CN1903878A - Anti SARS virus human source nature antibody IgG Fab section - Google Patents
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- CN1903878A CN1903878A CN 200510028155 CN200510028155A CN1903878A CN 1903878 A CN1903878 A CN 1903878A CN 200510028155 CN200510028155 CN 200510028155 CN 200510028155 A CN200510028155 A CN 200510028155A CN 1903878 A CN1903878 A CN 1903878A
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Abstract
The present invention belongs to the field of gene engineering technology, in particular, it relates to a humanized antibody for resisting SARS virus. Said invention utilizes recombinant protein technology and antibody bank technology to obtain the humanized gene engineering antibody Fab fragment AS3-3 for resisting coronavirus. The tests show that it has higher affinity with recombinant coronavirus S protein, and can specifically identify EU 14 cell infected by cororavirus, and can be used for diagnosing and curing SARS.
Description
Technical field
The invention belongs to the genetically engineered field, be specifically related to anti SARS virus human source IgG antibody Fab fragment.
Background technology
Severe acute respiratory syndrome in 2003 is serious respiratory complication (Severe AcuteRespiratory Syndrome, SARS) eruption and prevalence worldwide.This disease has long, characteristics such as infectivity is high, propagation is fast, severe symptoms, mortality ratio height, no specific medicament, no vaccine prevention in latent period mainly by droplet transmission.It is reported, this disease is by the coronavirus of novel species---SARS virus is caused, one of glycoprotein of confirmer coronavirus genes encoding E2 also claims S albumen (spike protein), can with surface epithelial cell acceptor interaction, the key that to be mediation virus absorption merge with cytolemma.Prove that in research the antibody at anti-S protein 41 7 to 547 amino acids positions can be blocked combining of this strain and target cell, the antiserum(antisera) of anti-this strain of cavy this viral infectivity that can neutralize to human corona virus E229 strain.The domestic serum that also has the medical worker who infects SARS virus to use SARS convalescent is successfully controlled the report of self state of an illness.Variation in view of the coronavirus strain exists prevents trouble before it happens, and seeks the emphasis that effective medicine or vaccine remain this area research.
Summary of the invention
The objective of the invention is to make up immunoglobulin G while gene library, recombinant expressed coronavirus S albumen, preparation anti SARS virus human source genetic engineering antibody is used for diagnosis and the treatment of SARS.
Purpose of the present invention realizes by following method and step,
1. structure antibody library
Get convalescent's peripheral blood, isolated lymphocytes is extracted total RNA, and reverse transcription synthesizes cDNA, and amplification obtains immunoglobulin G while heavy and light chain variable region gene, makes up immunoglobulin G while Fab antibody library.
2. recombinant expressed S albumen
The construction expression plasmid, the IPTG abduction delivering is purified renaturation.Enzyme-linked immunosorbent assay detects recombinant s protein specificity and susceptibility.
3. screen the anti-coronavirus immunoglobulin G while
Immunoglobulin G while Fab antibody library DNA transformed into escherichia coli, the IPTG abduction delivering as antigen, obtains positive colony AS3-3 by clone's blotting, ELISA method and indirect immunofluorescence reaction screening with recombinant s protein.
4. the specificity analysis of anti-coronavirus Fab antibody fragment AS3-3
AS3-3 checks order to the gained positive colony, putative amino acid sequence, and carry out homology analysis.The gained antibody fragment is carried out purifying, with the kit measurement protein content, detect its purity by SDS-PAGE, detect this Fab antibody and the proteic reactivity of recombinant antigen S with Western blot simultaneously, and carry out avidity and detect.
Experiment confirm is used the anti-coronavirus humanized genetic engineering antibody Fab Segment A S3-3 that recombinant protein technology and antibody library technology obtain, and has higher avidity with reorganization coronavirus S albumen, but and EU 14 cells of specific recognition coronavirus infection.Can by further experiment confirm this Fab fragment and the proteic height of coronavirus S affine bring into play suppress coronavirus to the absorption of host cell with penetrate, thereby in and the communicable effect of coronavirus, the treatment of SARS and prevention work are had important use are worth.
Though animal derived Monoclonal Antibody technology is universal, can cause when being applied to human body the anti-heterogenetic antibody of people send out should, though can be improved by antibody humanization's technology, process is numerous and diverse, takes time and effort, short-term is difficult to obtain effect.The present invention has overcome the defective of prior art, humanized's anti-coronavirus of acquisition is because of engineered antibody Fab fragment, because no Fc section, in performance neutralization virus and host cell effect, can activating complement and cause pathological lesion such as human immunity reaction, it is safe and reliable to be applied to human body.
Description of drawings
Fig. 1. human normal immunoglobulin Fab expression vector pFab-His2
Fig. 2. the indirect immunofluorescence of positive colony AS3-3 detects
Wherein, the reaction of the indirect immunofluorescence of A-positive colony AS3-3 and sars coronavirus cells infected (species EU 14),
B-positive colony AS3-3 reacts with the indirect immunofluorescence of no sars coronavirus cells infected (species EU14).
Fig. 3 .SDS-PAGE detects Fab antibody purity
Wherein, A-Coomassie brilliant blue dyeing Fab
The anti-people κ of B-horseradish peroxidase labelled goat chain antibody
The nitrilotriacetic acid (Qiagen) of C-horseradish peroxidase mark
The Fab fragment that 1-intestinal bacteria JM 109 expresses
The reorganization Fab fragment of 2-purifying
Fig. 4 .Western blot detects Fab antibody and the proteic reactivity of recombinant antigen S
Wherein, 1-Coomassie brilliant blue dyeing.
2-recombinant human SARS coronary virus resistant Fab
The 3-human Fab
4～7 SARS patient's convalescent phase serums
The 8-normal human serum
9-anti-SARS virus S protein polyclone antibody (IMG-542)
The normal rabbit igg of 10-.
Embodiment
1. structure antibody library
Get 2 SARS convalescent's periphery anticoagulations (everyone 5ml) (Shanghai City Center for Disease Control), with Ficoll-Paque (Pharmacia, Uppsala, Sweden) isolated lymphocytes, (QIAGEN GmbH, Hilden Germany) extract total RNA with test kit.With total RNA Gene-Amp RNA PCR test kit (Perkin-Elmer Cetus, Norwalk, Conn) become cDNA with Oligo (dT) 16 reverse transcriptions, and with upstream and downstream primer (Invitrogen) (κ chain 5 ' end primer-sequence 3, sequence 4, sequence 5, the sequence 6 of human IgG weight chain variable region conserved sequence; κ chain 3 ' end primer-sequence 7; λ chain 5 ' end primer-sequence 8, sequence 9, sequence 10, sequence 11, sequence 12, sequence 13; λ chain 3 ' end primer-sequence 14; γ chain 5 ' end primer-sequence 15, sequence 16, sequence 17, sequence 18, sequence 19, sequence 20; γ chain 3 ' end primer-sequence 21; Sequence 22, sequence 23, sequence 24) carry out the pcr amplification of immunoglobulin (Ig) γ, κ, λ chain gene.(QIAGEN GmbH, Hilden is Germany) after the purification, respectively with AscI and NheI (NEW ENGLAND BioLabs) double digestion κ chain and λ chain product through test kit for the PCR product.κ chain after enzyme is cut (Am.J.Trop.Med.Hyg.60:35-40.1999) is connected with human normal immunoglobulin Fab expression vector pFab-His2 (Fig. 2) with λ chain product, and electricity changes in the e. coli jm109 (Takara) subsequently, constitutes the light chain storehouse.With SfiI and NotI (NEW ENGLAND BioLabs) light chain storehouse and γ chain PCR product are carried out enzyme respectively and cut modification, after the ligation, the electricity transduction is gone in the e. coli jm109, constitutes 9.8 * 10
6Dependent/non-dependent clone titer antibody storehouse.
2. recombinant expressed S albumen
Spinous process albumen (S albumen) is the outside glycoprotein of coronavirus, has multiple function, with the pathogenic of virus and organize close preferendum closely related.Total length S albumen is excessive, may influence and the combining of antibody, the present invention is the required dna fragmentation of composite coding S albumen (21477～25244) at first, then with pcr amplification 772～1719 bit bases (Invitrogen is synthetic), obtains S protein part gene fragment (amino acid sites 257～573).After this gene fragment process BamHI and EcoRI enzyme are cut, be connected with expression vector pET21b (Novagen), be transformed in the e. coli jm109, the positive colony that obtains is carried out sequential analysis, the plasmid that will contain correct sequence changes over to expresses bacterium BL21 (DE3) (Novagen), through isopropyl-(IPTG) (worker is given birth in Shanghai) abduction delivering, extract inclusion body protein, and carrying out protein renaturation (according to the inclusion body protein method of purification of Novagen), its molecular weight is 30kDa.With the recombinant protein 0.1 μ g/100 μ l coated elisa plate of purifying, detect the specificity and the susceptibility of recombinant protein.When extent of dilution is 1: 400, the OD of patients serum's reaction
490Value is 1.744～1.945, and normal control is 0.424～0.492.When extent of dilution was 1: 800, patients serum's reading was 1.067～1.381, and normal control is 0.132～0.147.Point out this recombinant antigen to can be used as the proteic part fragment of SARS virus spinous process, be used for the screening in anti-SARS immune globulin antibody storehouse.
3. screen the anti-coronavirus immunoglobulin G while
Get 9.8 * 10
6Dependent/non-dependent clone titer antibody storehouse DNA 10ng, transform 100 μ lJM109 intestinal bacteria, bacterium liquid is coated on Luria broth (10g sodii chloridum, 10gtryptone, 5g yeast extract/L, PH7) on dull and stereotyped (containing penbritin 50 μ g/ml), cultivated 7 hours for 37 ℃, treat clone (about 5 * 10
3Individual clone/90mm diameter flat board) when diameter is 0.1～0.2mm, with the diameter is that 82mm nitrocellulose filter (Armacia/Pharmacia) is overlying on the flat board, treat that the clone is transferred on the film fully, film is placed on the LB flat board that contains 1.0mM IPTG, 30 ℃ of abduction deliverings 6 hours, then use N,O-Diacetylmuramidase, DNA enzyme and bovine serum albumin (100mMTris-HCl[pH7], 150mM NaCl, 5mM MgCl
2, 1.5% BSA, 1mg of DNase 40mglysozyme/ml) carries out bacteriolyze to film.Remove bacterial debris residual on the striping etc. through washing, seal with bovine serum albumin (BSA), recombinant protein reaction with 125 μ g purifying, subsequently respectively with the anti-human IgG Fc antibody of positive serum, horseradish peroxidase (HRP) mark (ICNPharmaceuticals, Aurora, Ohio) reaction, colour developing (HRP-1000, Konica Co, Tokyo, Japan).Screen with 4～8 films, the clone who finds engrain on film is as positive colony at every turn, and this is clone's blotting.
Or from antibody library, get 10ng DNA, transform 100 μ lJM109 intestinal bacteria, each single 2ml of being cloned into of picking conversion gained contains the super broth substratum (30gtryptone of penbritin, 20g yeast extract, 10g 3-(N-morpholino) propanesulfonic acid[MOPS]/L, pH7) in, treat OD
600Be 0.6～0.8 o'clock adding IPTG, making its ultimate density is 0.1mM, 30 ℃ of abduction deliverings 10～12 hours, 2 minutes centrifugal collection bacteriums of 14000rpm, add the PBS that 100 μ l contain 1mM PMSF (phenylmethylsulfonyl fluoride) in the bacterial precipitation, the suspension bacterium, 4 ℃ are carried out ultrasonication, and then 14000rpm is centrifugal 10 minutes, get supernatant liquor, adding is coated with on the enzyme plate in 0.1 μ g recombinant s protein/hole, carries out enzyme linked immunoassay, with anti-human IgG Fab (the ICN Pharmaceuticals of HRP mark, Aurora, Ohio) be two anti-, with O-Phenylene Diamine (o-phenylene-diamin, OPD) colour developing.All add the positive contrast of positive serum on the every enzyme plate, OD
490It is promptly positive that reading is higher than certain numerical value.
With the positive colony of clone blotting or ELISA screening gained choose 25ml contain penbritin the SB nutrient solution in, 37 ℃ are cultured to OD
600It is 0.6～0.8 o'clock, add IPTG, making its ultimate density is 0.1mM, 30 ℃ of abduction deliverings 10～12 hours, 2 minutes centrifugal collection bacteriums of 14000rpm, add the PBS that 500 μ l contain 1mM PMSF in the bacterial precipitation, the suspension bacterium, 4 ℃ are carried out ultrasonication, and then 14000rpm is centrifugal 10 minutes, gets supernatant liquor, adding every hole is coated with on the enzyme plate of 0.1 μ g recombinant protein or other irrelevant albumen such as BSA, reorganization entamoeba histolytica 150kDa surface protein etc., carrying out enzyme linked immunoassay, is two anti-with the anti-human IgG Fab of HRP mark, colour developing.As recombinant s protein and other irrelevant proteic OD
490When reading has the significance difference, be about to this clone's the bacterial suspension and the cell (species EU 14) (EUROIMMUNU LLC buys) of sars coronavirus infection and carry out the indirect immunofluorescence reaction, when the specificity fluorescent reaction appears in cell, be positive colony.
Accumulative total screens about 4.8 * 10
4Individual clone, wherein positive colony AS3-3 and coronavirus infection cell are tangible specificity association reaction.
4. the specificity analysis of anti-coronavirus Fab antibody fragment AS3-3
Get the plasmid of positive colony AS3-3, respectively with AscI-NdeI and SfiI-NotI digestion with restriction enzyme, obtain light chain and heavy chain gene, be connected with CV-1 with the sequencing vector CV-2 that cuts modification through enzyme again, transformed into escherichia coli JM109, extract the plasmid DNA that contains light chain or heavy chain gene respectively, with M13Reverse primer (5 '-GGATAACAATTTCACACAGG-3 '), and BigDyeFerminatorV3.1 cycle sequencing kit (Applied Biosystems, Foster City Calif) check order, sequencing reaction carries out on ABIPrism3100 geneticAnalyzer (Applied Biosystems).And carry out aminoacid sequence with GENETYX-MAC Verll and infer (sequence 1, sequence 2), carry out homology analysis with IgBlast.
Table 1 is the homology comparative result of positive colony AS3-3 gene order.
Table 1.
| The clone | The V fragment | The D fragment | The J fragment | ||
| Family | System | Homology % | |||
| AS3-3 | VK1 VH3 | A17 VH3-30 | 92 85 | - D2-2 | JK1 JH6 |
Because Fab antibody contains 6 His-Tag, and heavy chain links to each other with His, and (Novagen, Madison Wis.) purify so select the His binding resin for use.Positive colony AS3-3 cultivate in a large number with abduction delivering after, collect bacterium, carry out bacteriolyze and ultrasonication, the centrifuging and taking supernatant liquor carries out purifying.With the Fab fragment of 1M imidazoles stripping purifying, measure protein content with DC Protein Assay (BioRad), and detect its purity (Fig. 3) by SDS-PAGE (SDS-PAGE).Detect Fab antibody and the proteic reactivity of recombinant antigen S (Fig. 4) with Western blot simultaneously.Antibody purified is with Biocore3000 (Biocore AB, Uppsala Sweden) detects its avidity, behind the recombinant antigen S albumen bag quilt with purifying, be 2.5 μ g/ml with concentration respectively, 1.25 μ g/ml, 0.625 μ g/ml, the Fab antibody fragment of 0.3125 μ g/ml reacts with it, obtains combination rate (KA) and dissociation yield (KD) is respectively 5.05 * 10 with BIAevalution3.1 software
7(1/M) with 1.98 * 10
-8(M) this Fab antibody of results suggest has higher avidity with reorganization coronavirus S albumen.
Table 2 is anti-reorganization coronavirus S albumen Fab antibodies rate (KA) and dissociation yield (KD).
Table 2
| ka(1/Ms) | kd(1/s) | KA(1/M) | KD(M) |
| 1.54×10 5 | 3.06×10 -3 | 5.05×10 7 | 1.98×10 -8 |
SEQUENCE LISTING
＜110〉Fudan University
＜120〉anti SARS virus human source IgG antibody Fab fragment
＜130〉anti-SARS virus 20050622
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Ala Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Ser His Asp Gly Ser Gln Thr His Tyr Ala Asp Ser Val
50 55 60
Gln Gly Arg Phe Gly Val Ser Arg Asp Asn Ser Asn Tyr Thr Ala Tyr
65 70 75 80
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Claims (5)
1, anti SARS virus human source IgG antibody Fab fragment is characterized in that having sequence 1, the aminoacid sequence of sequence 2.
2, by the described anti SARS virus human source IgG antibody of claim 1 Fab fragment, the protein that it is characterized in that described antibody fragment comprises heavy chain and light chain, wherein the heavy chain part is made up of 121 amino acid, contain three complementary determining region CDR1, CDR2, CDR3, with four skeleton district FR1, FR2, FR3, FR4; Wherein the light chain part is made up of 110 amino acid, contains three complementary determining region CDR1, CDR2, CDR3 and four skeleton district FR1, FR2, FR3, FR4.
3, by the described anti SARS virus human source IgG antibody of claim 2 Fab fragment, wherein the FR1 of heavy chain part contains 30 amino acid, CDR1 contains 5 amino acid, FR2 contains 14 amino acid, CDR2 contains 17 amino acid, FR3 contains 32 amino acid, and CDR3 contains 12 amino acid, and FR4 contains 11 amino acid; The FR1 of light chain part contains 22 amino acid, and CDR1 contains 16 amino acid, and FR2 contains 15 amino acid, and CDR2 contains 7 amino acid, and FR3 contains 30 amino acid, and CDR3 contains 9 amino acid, and FR4 contains 11 amino acid.
4, the purposes of the described anti SARS virus human source IgG antibody of claim 1 Fab fragment in the preparation of preparation detection coronavirus infection.
5, the purposes of the described anti SARS virus human source IgG antibody of claim 1 Fab fragment in the medicine of preparation treatment coronavirus infection.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892525A (en) * | 2010-07-16 | 2010-11-24 | 复旦大学 | Method for constructing natural humanized IgG Fab phage antibody library |
| CN112630444A (en) * | 2020-12-11 | 2021-04-09 | 珠海碳云智能科技有限公司 | Polypeptide screening method based on target protein |
| CN113388029A (en) * | 2020-03-12 | 2021-09-14 | 中国科学院武汉病毒研究所 | Neutralizing human monoclonal antibody against novel coronavirus and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100467485C (en) * | 2003-05-26 | 2009-03-11 | 中国科学院上海生命科学研究院 | SARS coronary virus related polypeptide and its uses |
| CN1488645A (en) * | 2003-07-08 | 2004-04-14 | 中国人民解放军空军广州医院肝病研究 | Specific antibody IgY for anti SARS virus, and preparing method thereof and composition containing same and its use |
| CN100374464C (en) * | 2003-07-10 | 2008-03-12 | 上海单抗制药技术有限公司 | Anti-SARS monoclonal antibody, encoding sequence and application thereof |
| WO2005042579A1 (en) * | 2003-10-31 | 2005-05-12 | Fujirebio Inc. | Anti-sars virus antibody, hybridoma producing the antibody and immunoassay reagent using the antibody |
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2005
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892525A (en) * | 2010-07-16 | 2010-11-24 | 复旦大学 | Method for constructing natural humanized IgG Fab phage antibody library |
| CN113388029A (en) * | 2020-03-12 | 2021-09-14 | 中国科学院武汉病毒研究所 | Neutralizing human monoclonal antibody against novel coronavirus and application thereof |
| CN112630444A (en) * | 2020-12-11 | 2021-04-09 | 珠海碳云智能科技有限公司 | Polypeptide screening method based on target protein |
| CN112630444B (en) * | 2020-12-11 | 2024-05-24 | 深圳碳云智肽药物科技有限公司 | Polypeptide screening method based on target protein |
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