CN1176948C - Single-chain antibody resisting HIV-1 outer membrane protein and recombinant immunotoxin - Google Patents
Single-chain antibody resisting HIV-1 outer membrane protein and recombinant immunotoxinInfo
- Publication number
- CN1176948C CN1176948C CNB011202408A CN01120240A CN1176948C CN 1176948 C CN1176948 C CN 1176948C CN B011202408 A CNB011202408 A CN B011202408A CN 01120240 A CN01120240 A CN 01120240A CN 1176948 C CN1176948 C CN 1176948C
- Authority
- CN
- China
- Prior art keywords
- ggc
- gga
- act
- cag
- ggt
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The present invention provides a single-chain antibody resisting HIV-1 outer membrane protein and two kinds of recombinant immunotoxin resisting HIV-1, wherein the single-chain antibody resisting HIV-1 outer membrane protein has a specific gene sequence; the two kinds of recombinant immunotoxin are prepared by using the single-chain antibody resisting HIV-1 outer membrane protein to respectively replace pseudomonas aeruginosa exotoxin and the receptor bonding area of diphtheria toxin. The single-chain antibody resisting HIV-1 outer membrane protein can be applied to the diagnosis and the immune treatment of AIDS virus infection; the recombinant immunotoxin can carry out specific killing and damage to target cells infected by HIV-1 and can be used as treating medicaments of AIDS virus infection.
Description
Technical field:
The invention provides single-chain antibody gene and two kinds of recombinant immunotoxins of anti-people I type immunodeficiency virus (HIV-1) outer membrane protein gp120, belong to biological technical field at the HIV-1 cells infected.
Background technology:
Monoclonal antibody plays a significant role in the diagnosis of acquired immune deficiency syndrome (AIDS) and treatment, but there are two difficult problems in mouse monoclonal antibody in actual applications as medicine: the one, and mouse source antibody is heterology albumen to the people, uses the people repeatedly and knows from experience that to produce anti-mouse antibody be HAMA (human anti-mouse antibody); The 2nd, the monoclonal antibody molecular weight is big, and tissue penetration is poor.In addition, because the hybridoma cell strain of monoclonal antibody can cause heritable variation in the prolonged preservation process, make the ability drop of its secrete monoclonal antibody, people-people's hybridoma exist again can not be arbitrarily immunity, build the strain difficulty, yield poorly, difficulty such as instability, therefore limited monoclonal antibody as medicine application clinically.In order to make antibody humanization and miniaturization, gene work post antibody arises at the historic moment, and people-mouse chimeric antibody (human-mouse chimeric antibody), reshaping antibody (reshaped antibody), small molecular antibody (as Fv, Fab, scFv), recombinant phages antibody library technology (recombinant bacteriophage antibody repertoire) several stages have roughly been experienced in its evolution.
The focus of genetic engineering antibody research at present is a small molecular antibody, and especially (single chainFv fragment scFv) is favored single-chain antibody.Single-chain antibody is the small molecular antibody that utilizes the genetic engineering means preparation, it is that connection peptides Linker by one section synthetic couples together the weight chain variable region gene of natural antibody, it is little to have molecular weight, penetrance is strong, antigenicity is low, is beneficial to advantages such as carrying out genetically engineered operation and great expression.ScFv has the following advantages: gene structure is simple, and construction expression is convenient, and it is formed by connecting by the small peptide of one 15 peptide linker (Gly4Ser) 3 by chain variable region gene VL and heavy chain variable region gene VH.ScFv can express in intestinal bacteria and eukaryotic cell, is considered to have the minimum antibody fragment of high-affinity; (1) have only the V district, more much smaller than corresponding mouse source monoclonal antibody immunogenicity; (2) molecular weight is about 27kDA, and tissue penetration is strong, is fit to targeted therapy; (3) do not have the constant region fc fragment, thus can not with the Fc receptors bind on the non-target cell, specificity strengthens;
The range of application of single-chain antibody is wider, utilize the means of gene therapy, the ScFv of anti-HIV-1 gp120 of preparation is expressed in cell, can produce intracellular antibody with critical treatment meaning, because HIV-1gp120 albumen duplicating in virus, in the invasion procedure, play crucial effect.Therefore the intracellular antibody at HIV-1 gp120 can produce the stronger resistibility of HIV-1 infection, thereby virus titer is reduced.For the antiviral efficacy of its generation, depend on the position of expressing in its affine active height and the born of the same parents.Therefore can on strand is anti-, add nucleus signal for locating (NLS) or endoplasmic reticulum stick signal (KDEL) etc.Or respectively introduce the disulfide linkage fixed FV section that a halfcystine forms in the appropriate location, variable region of the weight chain of ScFv, be built into and have the more DsFv antibody of high-affinity.In addition, at the V of single-chain antibody
LEnd adds more relevant toxin genes; As PE40, DT etc. can be built into the recombinant toxin class " suicide molecule " with killing ability, finish the destruction of killing and wounding to the target cell that carries HIV-1 viral protein mark, thereby reduce virus hiding in vivo.Aspect the HIV-1 diagnosis of infection.Can be by the different marker gene of further coupling, by indirect ELISA or strive blocking-up ELISA method unexpectedly and finish diagnosis to HIV-1 antigen or antibody.
Utilize the recombinant immunotoxin of anti-HIV-1 outer membrane protein single-chain antibody preparation, can be used for medicine as AIDS viral infection, if be used for the drug main chemicals of HIV-1 treatment at present both at home and abroad, the nucleoside medicine that at first began in 1987, as zidovudine (ADV), didanosine (ddI), Zha Xita river in Henan Province shore (ddC), Boyan Radev fixed (3TC) etc., but clinical application, nucleoside medicine is unsatisfactory to the curative effect of HIV-1; 1994, research through 4-5, non-nucleoside reverse transcriptase inhibitors and proteinase inhibitor begin to be used to clinical, mainly comprise nevirapine, Saquinavir, Ritonavir and Indinavir, these medicines can reduce virus quantity, increase the cd4 cell counting, but single medicine unsatisfactory curative effect, nineteen ninety-five is brought into use the combination therapy of multiple medicine, be highly efficient anti-virus therapy (Highly active anti-retroviral therapyHAART), discover at present, the highly efficient anti-virus therapy can obtain better therapeutic effect, can effectively suppress virus replication, play crucial effect for the treatment that HIV-1 infects, it can remove the free HIV-1 virus particle in the blood basically fully.However, this method is treatment of AIDS fully still, its basic reason is, the latent infection that in immobilized CD4+ memory T lymphocyte, has HIV-1, therefore when stopping using the curative drug of highly efficient anti-virus therapy, the HIV-1 of this latent infection can duplicate once more along with the lymphocytic activation of CD4+ memory T that originally remains static, cause the bounce-back of HIV-1 viral load in the body, for this situation, people have proposed two kinds of feasible assisting therapy approach, and first kind of approach is exactly to utilize IL2 to activate the HIV-1 infection CD4 that script remains static
+The memory T lymphocyte, activated CD4
+The memory T lymphocyte can be discerned by the ctl response of body and kill and wound, and the HIV-1 of Gan Raning itself also can be to activated CD4 in addition
+The memory T lymphocyte causes certain destruction, thereby these cell disruptions are loose and discharges the HIV-1 virus particle, these virus particle then by the neutralizing antibody of body and HAART medicine kill; Another therapeutic strategy is that the target cell that utilizes recombinant toxin that HIV-1 is infected carries out specific killing destruction, this recombinant toxin can directly destroy the static CD4+ memory T lymphocyte that is infected by HIV-1 on the one hand, also can make it in addition to activate, and can quicken activated HIV-1 and infect the lymphocytic disintegration of T.
The structure of recombinant toxin is former at present is to utilize certain specific carrier is arranged, medicine or other active substances that can kill and wound the HIV-1 target cell infection optionally are transported to the target cell position that HIV-1 infects, to kill and wound by the parasitic cell of virus, remove hiding of virus, thereby reach a kind of treatment approach and the method that improves curative effect.The targeted drug great majority of the anti-HIV-1 of development can utilize the beautiful property monoclonal antibody of people CD4 surface antigen or anti-HIV-1 structural protein as oriented carrier at present.These monoclonal antibodies at target be generally the VAA on HIV-1 target cell infection surface or specific acceptor.Material as " bullet " mainly contains three classes, i.e. radionuclide, medicine and toxin.Experiment in vitro proves that recombinant toxin can kill and wound the target cell that is infected by HIV-1 specifically at present, suppresses virus replication.
Summary of the invention:
The object of the present invention is to provide the single-chain antibody gene and the correlated series of anti-HIV-1 outer membrane protein, the outer membrane protein at HIV-1 is provided, can effectively discern and kill and wound two kinds of recombinant immunotoxins of HIV-1 target cell infection, be used for the novel biological engineering medicine of the treatment of acquired immune deficiency syndrome (AIDS).
The present invention program's implementation method: with the single-chain antibody gene of anti-HIV-1, be cloned among the related vector, be used to carry out the gene therapy of AIDS viral infection; With the single-chain antibody gene of anti-HIV-1, to express at protokaryon and eukaryotic system, the recombinant single chain antibody that obtains can be used for the passive immunization preparation as AIDS viral infection; The recombinant immunotoxin of anti-HIV-1 can be directly used in the adjuvant therapy medicaments as patients infected hiv.
Below narrate main contents of the present invention:
The single-chain antibody of a kind of anti-HIV-1 outer membrane protein gp120, the gene order that it is characterized in that this single-chain antibody are the gene orders of Figure of description 1 indication of the present invention.Utilize the single-chain antibody of this anti-HIV-1 outer membrane protein gp120 to can be used as the diagnosis of acquired immune deficiency syndrome (AIDS), the ways and means of treatment.
A kind of recombinant immunotoxin of anti-HIV-1, it is characterized in that this recombinant immunotoxin is made of single-chain antibody and the Pseudomonas aeruginosa exotoxin A of anti-HIV-1 outer membrane protein gp120, wherein, the receptor binding domain of Pseudomonas aeruginosa exotoxin A is replaced by the single-chain antibody of anti-HIV-1 outer membrane protein gp120.Remove the Pseudomonas aeruginosa extracellular toxin PE40 nucleotide sequence of receptor binding domain and see Fig. 3, the building process of this recombinant toxin scFv-PE as shown in Figure 6.This recombinant immunotoxin of the present invention can be used as the medicine of acquired immune deficiency syndrome (AIDS).
A kind of recombinant immunotoxin of anti-HIV-1, it is characterized in that this recombinant immunotoxin is made of single-chain antibody and the diphtheria toxin of anti-HIV-1 outer membrane protein gp120, wherein, the receptor binding domain of diphtheria toxin is replaced by the single-chain antibody of anti-HIV-1 outer membrane protein gp120.Remove the nucleotide sequence of the diphtheria toxin DT of receptor binding domain and see Fig. 4, the building process of this recombinant toxin DT-scFv as shown in Figure 5.This recombinant immunotoxin of the present invention can be used as the medicine of acquired immune deficiency syndrome (AIDS).
Experiment showed, that the present invention has following advantage:
1, the anti-HIV-1 single-chain antibody gene that obtains of the present invention, proof not only has very strong antigen binding characteristic after expression, and can effectively discern the HIV-1 outer membrane protein of different isolates.
2, recombinant immunotoxin involved in the present invention, experiment in vitro proves, but the target cell that direct killing is infected by HIV-1, and to healthy cell and the no destruction of tissue.
3, recombinant immunotoxin involved in the present invention does not have obvious toxic-side effects to laboratory animal, does not produce the specificity pathological change.
Description of drawings:
Fig. 1 is the gene order of anti-HIV-1 outer membrane protein gp120 single-chain antibody scFv of the present invention
Fig. 2 is the preparation principle figure of anti-HIV-1 recombinant toxin of the present invention
Fig. 3 is a Pseudomonas aeruginosa extracellular toxin PE40 nucleotide sequence of the present invention
Fig. 4 is the nucleotide sequence of diphtheria toxin DT of the present invention
Fig. 5 is the building process synoptic diagram of recombinant toxin DT-scFv of the present invention
Fig. 6 is the building process synoptic diagram of recombinant toxin scFv-PE of the present invention
Fig. 7 is that the different concns recombinant toxin infects the lymphocytic cytotoxic effect of MT4 to HIV-1
Embodiment:
Preparation and evaluation narration implementation method of the present invention below by separation of anti-HIV-1 outer membrane protein single-chain antibody gene and recombinant toxin.
1. the cultivation of monoclonal antibody hybridoma cell strain, preservation and recovery
Ordinary method is adopted in the cultivation of hybridoma cell strain, preservation and recovery, " modern immunological experiment technology " Hubei science tech publishing house that concrete steps are write with reference to Shen Guanxin, with your unicorn etc., 24-25 page or leaf.It is the M-1640 perfect medium that hybridoma cell strain is cultivated used substratum.
2. the genetic manipulation of molecular biology routine
The recovery of the extraction of the preparation of competent escherichia coli cell and conversion, plasmid and digestion with restriction enzyme, dna fragmentation, linear DNA segmental is connected, screening and evaluation, pcr amplification reaction, SDS-polyacrylamide gel electrophoresis, the protein blot experiment of recombinant plasmid are translated " molecular cloning experiment guide " second edition with reference to Jin Dongyan, Li Mengfeng etc., and Science Press (1992) related Sections carries out.
3. the separation of mouse source single-chain antibody gene and sequencing
(1) extraction of the total RNA of hybridoma
Cultivating the monoclonal antibody hybridoma cell strain 102 (cell strain is preserved in this chamber preparation) of secretion anti-HIV-1 outer membrane protein gp120 to the optimum growh state, calculates the cell total amount and is approximately 2～3 * 10
7Scrape with the cell sleaker and to get cell, centrifugal collection utilizes total RNA extraction reagent box extraction separation to go out total RNA, does not have the RNase water dissolution with 500ul, and 20 ℃ of placements are standby.
(2) separation of hybridoma mRNA
Total RNA that the last step was extracted utilizes vitamin H polymer-magnetic bead mRNA purification kit separation and purification mRNA, and resuspended with 100ul water ,-70 ℃ frozen.
(3) cDNA article one chain is synthetic
With the method that the mRNA that extracts provides by Mouse ScFv Module Recombinant Phage Autibodysystem, synthetic cDNA under the effect of ThermoScript II.
(4) rat immune globulin V
HAnd V
LThe pcr amplification of gene and product purification thereof
Get above-mentioned synthetic cDNA product respectively and use V respectively
L, V
HGene primer (the Mouse ScFv Module Recombinant phage Antibody System that is produced by pharmacia company provides), with 94 ℃ of 1min, 55 ℃ of 2min; The PCR art formula of 72 ℃ of 2min, light, the heavy chain variable region gene fragment of 30 cyclic amplification antibody of effect.
(5) assembling of ScFv
The V that reclaims
HAnd V
LGene fragment and Lirker-primer Mix with etc. volumetric molar concentration mix, use RSprimer Mix, and add dNTPs, Tag archaeal dna polymerase 5 units carry out 7 circulations, reaction conditions is (94 ℃ of 1min, 63 ℃ of 4min), reacts V with this
H, V
LGene splicing is the ScFv gene.This reaction can add ScFv gene two ends respectively SfiI and NotI restriction enzyme site.Adopt Wizard PCR prepsDNA Purification system to reclaim the ScFv gene product.
(6) clone of ScFv gene and order-checking
With the add-on PCR product of above-mentioned purifying, at T
4Under the effect of dna ligase, be connected with pGEM-T carrier system (Promega company product), be converted among the recipient bacterium DH5a, the recombinant plasmid that extraction and purifying contain ScFv checks order with the full-automatic fluorescent DNA sequenator of ABI377.
4. mouse source single-chain antibody gene is humanization modified
Utilize computer to carry out aided design, find out the pivot frame district, mouse source of single-chain antibody, it is replaced to utilize human antibody pivot frame district gene pairs.
5, the preparation of single-chain antibody and recombinant immunotoxin
(1) expression in intestinal bacteria of single-chain antibody and recombinant immunotoxin is (except that utilizing pET28 prokaryotic expression carrier of the present invention, also can use other carrier system express recombinant immunotoxin, the sex change of inclusion body, renaturation and purification process, except that the method for being stated herein, also available other method)
Utilize a pair of primer of artificial design, the Humanized single chain antibody gene is carried out the add-on PCR amplification, the receptor binding domain that the single-chain antibody gene that obtains is replaced Pseudomonas aeruginosa extracellular toxin PE and diphtheria toxin DT gene respectively, obtain recombinant toxin gene, single-chain antibody and recombinant toxin gene are cloned among the escherichia coli prokaryotic expression carrier pET28 (Novagen company product), obtain recombinant expression plasmid, in the recombinant plasmid transfection acceptor e. coli bl21 (DE3) (Novagen company product), carry out the IPTG abduction delivering, expression product utilizes the SDS-polyacrylamide gel electrophoresis to measure the expression amount of single-chain antibody and recombinant immunotoxin, single-chain antibody N ' the end of wherein expressing merges the purifying sign that His-Tag is arranged, and this phraseology can be convenient to single-chain antibody is carried out determination of activity.
Preparation process below in conjunction with accompanying drawing 5 narrations recombinant toxin DT-ScFv of the present invention.
The primer sequence that the DT recombinant toxin uses:
Primer A:
5’---AGA?TAT?
ACC?ATG?GGC?GCT?GAT?GAT?GTT?GTT?GAT---3’
NcoI
Primer B:
5’---A?GTG?CCT?GAA?TTC?
GGA?TCC?GGA?AGC?AAA?TGG?CGT?T---3’
BamHI
Primer C:
5’---CCA?TTT?GCT?TCC?GGA?GGT?CCT?GAG?CCA?GGT?CAA?GCT?GCA?GGA?G---3’
Primer D:
5’---CCC?
AAG?CTT?CTA?CTA?TAT?TTC?CAG?CTT?GGT?CC---3’
HindIII
Recombinant toxin DT-ScFv is prepared as the receptor binding domain that the ScFv gene is replaced the DT gene, its concrete building process is, utilize PCR method amplification DT gene, use primer (A and B), among the primer A, NcoI site and ATG sequence have been added at the DT upstream region of gene, primer B has added coding A, S, three amino acid whose nucleotide sequences of G and BamHI site in DT gene (389 amino acids) downstream, after the DT gene that amplifies uses NcoI and BamHI enzyme to cut digestion, be cloned in the pET28 carrier construction recombination plasmid pET28-DT; Utilize PCR method amplification ScFv gene, the primer (C and D) that uses, contain 388,389 amino acids sequences and coding A, S, G, G, the nucleotide sequence of P, E and the upstream sequence of ScFv among the primer C, primer D has introduced HindIII restriction enzyme site and terminator codon in ScFv gene downstream;
With DT and ScFv gene behind pcr amplification, reclaim purifying, as template, through the overlapping pcr amplification PCR reaction of chain (Overlap extension SOE), amplification DT-ScFv mosaic gene, use ClaI and HindIII to carry out enzyme the mosaic gene that obtains and cut digestion, be cloned into ClaI and the HindIII site of pET28-DT, obtain recombinant plasmid pET28-DT-ScFv.
Preparation process below in conjunction with accompanying drawing 6 narrations recombinant toxin ScFv-PE of the present invention.
The primer sequence that the ScFv-PE recombinant toxin uses:
Primer I:
5’-AGA?TAT?
ACC?ATG?AGG?TCA?AGC?TGC?AGG?AG-3’
NcoI
Primer I I:
5’-G?GAA?TTC?
CAT?ATG?TCC?GGA?AGC?TAT?TTC?CAG?CTT?GGT?CCC?CCC?TC-3’
NdeI
Recombinant toxin ScFv-PE is prepared as the receptor binding domain that the ScFv gene is replaced the PE gene, its concrete building process is, utilize PCR method amplification ScFv gene, use primer (I and II), in the ScFv gene, introduce the NcoI site in the primer I, introduced coding A, S, three amino acid whose nucleotide sequences of G and NdeI site among the primer I I in ScFv gene downstream.Behind pcr amplification ScFv gene, cut back to close with NcoI and NdeI enzyme, be cloned into NcoI and the NdeI site of pET28-PE, obtain recombinant plasmid pET28-ScFv-PE.
(2) preparation of single-chain antibody and recombinant toxin
With 1/10 culture volume 50mmol/L Tris-Cl (pH8.0), the resuspended expression thalline of 2mmol/L EDTA (pH8.0) solution, add N,O-Diacetylmuramidase (10mg/ml) to final concentration 100 μ g/ml, the 1%TritonX-100 that adds 1/10 volume, vibration mixing, 30 ℃ of water-bath 15min; Put in the ice bath ultrasonication or add DNA enzyme I (1mg/ml) and be placed to no longer thickness to final concentration 20 μ g/ml room temperatures;
Utilize twice of the about 30ml washing of 5%TritonX100 inclusion body respectively, utilize 2%DOC (deoxycholate salt) by every gram bacterium/6ml washing inclusion body once again, with the Tris-Cl that contains 1M Nacl (pH8.0) damping fluid washing inclusion body, by every gram bacterium/6ml urea dissolving inclusion body, 4 ℃ of 12000rpm are after centrifugal 20 minutes, the sex change inclusion body is carried out renaturation, build and produce following renaturation system: proteic starting point concentration is 30-50ug/ml, 50mMTris-Hcl PH8.5,1M urea, 1mMGSH, 0.1MGSSG 2mM-MT was put in the 10-20 ℃ of cold house renaturation 20 hours, with 2000ml 50mM Tris-Hcl (pH8.0) damping fluid in 4 ℃ of dialysis, changed liquid once in per 12 hours, totally 48 hours, ultrafiltration and concentration, utilize ion exchange resin to carry out renaturation product and carry out purifying, purified product-20 is ℃ frozen standby.
6. the antigen-binding activity of single-chain antibody is measured
Utilize HIV-1 serum antibody standard detection film bar, adopt Western blotting that single-chain antibody is carried out determination of activity, used two anti-are anti-His-Tag monoclonal antibody, concrete operations are as follows: utilize confining liquid (10mmol/LTris-Cl pH7.5,150mmol/L NaCl, 2%Tween20,3%BSA) to soak HIV-1 serum antibody standard detection film bar, room temperature sealing 2 hours, with sealing irrelevant protein binding site, reclaim confining liquid; With lavation buffer solution (10mmol/L TrisCl pH7.5,0.15mol/L NaCl, 0.05%Tween20) rinsing film bar 3 times, each 5 minutes; With 1: 10 dilution single-chain antibody to be checked of the lavation buffer solution that contains 0.4%BSA, react with HIV-1 serum antibody standard detection film bar; Room temperature reaction 2 hours; Reclaim antibody, wash HIV-1 serum antibody standard detection film bar film 3 times with lavation buffer solution, each 5 minutes; Back and second antibody (anti-His-Tag monoclonal antibody) reaction, room temperature effect 2 hours, with the lavation buffer solution that contains 0.4%BSA dilution in 1: 300 alkali phosphatase enzyme mark rabbit anti-mouse antibody, react room temperature effect 2 hours with HIV-1 serum antibody standard detection film bar film; Reclaim antibody, use lavation buffer solution rinsing nitrocellulose filter 3 times, each minute.Get 0.5g NBT and be dissolved in preparation NBT liquid in 10ml 70% dimethyl formamide solution; Get 0.5gBCIP and be dissolved in preparation BCIP liquid in 10ml 100% dimethyl formamide solution.33 μ l NBT liquid and 16.5 μ l BCIP liquid are added alkaline phosphatase damping fluid (100mmol/L NaCl, 5mmol/L MgCl successively
2, mixing 100mmol/L TrisCl pH9.5), preparation NBT/BCIP colour developing liquid.Behind alkaline phosphatase damping fluid rinsing nitrocellulose filter, add colour developing liquid and develop the color.After colour developing is finished, nitrocellulose filter moved to heat up in a steamer color development stopping in the water.Experimental result proves, the single-chain antibody of preparation can with HIV-1 gp120 standard antigen generation specific immune response, specificity colour developing band can appear in the gp120 position of HIV-1 serum antibody standard detection film bar film.
7. the Determination of biological activity of recombinant toxin
In order to verify that resulting product is a biologically active substance safely and effectively, the present invention has carried out general pharmacology, drug effect, and acute and subacute animal toxicity test to target protein.
(1) cell killing effect
At first prepare the target cell model that HIV infects, (HIV-1 is strong, and the poison operation needs to carry out in P3 level laboratory), with the lymph tumor cell MT4 that goes down to posterity, adjusting cell density is 10
6/ ml inserts B hypotype HIV-1, and sense is done to remove virus after 2 hours, changes the complete cell culture medium of healthy M-1640, in 37 ℃, 5%CO
2Following overnight incubation is used to measure the kill and wound destruction of recombinant toxin to the HIV-1 cells infected.Recombinant toxin behind the purification renaturation is carried out heat sterilization, carry out acting on the HIV-1 cells infected respectively after the concentration determination,, measure the cytotoxic effect of recombinant toxin these two kinds of cells by morphological observation and cell counting.Measurement result proves that the recombinant toxin of preparation has the good cell killing activity, the target cell that can specific killing HIV-1 infects.Concrete outcome is seen Fig. 7.Fig. 7 uses the cytotoxic effect of the recombinant toxin of different concns to the HIV-1 cells infected, when the concentration of recombinant toxin ScFv-PE and DT-ScFv is following less than 0.4, the growth of HIV-1 cells infected is unaffected, and when the concentration of recombinant toxin reaches 4ng/m, the decreased growth of HIV-1 cells infected also the death of cell occurs, when the concentration of recombinant toxin reached 40ng/ml, the cell more than 90% occurred dead.(action effect of recombinant toxin is added up by morphological observation and cell counting with in the recombinant toxin adding cell 24 hours)
(2) antigen avidity is measured
Utilize indirect ElISA method, " modern immunological experiment technology " Hubei science tech publishing house that concrete steps are write with reference to Shen Guanxin, with your unicorn etc., 121-123 page or leaf.The antigen avidity that compares anti-HIV-1 recombinant toxin and original HIV-1 gp120 monoclonal antibody, respectively recombinant toxin and anti-HIV-1 gp120 monoclonal antibody are measured, used antigen has the affine activity of the antigen identical with parental antibody for reorganization HIV-1gp 120 albumen, measurement result proof recombinant toxin.
(3) toxicology inspection
The animal intravenous injection quite and the anti-HIV-1 recombinant toxin of 100 times of human dosage, upgrowth situation and pathological examination by animal are measured the toxic side effect of recombinant toxin to laboratory animal, the result shows that the anti-HIV-1 recombinant toxin does not demonstrate obvious toxic and side effects (tangible pathological change does not appear in main organs).
Result of study proves that the anti-HIV-1 recombinant toxin is a kind of safe and effective new fusion protein, has the good prospect that development and use become new treating AIDS medicine.
Claims (4)
1, the single-chain antibody of a kind of anti-HIV-1 outer membrane protein gp120 is characterized in that this single-chain antibody has the gene order of the amino acid as follows and this antibody of encoding:
5′-CCA?GGT?CAA?GCT?GCA?GGA?GTC?AGG?ACT?GAA?CTT?GTG?AGG?CCA?GGG 45
P G Q A A G V R T E L V R P G
GCC?TCA?GTC?AAG?TTG?TCC?TGC?ACA?GCT?TCT?GGC?TTT?AAC?ATT?AAA 90
A S V K L S C T A S G F N I K
GAC?GAC?TAT?ATG?CAC?TGG?GTG?AAG?CAG?AGG?CCT?GAA?CAG?GGC?CTG 135
D D Y M H W V K Q R P E Q G L
GAG?TGG?ATT?GGA?TGG?ATT?GAT?CCT?GAA?AAT?GGT?GAT?ACT?GAA?TAT 180
E W I G W I D P E N G D T E Y
GCC?TCG?AAG?TTC?CAG?GGC?AAG?GCC?ACA?ATA?ACA?CCA?GAC?ACA?TCC 225
A S K F Q G K A T I T P D T S
TCC?AAC?ACA?GCC?TAC?CTG?CAG?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC 270
S N T A Y L Q L S S L T S E D
ACT?GCC?GTC?TAT?TAC?TGT?ATT?ACA?AGG?GGT?AAC?TGG?GGC?CAA?GGG 315
T A V Y Y C I T R G N W G Q G
ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGA?GGC?GGT?TCA?GGC?GGA?GGT 360
T T V T V S S G G G G S G G G
GGC?TCT?GGC?GGT?GGC?GGA?TCG?GAC?ATC?GAG?CTC?ACT?CAG?TCT?CCG 405
G S G G G G S D I E L T Q S P
TCC?TAC?TTG?TCT?GTA?TCT?CTA?GGA?GGC?AGA?GTC?ACC?ATT?ACT?TGC 450
S Y L S V S L G G R V T I T C
GAG?GCA?AGT?GAC?CAC?ATT?AAT?AAT?TGG?TTA?GCC?TGG?TAT?CAG?CAG 495
E A S D H I N N W L A W Y Q Q
AAA?CCA?GGA?AAT?GCT?CCT?AGG?CTC?TTA?ATA?TCT?GGT?GCA?ACC?ACT 540
K P G N A P R L L I S G A T T
TTG?GAA?ACT_GGG?GTT?CCT?TCA?AGA?TTC?AGA?GGC?AGT?GGA?TCT?GGA 585
L E T G V P S R F R G S G S G
AAA?GAT?TAC?ACT?CTC?AGC?ATT?ACC?AGT?CTT?CAG?ACT?GAA?GAT?GTT 630
K D Y T L S I T S L Q T E D V
GCT?ACT?TAT?TAC?TGT?CAA?CAG?TAT?TGG?AGT?CCT?CCG?TAC?ACG?TTC 675
A T Y Y C Q Q Y W S P P Y T F
GGA?GGG?GGG?ACC?AAG?CTG?GAA?ATA?AAA?TGG-3′ 705
G G G T K L E I K W 。
2, a kind of recombinant immunotoxin of anti-HIV-1, this recombinant immunotoxin is made of single-chain antibody and the Pseudomonas aeruginosa exotoxin A of anti-HIV-1 outer membrane protein gp120, it is characterized in that being that Pseudomonas aeruginosa exotoxin A receptor binding domain is replaced by the single-chain antibody of gene order coding as follows:
5′-CCA?GGT?CAA?GCT?GCA?GGA?GTC?AGG?ACT?GAA?CTT?GTG?AGG?CCA?GGG 45
GCC?TCA?GTC?AAG?TTG?TCC?TGC?ACA?GCT?TCT?GGC?TTT?AAC?ATT?AAA 90
GAC?GAC?TAT?ATG?CAC?TGG?GTG?AAG?CAG?AGG?CCT?GAA?CAG?GGC?CTG 135
GAG?TGG?ATT?GGA?TGG?ATT?GAT?CCT?GAA?AAT?GGT?GAT?ACT?GAA?TAT 180
GCC?TCG?AAG?TTC?CAG?GGC?AAG?GCC?ACA?ATA?ACA?CCA?GAC?ACA?TCC 225
TCC?AAC?ACA?GCC?TAC?CTG?CAG?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC 270
ACT?GCC?GTC?TAT?TAC?TGT?ATT?ACA?AGG?GGT?AAC?TGG?GGC?CAA?GGG 315
ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGA?GGC?GGT?TCA?GGC?GGA?GGT 360
GGC?TCT?GGC?GGT?GGC?GGA?TCG?GAC?ATC?GAG?CTC?ACT?CAG?TCT?CCG 405
TCC?TAC?TTG?TCT?GTA?TCT?CTA?GGA?GGC?AGA?GTC?ACC?ATT?ACT?TGC 450
GAG?GCA?AGT?GAC?CAC?ATT?AAT?AAT?TGG?TTA?GCC?TGG?TAT?CAG?CAG 495
AAA?CCA?GGA?AAT?GCT?CCT?AGG?CTC?TTA?ATA?TCT?GGT?GCA?ACC?ACT 540
TTG?GAA?ACT_GGG?GTT?CCT?TCA?AGA?TTC?AGA?GGC?AGT?GGA?TCT?GGA 585
AAA?GAT?TAC?ACT?CTC?AGC?ATT?ACC?AGT?CTT?CAG?ACT?GAA?GAT?GTT 630
GCT?ACT?TAT?TAC?TGT?CAA?CAG?TAT?TGG?AGT?CCT?CCG?TAC?ACG?TTC 675
GGA?GGG?GGG?ACC?AAG?CTG?GAA?ATA?AAA?TGG?-3′ 705。
3, a kind of recombinant immunotoxin of anti-HIV-1, this recombinant immunotoxin is made of single-chain antibody and the diphtheria toxin of anti-HIV-1 outer membrane protein gp120, it is characterized in that the receptor binding domain that is diphtheria toxin is replaced by the single-chain antibody of gene order coding as follows:
5′-CCA?GGT?CAA?GCT?GCA?GGA?GTC?AGG?ACT?GAA?CTT?GTG?AGG?CCA?GGG?45
GCC?TCA?GTC?AAG?TTG?TCC?TGC?ACA?GCT?TCT?GGC?TTT?AAC?ATT?AAA?90
GAC?GAC?TAT?ATG?CAC?TGG?GTG?AAG?CAG?AGG?CCT?GAA?CAG?GGC?CTG?135
GAG?TGG?ATT?GGA?TGG?ATT?GAT?CCT?GAA?AAT?GGT?GAT?ACT?GAA?TAT?180
GCC?TCG?AAG?TTC?CAG?GGC?AAG?GCC?ACA?ATA?ACA?CCA?GAC?ACA?TCC?225
TCC?AAC?ACA?GCC?TAC?CTG?CAG?CTC?AGC?AGC?CTG?ACA?TCT?GAG?GAC?270
ACT?GCC?GTC?TAT?TAC?TGT?ATT?ACA?AGG?GGT?AAC?TGG?GGC?CAA?GGG?315
ACC?ACG?GTC?ACC?GTC?TCC?TCA?GGT?GGA?GGC?GGT?TCA?GGC?GGA?GGT?360
GGC?TCT?GGC?GGT?GGC?GGA?TCG?GAC?ATC?GAG?CTC?ACT?CAG?TCT?CCG?405
TCC?TAC?TTG?TCT?GTA?TCT?CTA?GGA?GGC?AGA?GTC?ACC?ATT?ACT?TGC?450
GAG?GCA?AGT?GAC?CAC?ATT?AAT?AAT?TGG?TTA?GCC?TGG?TAT?CAG?CAG?495
AAA?CCA?GGA?AAT?GCT?CCT?AGG?CTC?TTA?ATA?TCT?GGT?GCA?ACC?ACT?540
TTG?GAA?ACT?GGG?GTT?CCT?TCA?AGA?TTC?AGA?GGC?AGT?GGA?TCT?GGA?585
AAA?GAT?TAC?ACT?CTC?AGC?ATT?ACC?AGT?CTT?CAG?ACT?GAA?GAT?GTT?630
GCT?ACT?TAT?TAC?TGT?CAA?CAG?TAT?TGG?AGT?CCT?CCG?TAC?ACG?TTC?675
GGA?GGG?GGG?ACC?AAG?CTG?GAA?ATA?AAA?TGG?-3′ 705。
4, the application in the medicine of preparation treatment acquired immune deficiency syndrome (AIDS) according to claim 2 or 3 described recombinant immunotoxins.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011202408A CN1176948C (en) | 2001-07-11 | 2001-07-11 | Single-chain antibody resisting HIV-1 outer membrane protein and recombinant immunotoxin |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011202408A CN1176948C (en) | 2001-07-11 | 2001-07-11 | Single-chain antibody resisting HIV-1 outer membrane protein and recombinant immunotoxin |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1330081A CN1330081A (en) | 2002-01-09 |
| CN1176948C true CN1176948C (en) | 2004-11-24 |
Family
ID=4664006
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011202408A Expired - Fee Related CN1176948C (en) | 2001-07-11 | 2001-07-11 | Single-chain antibody resisting HIV-1 outer membrane protein and recombinant immunotoxin |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1176948C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100424095C (en) * | 2003-11-26 | 2008-10-08 | 中国人民解放军军需大学军事兽医研究所 | Recombinant targeted toxin against HIV-1 |
| WO2025050404A1 (en) * | 2023-09-08 | 2025-03-13 | 成都维瑾柏鳌生物医药科技有限公司 | Pharmaceutical composition and use thereof |
-
2001
- 2001-07-11 CN CNB011202408A patent/CN1176948C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1330081A (en) | 2002-01-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4294596B2 (en) | Compositions and methods for the treatment of glomerulonephritis and other inflammatory diseases | |
| ES2582001T3 (en) | Molecule that binds to antigen and uses thereof | |
| CN109071679A (en) | The composition and method that cell factor for targeting delivers | |
| ZA200109746B (en) | Chimeric polypeptides, method for production and uses thereof. | |
| CN108431018A (en) | The method of protein modification in medicinal application | |
| CN103483421A (en) | Polypeptides and antibodies for treating HBV infection and related diseases | |
| CN101970005A (en) | Breaking immunological tolerance with a genetically encoded unnatural amino acid | |
| CN102791293A (en) | Liver targeting molecules | |
| NL8701950A (en) | MONOCLONAL ANTIBODIES AND PEPTIDES, SUITABLE FOR THE TREATMENT AND DIAGNOSIS OF HIV INFECTIONS. | |
| Bera et al. | Specific killing of HIV-infected lymphocytes by a recombinant immunotoxin directed against the HIV-1 envelope glycoprotein | |
| McCartney et al. | Engineering disulfide-linked single-chain Fv dimers [(sFv') 2] with improved solution and targeting properties: anti-digoxin 26–10 (sFv') 2 and anti-c-erbB-2 741F8 (sFv') 2 made by protein folding and bonded through C-terminal cysteinyl peptides | |
| JP2023540486A (en) | Immunogenic coronavirus fusion proteins and related methods | |
| CN1154659C (en) | Novel anti-AIDS immunotoxins | |
| EP2968453A2 (en) | Stabilized single human cd4 domains and fusion proteins | |
| TWI296930B (en) | Recombinant enterovirus 71 neutralizing antibodies and use thereof | |
| CN1176948C (en) | Single-chain antibody resisting HIV-1 outer membrane protein and recombinant immunotoxin | |
| WO2016026354A1 (en) | Tachyplesin-antibody fusion protein and preparation method | |
| CN111116743A (en) | Hsp90 antibodies and their use against fungal infections | |
| CN117126271B (en) | Human neutralizing antibodies and bispecific antibodies targeting the envelope protein of fever with thrombocytopenia syndrome virus | |
| US7488481B2 (en) | Polypeptides derived from anti-HIV-1 gp120 antibodies that abrogate gp120 binding to CCR5 | |
| Li et al. | Canine distemper virus (CDV)-neutralizing activities of an anti-CDV canine-derived single-chain variable antibody fragment 4-15 (scFv 4-15) screened by phage display technology | |
| CN110483624B (en) | C-terminal peptide of OspA protein of Borrelia gallini and its application | |
| AU763118B2 (en) | Recombinant immunotoxin directed against the HIV-1 gp120 envelope glycoprotein | |
| CN1966529A (en) | Recombinant toxin agent containing gonadorelin and pyocyanin functional frament | |
| CN101851289A (en) | Humanized antibody of H5 subtype avian influenza virus hemagglutinin protein and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20041124 Termination date: 20120711 |