CN104130331B - A kind of virus drugs of AntiHIV1 RT activity 1 and its preparation and application - Google Patents

Abstract

The present invention provides a kind of virus drugs of AntiHIV1 RT activity 1（Also known as chimeric vector Rev Vif C）, it is to spell and be formed by connecting by the binding site of Rev albumen and the C-terminal of Vif albumen, described connection is the N-terminal that the multimerization domain of Rev is replaced Vif.Then this research prove that Rev Vif C carriers have good antiviral effect by designing and building Rev Vif C carriers by kinds of experiments, it is proposed that a kind of antivirus technology of the new Rev albumen for HIV 1.Successful Application of the Vif C carriers in Rev albumen of degrading, makes it be expected to turn into a kind of new gene knockout means.Vif C carriers can specifically degrade target proteinses, it is particularly well-suited to easily to produce the medicament research and development of the viral target protein that high frequency is mutated, as long as the binding site of albumen itself is not mutated, our chimeric vector will be permanently effective, such that it is able to reduce the drug resistance of antiviral drugs.

Description

A kind of anti-HIV-1 virus drugs and its preparation and application

Technical field

The present invention relates to antiviral drugs field, more particularly, to a kind of anti-HIV-1 virus drugs and its preparation and should With.

Background technology

At beginning of the eighties late 1970s, Europe and the U.S. occur in that one kind is main with immune system dysfunction The disease of feature.Then, scientists from all over the world have begun to seek its pathogenesis and therapeutic scheme.Until nineteen eighty-three, French Pasteur research group has been successfully separated out after this new retrovirus at first, theory of the people for HIV-1 Research and therapeutic treatment are also more and more therewith.At present, the medicine for HIV-l is mainly acted in the vial life cycle not Same link, especially some necessary enzyme such as reverse transcriptases and protease.

Although there is the medicine of many anti-HIV-1s in the market, the extensive use of HARRT, the resistance for producing therewith The problems such as property, huge medical expenses, side effects of pharmaceutical drugs, also should not be underestimated.Although HAART can make quite a few patient Plasma viral load is down to can be detected below horizontal, but bounce-back and serious toxic and side effect can not yet be solved after being discontinued；Base Although because treatment shows its antiviral potentiality, and having part achievement in research to enter clinical experimental stage, its efficiency is low, outer Adverse reaction that source carrier may bring etc. is still a major obstacle of research and development.At present, the research and development of anti-HIV-1 medicines in the world Mainly there are four focuses：One is to enter cytostatics；Two is neutralizing antibody；Three is integrase inhibitor；Four is chemotactic Cytokine receptor antagonist.Scientists are still in the exploration antiviral agent more safely and effectively, more economically applicable that keeps punching Thing.

Virion protein Expression modulation factor R ev（regulation of virion protein expression） It is indispensable modulin in HIV-1 transcriptions.The RRE of Rev and virus mRNA interacts, so as to help not shear Or the HIV-1 mRNA of Partial Shear are transported to outside core.If the expression of Rev is suppressed, the HIV- of non-montage or part montage 1 mRNA can not cause it degradable in core to being transported outside core, further the duplication of blocking HIV-1.Therefore, how Suppress the expression of Rev albumen, will be an important target spot for researching and developing anti-HIV-1 medicines.

The content of the invention

The present invention provides a kind of anti-HIV-1 virus drugs（Also known as chimeric vector Rev-Vif-C）, described anti-HIV-1 disease Cytotoxic drug is primarily directed to the Rev albumen of HIV-1 itself.

A kind of anti-HIV-1 virus drugs are provided in addition, and described anti-HIV-1 virus drugs are by Rev albumen and Vif Albumen is formed by connecting.

Described connection is the N-terminal that the multimerization domain of Rev is replaced Vif.

A kind of application of Vif albumen in anti HIV-1 virus medicine is prepared is provided again.

A kind of preparation method of above-mentioned anti-HIV-1 virus drugs is provided in addition, is comprised the following steps：

S1. there are two oligomerization domains on the protein structure of Rev, the two oligomerization domains are replaced into Vif respectively The 1-79 amino acids sequences of albumen n end, so as to construct three new chimeric protein ROL1-Vif-C, ROL2-Vif-C and ROL12-Vif-C, described ROL1-Vif-C, the oligomerization domain comprising Rev N-terminals, described ROL2-Vif-C is included The oligomerization domain of Rev C-terminals, described ROL12-Vif-C includes two oligomerization domains of Rev N-terminals and C-terminal,

The oligomerization domain of described Rev N-terminals is the 9-26 amino acids of Rev protein amino acid sequences,

The oligomerization domain of described Rev C-terminals is the 51-65 amino acids of Rev protein amino acid sequences,

Described two oligomerization domains of Rev N-terminals and C-terminal are the 9-65 bit aminos of Rev protein amino acid sequences Acid.

Described Vif-C parts are the 80-193 amino acids of Vif protein amino acid sequences

S2. these three chimeric vectors are cloned on expression vector pcDNA3.1 respectively, instantaneous table is carried out by transfection Reach, wherein, in S1, the described following SEQ NO of 3 carrier sequences:1、SEQ NO:2 and SEQ NO:Shown in 3, carrier is connected Restriction enzyme site used is bamH I and Xhol I on to pcDNA3.1.

Demand according to more than provides three kinds of applications, a kind of for above-mentioned anti-HIV-1 virus drugs are preparing anti-HIV-1 medicine Application in thing.

Another is that above-mentioned anti-HIV-1 virus drugs are preparing the application suppressed during HIV-1 replicates medicine.

A kind of application of above-mentioned anti-HIV-1 virus drugs in the Rev albumen of degraded HIV-1.

Vif albumen is an important albumen of HIV-1 itself, can combine target proteinses, and they are attached to an E3 Ligase（ligase）Compound（Vif-SCF-CUL5 complex）On, then this compound will make target proteinses ubiquitin Change, so as to cause it in proteasome（Proteasome）In degraded.The main ubiquitination work(according to Vif albumen of the invention Can, the multimerization domain of Rev is replaced the N-terminal of Vif, can specifically with reference to the Rev albumen of HIV-1, Ran Houtong The ubiquitination function degraded Rev of Vif is crossed, oneself shield is attacked with oneself lance, suppress the mesh ground that HIV-1 is replicated so as to reach, with very Strong novelty and specificity, this is also for research and development anti-HIV-1 medicines provide a kind of brand-new thinking and method.

（1）In order to overcome the shortcoming and deficiency of prior art, it is an object of the invention to provide a kind of new chimeric load The method of the structure of body and its application in life science and clinical treatment.

（2）It is the invention provides a kind of new tool for protecting cell not attacked by HIV-1, the CD4+T in patient's body is thin After born of the same parents sort out amplification in vitro, expression Rev-Vif-C carriers are stabilized it, then fed back in patient's body, can not only protected Patient from HIV-1 attack again, while patient can also be aided in understand internal HIV-1 viruses.

（3）The present invention proposes a kind of new support of brand-new anti-HIV-1 spontaneous mutation --- the drug resistance of HIV-1 it is many because Its drug-induced self-break, and our chimeric vector is built using be combineding with each other for Rev self structures domain, can be with Possible various mutation are avoided, thus there can be good inhibition to multiple HIV-1 mutant strains

（4）The present invention proposes a kind of new technology of degraded Rev protein expressions

（5）The present invention proposes a kind of new technology of new degraded multiple protein --- being combined into for certain albumen a little inserted Enter the N-terminal to Vif genes, so as to realize the degradation process of specific proteins by the ubiquitination path of Vif C-terminals.

Brief description of the drawings

Fig. 1：The structure model of chimeric vector Rev-Vif-C.

Fig. 2：Chimeric vector Rev-Vif-C is by ubiquitination path degraded Rev albumen.

Fig. 3：Chimeric vector Rev-Vif-C suppresses various wild type HIV-1 viruses by suppressing the kernel function that of Rev-RRE The duplication of strain.

Specific embodiment

Below in conjunction with the accompanying drawings the present invention is further described with specific embodiment.Unless stated otherwise, the present invention is used Reagent, apparatus and method be the art reagent routinely purchased in market, equipment and conventional use of method.

The structure model of the chimeric vector Rev-Vif-C of embodiment 1

By two oligomerization binding structural domains on HIV-1 Rev genes（9-26 amino acids and 51-65 amino Acid）The N-terminal of Vif genes is cloned into respectively, is substituted into the binding site of APOBEC3G（1-79 amino acids）, it is then attached to Formd on the carrier of pcDNA3.1 3 it is different chimeric, be respectively designated as ROL1-Vif-C, ROL2-Vif-C, ROL12-Vif-C.Wherein ROL1 represents the oligomerization binding structural domain of the N-terminal portion of Rev, i.e. 9-26 amino acids；ROL2 Represent the oligomerization binding structural domain of the C-terminal part of Rev, i.e. 51-65 amino acids；ROL12 represents the N-terminal and C of series connection Rev Two oligomerization binding structural domains at end, i.e. 9-26 amino acids and 51-65 amino acids.

Comprise the following steps that：

（1）There are two oligomerization domains on the protein structure of Rev, the two oligomerization domains are replaced into Vif respectively The 1-79 amino acids sequences of albumen n end, so as to construct three new chimeric protein ROL1-Vif-C（Comprising Rev N-terminals Oligomerization domain, i.e. 9-26 amino acids）、ROL2-Vif-C（Oligomerization domain comprising Rev C-terminals, i.e. 51-65 Amino acids）And ROL12-Vif-C（Comprising two oligomerization domains of Rev N-terminals and C-terminal, i.e. 9-65 amino acids）

（2）These three chimeric vectors are cloned on expression vector pcDNA3.1 respectively, transient expression is carried out by transfection

Wherein, step（1）In, described vector construction is comprised the following steps：Vif albumen and Rev albumen according to HIV-1 4 pairs of primers are respectively synthesized, then the PNL4-3 plasmid sequences with HIV-1 viruses enter performing PCR and expand as template using above-mentioned primer Increase；Then pcr amplification product is cloned on pcDNA3.1 using different restriction enzyme sites, the fragment insertion position of wherein Rev In the N-terminal of Vif fragments.

The chimeric vector Rev-Vif-C of embodiment 2 is by ubiquitination path degraded Rev albumen

By HIV-1 Rev genes and RFP amalgamation and expressions and it is cloned on the carrier of pcDNA3.1, so can be by observation RFP's expresses to reflect the expression of Rev；Rev genes and HA tag fusions are expressed simultaneously, is conveniently western blot Further to verify the expression of Rev.

（1）In the 293t cells of 24 orifice plates, cotransfection Rev-RFP and ROL1-Vif-C（Or ROL2-Vif-C or ROL12-Vif-C）Carrier, the amount of plasmid is respectively 1：0,1：1,1：2,1：3, the expression of RFP is observed after transfection 48h, while 6 In the 293t cells of orifice plate, cotransfection Rev-HA and ROL1-Vif-C carrier, the amount of plasmid is respectively 1：0,1：1,1：2,1：3, Transfected after transfection 48h and receive the expression that Western Blot detections Rev is in cell cracking

The description of test, three chimeric vectors can suppress the expression of Rev albumen, and with certain concentration ladder Degree dependence.

（2）In the 293t cells of 24 orifice plates, the ng Rev-RFP of cotransfection 200 and 200 ng ROL12-Vif-C carriers, Add the MG132 of 10 uM to process cell after 24 h, the expression of RFP is observed after 12 h for the treatment of

（3）In the 293t cells of 24 orifice plates, the ng Rev-RFP of cotransfection 200 and 200 ng ROL12-Vif-C carriers, Si-NC, si-ElonginB, si-ElonginC and the si-Culin5 of 50 nM are transfected after 6h again, RFP is observed after transfection 48h Expression

（4）In the 293t cells of 6cm plates, difference cotransfection 3 ug Ub-Flag, 3 ug Rev-HA and 2 ug ROL1- Vif-C, ROL2-Vif-C, ROL12-Vif-C carrier, receive cell cracking and do Co-IP enrichment Rev-HA albumen after transfection 48h, and Further detect the ubiquitination situation of Rev albumen

Three above description of test, chimeric vector is the ubiquitination path degraded Rev albumen mediated by Vif.

The chimeric vector Rev-Vif-C of embodiment 3 suppresses various wild type HIV- by suppressing the kernel function that of Rev-RRE The duplication of 1 Strain

There is SD and SA shearing sites on PDM628, when Rev albumen does not exist, carried on PDM 628 Luciferase genes cause luciferase trace expression by editing；When two kinds of plasmid corotation, Rev and RRE is combined, will Luciferase genetic fragments take nucleus out of, it is to avoid by SD and SA editings, make luciferase great expression.Therefore, when Rev eggs When white expression receives suppression, Rev-RRE is related to be gone out nuclear translocation system and will be suppressed, now the expression quantity of luciferase will under Drop.Based on this principle, it can be determined that whether the function of Rev albumen can be affected.

（1）In the 293t cells of 96 orifice plates, the Rev plasmids of the difference ng pDM628 plasmids of cotransfection 10 and 10 ng, so ROL1-Vif-C, ROL2-Vif-C, ROL12-Vif-C and M10 plasmid of cotransfection various concentrations again, receives thin after transfection 48h afterwards Cellular lysate detects the expression of luciferase

The description of test, three chimeric vectors can suppress Rev-RRE correlations and go out nuclear translocation system, and with one Fixed concentration gradient dependence, meanwhile, the effect of our chimeric vector will be substantially better than existing RevM10 mutant.

（2）In the 293t cells of 24 orifice plates, the ROL1- of the difference ng PNL4-3 plasmids of cotransfection 50 and various concentrations Vif-C, ROL2-Vif-C, ROL12-Vif-C plasmid（50 ng, 100 ng, 150 ng）, supernatant ELISA inspections are received after transfection 48h Survey the expression of P24（B）In the 293t cells of 24 orifice plates, respectively the ng PYU-2 plasmids of cotransfection 50 and various concentrations ROL1-Vif-C, ROL2-Vif-C, ROL12-Vif-C plasmid（50 ng, 100 ng, 150 ng）, supernatant is received after transfection 48h ELISA detects the expression of P24

The description of test, three chimeric vectors can suppress the duplication of various different wild type HIV-1, and have There is certain concentration gradient dependence.

SEQUENCE LISTING

<110>Zhongshan University

<120>A kind of anti-HIV-1 virus drugs and its preparation and application

<130>

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 405

<212> DNA

<213> ROL1-Vif-C

<400> 1

atggacagcg acgaagagct catcagaaca gtcagactca tcaagcttct ctatcaaagc 60

aaccatttgg gtcagggagt ctccatagaa tggaggaaaa agagatatag cacacaagta 120

gaccctgaac tagcagacca actaattcat ctgtattact ttgactgttt ttcagactct 180

gctataagaa aggccttatt aggacacata gttagcccta ggtgtgaata tcaagcagga 240

cataacaagg taggatctct acaatacttg gcactagcag cattaataac accaaaaaag 300

ataaagccac ctttgcctag tgttacgaaa ctgacagagg atagatggaa caagccccag 360

aagaccaagg gccacagagg gagccacaca atgaatggac actag 405

<210> 2

<211> 387

<212> DNA

<213> ROL2-Vif-C

<400> 2

atgatccatt cgattagtga acggatcctt ggcacttatc tgggacattt gggtcaggga 60

gtctccatag aatggaggaa aaagagatat agcacacaag tagaccctga actagcagac 120

caactaattc atctgtatta ctttgactgt ttttcagact ctgctataag aaaggcctta 180

ttaggacaca tagttagccc taggtgtgaa tatcaagcag gacataacaa ggtaggatct 240

ctacaatact tggcactagc agcattaata acaccaaaaa agataaagcc acctttgcct 300

agtgttacga aactgacaga ggatagatgg aacaagcccc agaagaccaa gggccacaga 360

gggagccaca caatgaatgg acactag 387

<210> 3

<211> 537

<212> DNA

<213> ROL12-Vif-C

<400> 3

atggcaggaa gaagcggaga cagcgacgaa gagctcatca gaacagtcag actcatcaag 60

cttctctatc aaagcaaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat 120

agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatcctt 180

ggcacttatc tgggacattt gggtcaggga gtctccatag aatggaggaa aaagagatat 240

agcacacaag tagaccctga actagcagac caactaattc atctgtatta ctttgactgt 300

ttttcagact ctgctataag aaaggcctta ttaggacaca tagttagccc taggtgtgaa 360

tatcaagcag gacataacaa ggtaggatct ctacaatact tggcactagc agcattaata 420

acaccaaaaa agataaagcc acctttgcct agtgttacga aactgacaga ggatagatgg 480

aacaagcccc agaagaccaa gggccacaga gggagccaca caatgaatgg acactag 537

Claims (4)

1. a kind of anti-HIV-1 virus drugs, it is characterised in that described anti-HIV-1 virus drugs are by Rev albumen And Vif

Albumen is formed by connecting, specially respectively by the 9-26 amino acids of Rev protein amino acid sequences, Rev albumen ammonia The 51-65 amino acids of base acid sequence or the 9-65 amino acids of Rev protein amino acid sequences replace Vif albumen The 1-79 amino acids sequences at N ends, so as to construct three new chimeric protein ROL1-Vif-C, ROL2-Vif- C and ROL12-Vif-C,

Oligomerization domains of the described ROL1-Vif-C comprising Rev N ends, described ROL2-Vif-C includes Rev C The oligomerization domain at end, described ROL12-Vif-C includes Rev N ends and two, C ends oligomerization domain,

The oligomerization domain at described Rev N ends is the 9-26 amino acids of Rev protein amino acid sequences, institute The oligomerization domain at the Rev C ends stated is the 51-65 amino acids of Rev protein amino acid sequences,

Described Rev N ends and two, C ends oligomerization domain are 9-65 ammonia of Rev protein amino acid sequences Base acid,

Described Vif-C parts are the 80-193 amino acids of Vif protein amino acid sequences.

2. a kind of preparation method of anti-HIV-1 virus drugs, it is characterised in that comprise the following steps：

S1. described anti-HIV-1 virus drugs are by Rev albumen and Vif

Albumen is formed by connecting, specially respectively by the 9-26 amino acids of Rev protein amino acid sequences, Rev albumen ammonia The 51-65 amino acids of base acid sequence or the 9-65 amino acids of Rev protein amino acid sequences replace Vif albumen The 1-79 amino acids sequences at N ends, so as to construct three new chimeric protein ROL1-Vif-C, ROL2-Vif- C and ROL12-Vif-C,

Oligomerization domains of the described ROL1-Vif-C comprising Rev N ends, described ROL2-Vif-C includes Rev C The oligomerization domain at end, described ROL12-Vif-C includes Rev N ends and two, C ends oligomerization domain,

The oligomerization domain at described Rev N ends is the 9-26 amino acids of Rev protein amino acid sequences, institute The oligomerization domain at the Rev C ends stated is the 51-65 amino acids of Rev protein amino acid sequences,

Described Rev N ends and two, C ends oligomerization domain are 9-65 ammonia of Rev protein amino acid sequences Base acid,

Described Vif-C parts are the 80-193 amino acids of Vif protein amino acid sequences,

S2. these three chimeric proteins are cloned on expression vector pcDNA3.1 respectively, transient expression are carried out by transfection, Wherein, in S1, three sequence difference of new chimeric protein ROL1-Vif-C, ROL2-Vif- C and ROL12-Vif-C Such as SEQ NO:1、SEQ NO:2 and SEQ NO:Shown in 3, three new chimeric proteins are connected to institute on pcDNA3.1 Restriction enzyme site is bamH I and Xhol I.

3. application of the anti-HIV-1 virus drugs described in a kind of claim 1 in anti-HIV-1 medicine is prepared.

4. the anti-HIV-1 virus drugs described in a kind of claim 1 are preparing answering in suppressing HIV-1 duplication medicines With.