 albumen NHS-rhodamine (NHS-Rhodanmine) of purification has carried out labelling, and labeled albumen processes PBMC and Jurkat cell enter cell with flow cytometer detection Tat state after 6 hours respectively.Tat-R5M4 has and good wears film activity.Cell is entered later in the distribution situation of kytoplasm with kytoplasm in order to detect Tat-R5M4, Immunofluorescence test is carried out with the antibody of Tat-R5M4 after the Tat-R5M4 albumen process of Tzm-b1 cell purification, we find Tat-R5M4 enter cell after major part be distributed in kytoplasm, only have a small amount of albumen to enter karyon.In order to detect the distribution situation of Tat-R5M4 albumen in Mice Body, the Tat-R5M4 albumen of rhodamine labelling enters in Mice Body by tail vein injection, after 6 hours, the tissue such as spleen, thymus, brain and small intestinal detects after making section, finds that Tat-R5M4 is obviously distributed in these tissues.
This experiment demonstrates that improved albumen is complete to be remained it and wear membrane property, efficiently can enter biological cells and tissues and play function.
The structure of the external latent infection model of embodiment four: HIV-1
With with
bcl-2the pseudovirus of gene infects the primary CD4 activated
+t cell, usual positive rate is between 5%-10%.Infect after 3 days, we by airflow classification the cell sorting of the GFP positive out, continue to cultivate by the PRM1640 culture medium that with the addition of IL-2, add CD3 and CD28 antibody again to activate simultaneously, change into after one week and do not add any cytokine PRM1640 culture medium, cultivate 3-4 week and drop back except IL-2 makes cell progressively enter quiescent condition.Namely the cell of these quiescent conditions can be used for hiding the detection of activator.
The CD4 that embodiment five: Tat-R5M4 is originated at the external latent infection model of HIV-1 and clinical patient
+activation in T cell
Detect by the ability of latent infection system to Tat-R5M4 activating dormant infection of external structure, use CD3/CD28 and SAHA process as positive control simultaneously, three days afterwards by the ratio of fluorescence microscope GFP positive cell.
With CD3/CD28 antibody and SAHA as positive control, simultaneously with the clinical sample CD4 that Tat-R5M4 or Tat-R5M4 is separated with SAHA coupling process
+t cell, got supernatant after 18 hours, extracted the HIV-1RNA content in RNA detection supernatant.
More than experiment proves that Tat-R5M4 can activate the CD4 of the latency of the external latent infection model of HIV-1 and clinical patient samples sources effectively
+t cell, can strengthen its trans-activation effect with SAHA coupling simultaneously.
Experiment six: acute toxicity testing and immunogenicity detect
In order to provide certain theories integration to follow-up zoopery, the toxicity of Tat-R5M4 albumen is further detected.First, show with the acute toxicity testing that Babl/c mice carries out, when tail vein injection dosage reaches 40mg/ml, mice still survives well.The alanine aminotransferase of mice or paddy third (ALT), aspartate aminotransferase or millet straw (AST), blood urea nitrogen (BREA) and CR(creatinine) etc. index be all in normal level after testing, pathological section also shows, except wild type Tat-86 albumen causes the infiltration of liver's local inflammatory cells, the heart of mice, liver, spleen, pulmonary and kidney all do not show obvious damage, show that Tat-R5M4 is safe for mouse experiment.Meanwhile, we find that tail vein injection Tat-86 is after mono-week, and the spleen of mice has obvious enlargement, illustrate that Tat-86 can cause obvious immunoreation, but Tat-R5M4 can not cause similar phenomenon.Then we use Tat-86 and Tat-R5M4 subcutaneous injection immune mouse respectively, at 7 days, 14 days, dock respectively and get the concentration that Tat antibody is surveyed in blood examination when 21 days, find that the antibody amount that Tat-R5M4 stimulation produces obviously will lower than Tat-86.
This experiment proves that improved Tat-R5M4 is comparatively safe to mice, and immunogenicity significantly reduces, and is more suitable for carrying out experiment in vivo than wild type Tat-86.
SEQUENCELISTING
<110> Zhongshan University
<120> Tat albumen and its preparation method and application
<130>
<160>4
<170>PatentInversion3.3
<210>1
<211>86
<212>PRT
<213>Tat albumen
<400>1
MetGluProValAspProArgLeuGluProTrpLysHisProGlySer
151015
GlnProLysThrAlaCysThrAsnCysTyrCysLysLysCysCysPhe
202530
HisCysGlnAlaCysPheIleThrLysAlaLeuGlyIleSerTyrGly
354045
ArgLysLysArgArgGlnArgArgArgProProGlnGlySerGlnThr
505560
HisAlaAlaAlaLeuSerLysGlnProThrSerGlnSerArgGlyAsp
65707580
ProThrGlyProLysGlu
85
<210>2
<211>86
<212>PRT
<213>Tat albumen
<400>2
MetGluProValAspProArgLeuGluProTrpLysHisProGlySer
151015
GlnProLysThrAlaCysThrAsnCysTyrCysLysLysCysCysPhe
202530
HisCysGlnValCysPheAlaThrLysAlaLeuGlyIleSerTyrGly
354045
ArgLysLysArgArgGlnArgArgArgProProGlnGlySerGlnThr
505560
HisAlaAlaAlaLeuSerLysGlnProThrSerGlnSerArgGlyAsp
65707580
ProThrGlyProLysGlu
85
<210>3
<211>86
<212>PRT
<213>Tat albumen
<400>3
MetGluProValAspProArgLeuGluProTrpLysHisProGlySer
151015
GlnProLysThrAlaCysThrAsnCysTyrCysLysLysCysCysPhe
202530
HisCysGlnValCysPheIleThrLysAlaLeuGlyIleSerTyrGly
354045
ArgLysLysArgArgGlnArgArgArgProProGlnGlySerGlnThr
505560
HisAlaAlaAlaLeuSerLysGlnProThrSerGlnAlaArgGlyAsp
65707580
ProThrGlyProLysGlu
85
<210>4
<211>86
<212>PRT
<213>Tat albumen
<400>4
MetGluProValAspProArgLeuGluProTrpLysHisProGlySer
151015
GlnProLysThrAlaCysThrAsnCysTyrCysLysLysCysCysPhe
202530
HisCysGlnAlaCysPheIleThrLysAlaLeuGlyIleSerTyrGly
354045
ArgLysLysArgArgGlnArgArgArgProProGlnGlySerGlnThr
505560
HisAlaAlaAlaLeuSerLysGlnProThrSerGlnAlaArgGlyAsp
65707580
ProThrGlyProLysGlu
85
Claims (3)
1. attenuation Tat albumen is preparing the application in inverase, it is characterized in that, described attenuation Tat protein amino acid sequence is as shown in SEQNO:4.
2. an attenuation Tat albumen, is characterized in that, described attenuation Tat protein amino acid sequence is as shown in SEQNO:4.
3. the preparation method of an attenuation Tat albumen according to claim 2, it is characterized in that, first the site of rite-directed mutagenesis is selected, on the gene of Tat, rite-directed mutagenesis is carried out to these sites, finally retained the trans-activation function of Tat in a large number, and removed the attenuated proteins of wherein apoptosis and other activity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410259996.8A CN104001155B (en) | 2014-06-12 | 2014-06-12 | A kind of Tat albumen and its preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410259996.8A CN104001155B (en) | 2014-06-12 | 2014-06-12 | A kind of Tat albumen and its preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104001155A CN104001155A (en) | 2014-08-27 |
| CN104001155B true CN104001155B (en) | 2016-04-13 |
Family
ID=51362243
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410259996.8A Active CN104001155B (en) | 2014-06-12 | 2014-06-12 | A kind of Tat albumen and its preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104001155B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1283121A (en) * | 1997-12-01 | 2001-02-07 | 高等健康研究院 | HIV-ITAT or derwatives thereof for prophylatic and therapeutic vacceination |
| CN1419456A (en) * | 2000-01-31 | 2003-05-21 | 史密丝克莱恩比彻姆生物有限公司 | Vaccine for the prophylactic or therapeutic immunization against HIV |
| CN102056618A (en) * | 2008-04-22 | 2011-05-11 | 地中海大学 | Compositions and methods for preventing or treating AIDS |
-
2014
- 2014-06-12 CN CN201410259996.8A patent/CN104001155B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1283121A (en) * | 1997-12-01 | 2001-02-07 | 高等健康研究院 | HIV-ITAT or derwatives thereof for prophylatic and therapeutic vacceination |
| CN1419456A (en) * | 2000-01-31 | 2003-05-21 | 史密丝克莱恩比彻姆生物有限公司 | Vaccine for the prophylactic or therapeutic immunization against HIV |
| CN102056618A (en) * | 2008-04-22 | 2011-05-11 | 地中海大学 | Compositions and methods for preventing or treating AIDS |
Non-Patent Citations (1)
| Title |
|---|
| Tat蛋白：治疗HIV-1感染的新靶点;白如珺 等;《生命的化学》;20051231;第25卷(第6期);457-460 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104001155A (en) | 2014-08-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bangham | Human T cell leukemia virus type 1: persistence and pathogenesis | |
| Suspène et al. | Somatic hypermutation of human mitochondrial and nuclear DNA by APOBEC3 cytidine deaminases, a pathway for DNA catabolism | |
| Van der Burg et al. | Association of cervical cancer with the presence of CD4+ regulatory T cells specific for human papillomavirus antigens | |
| Singh et al. | The first dominant co-circulation of both dengue and chikungunya viruses during the post-monsoon period of 2010 in Delhi, India | |
| CN105567738A (en) | Method for inducing CCR5-delta32 deletion with genome editing technology CRISPR-Cas9 | |
| CA2999912A1 (en) | Methods and compositions for rna-guided treatment of hiv infection | |
| Sung et al. | HIV-specific, ex vivo expanded T cell therapy: feasibility, safety, and efficacy in ART-suppressed HIV-infected individuals | |
| Alexander et al. | Determinants of increased replicative capacity of serially passaged simian immunodeficiency virus with nef deleted in rhesus monkeys | |
| O’Neil et al. | The role of tissue resident memory CD4 T cells in herpes simplex viral and HIV infection | |
| Sarabia et al. | HIV-1 latency and latency reversal: does subtype matter? | |
| Ventura | Focus: Sex & Reproduction: Human Immunodeficiency Virus 1 (HIV-1): Viral Latency, the Reservoir, and the Cure | |
| Song et al. | Mast cell activation triggered by retrovirus promotes acute viral infection | |
| BROWN et al. | Sequence Note: HIV Type 1 Subtypes in Malaysia Include B, C, and E | |
| Mao et al. | Novel antiviral efficacy of Hedyotis diffusa and Artemisia capillaris extracts against Dengue virus, Japanese encephalitis virus, and Zika virus infection and immunoregulatory cytokine signatures | |
| CN104001155B (en) | A kind of Tat albumen and its preparation method and application | |
| Jeong et al. | A new cell line derived from the caudal fin of the dwarf gourami (Trichogaster lalius) and its susceptibility to fish viruses | |
| Désaulniers et al. | Editing of the TRIM5 gene decreases the permissiveness of human T lymphocytic cells to HIV-1 | |
| Rappocciolo et al. | A comparative study of natural killer cell activity, lymphoproliferation, and cell phenotypes in nonhuman primates | |
| Morgan | HTLV-1 Associated Myelopathy/Tropical Spastic Paraparesis. | |
| US9790255B2 (en) | Transactivator of transcription (TAT) proteins and preparation method | |
| Olwenyi et al. | Diminished peripheral CD29hi cytotoxic CD4+ T cells are associated with deleterious effects during SIV infection | |
| Schmalen et al. | The N-terminus of the HIV-1 p6 Gag protein regulates susceptibility to degradation by IDE | |
| Adil et al. | Variations in genomic epidemiology and in-silico screening of potential phytochemicals to cure Monkeypox | |
| Hassan et al. | The incidence of HIV among blood donors in Kaduna, Nigeria | |
| CN102465117A (en) | Chimeric simian/human immunodeficency virus strain and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20190723 Address after: 518108 Chuangwei Innovation Valley B1113, No. 8 Tangtou No. 1 Road, Tangtou Community, Shiyan Street, Baoan District, Shenzhen City, Guangdong Province Patentee after: Shenzhen Renewal City Biomedical Technology Co., Ltd. Address before: 510006 Panyu District City, Guangdong Province, University of outer ring road, No., No. 132 Patentee before: Sun Yat-sen University |