CN101550189A - Anthropogenic antivirulin glycosidoprotein neutralizing genetic engineering antibody RD9 and preparation and application thereof - Google Patents
Anthropogenic antivirulin glycosidoprotein neutralizing genetic engineering antibody RD9 and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to an anthropogenic antivirulin glycosidoprotein neutralizing genetic engineering antibody RD9 and a preparation and an application thereof. The antibody is VH-connecting peptide-VK of a three-structural single-stranded antibody comprising a variable region of heavy chain and a variable region of light chain by using 12 sequences of amino acid of the 1 (CH1) 5' end of a constant region of a heavy chain as the connecting peptide AKTTAPSVYPL, and the antibody realizes efficient expression in a prokaryotic system. The biological characteristic studies show that the RD9 is an anthropogenic genetic engineering antibody which has high appetency and better stability and can specially centralize rabies virus as well as genes and gene products of the antibody can be used for preparing clinical drugs for preventing and treating the rabies; the preparation method of the antibody is easy for massive industrial production. The invention solves the problem in the other technology of the application of genes of the variable region of the heavy chain and the variable region of a light chain of the antibody and polypeptide coded by the genes in the drugs for preventing and treating the rabies.
Description
(1) technical field:
The invention belongs to biological technical field, relate to a kind of antibody and preparation thereof of rabies poison and use especially a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 and preparation thereof and application.
(2) background technology:
Rabies are the highest transmissible diseases of mortality ratio, and case fatality rate is 100%.Rabies worldwide extensively distribute, and there is that the people dies from rabies more than 60,000 an every year in the whole world.China also belongs to rabies country occurred frequently.The rubies epidemiology peak just forms in China again at present! Rabic control has become one of most important public health problem of China.It almost be impossible that the mankind will eliminate rabies from the earth, because the rabies symbiotic disease that is people and animals, and the human generation that can't control animal rabies so far.Therefore, the prevention after the rabies exposure is the major measure of rabies control.For serious resurrectionist (infection of II type III type), vaccine is used in World Health Organization's suggestion simultaneously and anti-rabies antibody carries out active and passive combination therapy.Be generally 1 to 3 months the latent period that the people infects rabies virus, but also can be as short as once the week morbidity.Therefore,, reach the crowd that hypoimmunity can not in time produce antibody, must use antibody to prevent and treat after the exposure for the children of immunity system developmental immaturity.
People source monoclonal antibody is the first-selection of clinical application, but the development of China's human antibody still is in the starting stage, awaits making up mainly due to various antibody technique platforms, and in addition, the limitation of eukaryotic system fermentation technique also is one of main bottleneck of Antibody Preparation.
The appearance of people source clone's display technique (cloning display) is that human antibody prepares the milestone on the history, it is the proteinic strong instrument of a kind of screening, this technology links together genotype and albumen phenotype, utilizes the specificity aglucon of target protein can filter out target protein from the protein display libraries and find corresponding gene order.In the past, using more display technique has phage display (phage display), bacterium surface displaying (bacterial surface display), yeast displaying (yeast display) etc.But these technology must dependent cells, owing to be subjected to bacterium transformation efficiency, phage packaging, stride the influence of limiting factors such as film secretion and proteasome degradation, storage capacity and molecular diversity thereof all are restricted.For overcoming above shortcoming, another kind of new external display technique-ribosomal display technology (ribosome display) was used and was given birth to the mid-90 in 20 century, because this technology is carried out external fully, remedied the deficiency of showing in the cell, can significantly increase storage capacity and molecular diversity, storage capacity can be up to 10
13～10
15, than phage display library (about 10
9) improved 10
4～10
6Doubly.In addition, the ribosomal display technology build the storehouse easy, be easy to the screening, injected new vitality for the preparation of specificity human antibody, demonstrated tempting development prospect.
The genetic engineering antibody of report is scFv at most at present, scFv-Fc and Fab etc., and scFv has made very big contribution in the diagnosis of multiple disease, but because the unstable of its space structure has limited its application in clinical treatment greatly.ScFv-Fc and Fab are because molecule is bigger; contain a plurality of glycosylation sites; be difficult to finish the maturation of antibody in the prokaryotic system, usually require in eukaryotic system to express preparation, but since at present in eukaryotic cell the limitation of fermentation technique make it to be difficult to form large-scale production.
Three domain antibodies can obviously improve the stability of scFv, and it changes the structure strategy is that IGg (CH1) 5 ' 12 amino acid of end (AKTTAPSVYPLA) of should choosing replace the Linker (Gly4Ser) of scFv
3, changing the later antibody stability of structure has obvious improvement, and analyzing reason may be because CH1 can provide and keep the useful space conformation of antibody molecule.And three domain antibodies have following characteristics: simple in structure, be easy to make up; From the ability that blood vessel enters its hetero-organization, comprise that the ability by hemato encephalic barrier all is better than full molecular antibody, make the removing that enters the rabies virus in the central nervous system become possibility; And, be easy to the activated antibody of preparation solubility in prokaryotic system.Therefore, three domain antibodies are a kind of very promising preventions and treat the rabic clinical small molecular antibody of using.
In all cases, develop and develop a kind of antibody of new rabies poison, make it then become the important topic that present stage biotechnology worker needs to be resolved hurrily as effectively preventing and treating rabic medicine.
(3) summary of the invention:
The object of the present invention is to provide a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 and preparation thereof and application, described antibody is that----ribosomal display technology screening that utilizes Protocols in Molecular Biology is to the human antibody gene RD9 of a strain at rabies virus glycoprotein (Gp), and realized this strain antibody efficiently expressing at prokaryotic system, its biological characteristic research shows that RD9 is a plant height avidity, stability better, simple in structure, be easy to make up can be special in and the human genetically engineered antibody of rabies virus, its gene and its gene product can be used for the preparation prevention and treat rabic clinical application; The preparation method of described antibody is easy to large-scale industrial production.
Another object of the present invention is to heavy chain and the chain variable region gene and the application of encoded polypeptides in prevention and treatment rabies medicine thereof of described antibody.
Technical scheme of the present invention:
A kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9, it is characterized in that: be human antibody, be by variable region of heavy chain VH, variable region of light chain VL with the three structural domain single-chain antibody VH-connection peptides-VKs of 12 amino acid whose sequences of CH1 (CH1) 5 ' end, be called for short VH/K as connection peptides AKTTAPSVYPLA formation.
Above-mentioned a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9, it is characterized in that: the heavy chain variable region gene of described antibody gene and chain variable region gene derive from the enrichment of human source anti-rabies virus rrna antibody library screening, and its heavy chain variable region gene, chain variable region gene have as sequence table＜400〉sequence shown in 1 and＜400〉3.
Above-mentioned a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9, it is characterized in that: described antibody gene encoded polypeptides sequence is as sequence table＜400〉sequence shown in 2 and＜400〉4.
Above-mentioned a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 is characterized in that: described aminoacid sequence changed, lacks or adds one or several amino acid, and in still keeping and the ability of rabies virus.
The preparation method of above-mentioned a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9, it is characterized in that: this method utilizes the method for gene recombination technology to prepare, and concrete steps comprise:
(1) structure of ribosomal display single-chain antibody gene storehouse (VH/k):
1-1) extraction of cell total rna and cDNA's is synthetic;
1-2) the pcr amplification of human antibody VH and VL gene;
1-3) clone of PCR product and sequential analysis;
1-4) the splicing of antibody weight, light chain DNA;
(2) structure in ribosomal display single-chain antibody gene storehouse;
(3) in-vitro transcription and translation;
(4) the affine screening of rrna mixture;
(5) clonal expression of VH/k antibody gene;
(6) ELISA identifies positive colony and carries out sequential analysis;
(7) human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9's efficiently expresses and purifying;
(8) specific antigens of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 detects in conjunction with activity;
(9) relative affinity of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 is measured;
(10) the relatively stable Journal of Sex Research of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9;
(11) among the human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 and active research.
The concrete steps that efficiently express with purifying of above-mentioned described step (7) human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 comprise:
(1) structure of antibody gene expression vector;
(2) in a suitable system or host, antibody gene is expressed;
(3) the antibody expression host that cultivates in a large number and increase;
(4) the antibody separation and purification to expressing.
Above-mentioned described antibody gene expression vector can be plasmid vector or virus vector, the genetically modified carrier of animal or universal support; The expression vector of described antibody gene changes among a suitable system or the host and is expressed, and wherein system of Shi Heing or host comprise the cell of animal or plant, cell strain, bacterium or yeast; This animal is transgenic animal.
The preparation method of above-mentioned a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 can also utilize chemical synthesising technology to prepare and produces.
The application of above-mentioned a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 is characterized in that: described antibody is used for diagnosing or treating the application of rabies medicine in preparation, comprises antibody gene and antibody gene encoded polypeptides.
Above-mentioned described antibody gene is used for diagnosing or treating the application of rabies medicine in preparation.
Above-mentioned described antibody gene encoded polypeptides is used for diagnosing or treating the application of rabies medicine in preparation.
Beneficial effect analysis of the present invention:
According to an aspect of the present invention, the present invention relates to a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9, be called for short VH/K, VH/K novel gene engineered antibody has been brought into play the advantage that the small molecules engineered antibody can enter hemato encephalic barrier, make the removing that enters the rabies virus in the central nervous system become possibility, simultaneously, its distinctive stability has overcome the unsettled drawback of modal small molecules single-chain antibody (single chainvariable region fragments scFv), for its specific antibody medicine that becomes prevention of clinical rabies poison and treatment has been established molecular basis.
According to another aspect of the present invention, the present invention relates to a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 gene and encoded polypeptides thereof are used for diagnosing or treating the rabies medicine in preparation application, the heavy chain variable region gene of anti-rabies virus antibody of the present invention and chain variable region gene be from three through the constructed antibody library of hydrophobia immune peripheral blood medium size lymphocyte, rabies virus glycoprotein with expression and purification screens the VH/K that obtains, pass through sequencing and in NCBI, after the BLAST comparative analysis, confirm described VH and VL gene and amino acid sequence coded thereof as shown in Figure 3.With this VH and VL gene, use on the Internet known antibodies geneseq database IMGT of specialty respectively and institute's calling sequence is carried out the homology comparison to NCBI and its germline gene source analysis shows, the gene order that is obtained is all not quite identical from the various antibody gene sequences of people's germline gene and existing report really.People's antibody variable region that resulting VH and VL gene codified are correct.
The present invention overcomes the deficiency of in the past cloning display technique, and first Application ribosomal display technology with three isolated lymphocytes in hydrophobia immune peripheral blood, makes up in the human antibody library, and storage capacity reaches 6.7 * 10
12With the rabies virus glycoprotein of expression and purification as the screening antigen selection, successfully obtain 1 plant height avidity, stability preferably can be special in and the people source novel gene engineered antibody of rabies virus.VH/K novel gene engineered antibody has been brought into play the advantage that the small molecules engineered antibody can enter hemato encephalic barrier, make the removing that enters the rabies virus in the central nervous system become possibility, simultaneously, its distinctive stability has overcome the unsettled drawback of modal small molecules single-chain antibody, for its specific antibody medicine that becomes prevention of clinical rabies poison and treatment has been established molecular basis.Realize its efficiently expressing in prokaryotic system simultaneously, overcome the technical bottleneck that most of human genetically engineered antibodies are difficult to large scale fermentation in eukaryotic expression system.Based on above-mentioned heavy chain of antibody, chain variable region gene, can adopt gene engineering method, make up and be expressed as the small molecules genetic engineering antibody of various ways, as Fab, F (ab)
2Antibody fusion protein etc., or external synthetic light, the nucleotide sequence in 6 CDR districts of heavy chain that antibody is identical therewith or the nucleotide sequence of the same amino acid of encoding, or the clone is prepared as other expression system, thereby obtain neutralizing antibody or the associated protein or the polypeptide product of rabies poison glycoprotein, be used for preventing clinically and treating rabic medicine with preparation, the acquisition of human source anti-rabies virus glycoprotein gene engineered antibody, will be to the rabic prevention of China, diagnosis and treatment provide new approach.
(4) description of drawings:
Fig. 1 is the pcr amplification in antibody gene storehouse.
Fig. 2 is the structure in ribosomal display VH/K antibody gene storehouse.
Fig. 3 efficiently expresses and purifying for human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9's.
Fig. 4 is that the antigen-specific of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 is in conjunction with activity.
Fig. 5 is that the relative affinity of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 detects.
Fig. 6 is the Detection of Stability of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9.
Fig. 7 is the active detection of the neutralization of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9.
(5) embodiment:
Embodiment 1: the structure in ribosomal display single-chain antibody gene storehouse (VH/k)
The structure in antibody gene storehouse mainly divides four big steps:
1-1) extraction of cell total rna and cDNA's is synthetic
The extraction of cell total rna is carried out fundamental immunity with rabies virus Vero vaccine (Lyons, France pasteur Mei Lie sensitized vaccine company) to three volunteers, and after 2 weeks, booster immunization once again.Extraction is through three volunteer's peripheral blood 40ml of immunity, and every milliliter of blood adds the 5U heparin.With equivalent PBS dilution peripheral blood, add gently the centrifuge tube that has filled lymphocyte separation medium (China Concord Medical Science University's Blood Research Institute) from tube wall, put the low speed desk centrifuge, 2500r/min, centrifugal 20min.With capillary pipet gentle aspiration buffy coat, place another 15ml centrifuge tube.Do the equivalent dilution with physiological saline, 1000r/min, centrifugal 10min carefully removes supernatant, so repeats to wash twice.The resuspended precipitation of ratio in every 10ml blood adds 1ml physiological saline is sub-packed in the EP pipe.Use total RNA separating kit (Promega company) to extract cell total rna then.
CDNA synthetic (RNA reverse transcription test kit is available from TaKaRa company) is template with the cell total rna, and HuIgGFor, HuIgMFor and HuCLFor are primer, and cDNA first chain is synthesized in reverse transcription, the reaction conditions of RT-PCR is: 30 ℃ of 10min, 42 ℃ of 30min, 99 ℃ of 5min, 5 ℃ of 5min.All cDNA will be used to prepare different VH of family and the VL gene that constitutes the VH/K gene pool.
The primer sequence of synthetic cDNA is as follows:
HuIgGFor：5’-GTC?CAC?CTT?GGT?GTT?GCT?GGG?CTT--3’
HuIgMFor：5’-TGG?AAG?AGG?CAC?GTT?CTT?TTC?TTT--3’
HuCLFor：5’-AGA?CTC?TCC?CCT?GTT?GAA?GCA?CTT--3’
1-2) the pcr amplification of human antibody VH and VL gene
It is template that the pcr amplification of VH and VL gene is got above-mentioned synthetic cDNA 4 μ l, each 1 μ mol of the upstream and downstream primer of amplification people antibody VH and VL gene, dNTP 200 μ mol/L, 10 * Taq buffer5 μ L, TaqDNA enzyme 5U, cumulative volume 50 μ l.The reaction conditions of PCR is: 94 ℃ of sex change 50sec, and 57 ℃ of renaturation 50sec, 72 ℃ are extended 50sec, and after 30 circulations, 72 ℃ are extended 10min.Amplification is seen Fig. 1.
Amplification people heavy chain variable region gene primer sequence is as follows:
VHup：5’ga?gat?ata?tcc?atg(c/g)ag?gt(g/c)ca(g/c)ctc?gag(c/g)agtct?gg?3’
VHdown：5’gga?gac?tg(c/g)gtc?atc(a/g)c(c/a)at(t/g)tcg?gag?acg?aggggg?aaa?ag?3’
VH 3 ' the end of amplification has 12 amino acid whose gene orders of CH1 (CH1) 5 ' end coding AKTTAPSVYPLA.
3 ' end primer (degeneracy) sequence of amplification people heavy chain variable region gene VH is as follows:
VLup?5’ga(c/a)at(t/g)g(t/c)g?atg?ac(c/g)cag?tct?cc3’
VLdown?5’gct?cta?gaa?cac?tct?ccc?ctg?ttg?aag?ct?3
1-3) clone of PCR product and sequential analysis
Pcr amplification product is reclaimed test kit with Promega company pillar reclaim purifying fast, then the different VH of family, VL gene are cloned into respectively in the PMD-18T carrier, transformed competence colibacillus cell E.coliDH5 α is by the blue white screening random choose recon of α-Hu Bu.Recombinant plasmid send the order-checking of Bo Ya company, itself and known sequence is compared, and analyze with related software.
1-4) the splicing of antibody weight, light chain DNA
The VH and the VL gene of the different families that sequential analysis is correct are assembled in twos, and molecular form is VH-linker-VL, Linker be 1.2 prepared V H gene ends with the sequence of (CH1) 5 ' 12 amino acid of end (AKTTAPSVYPLA).The weight of purifying, light chain product such as are quantitatively determined at molar ratio, VH DNA 6 μ L, VL DNA 6 μ L, TaqDNA polymerase 5U, cumulative volume 100 μ l, 94 ℃ of 60s, 50 ℃ of 80s, 72 ℃ of 120s are after 7 circulations of increasing, add primer VHup and VLdown, add the 1UDNA polysaccharase, 94 ℃ of 90s, 58 ℃ of 80s, 72 ℃ of 140s carry out 30 circulations again.Successfully made up VH/K antibody gene storehouse thus.
Embodiment 2: the structure in ribosomal display single-chain antibody gene storehouse
The structure that is used for the single-chain antibody gene of ribosomal display is finished by assembling PCR, VH/K antibody gene with purifying is a template, be that primer carries out two-step pcr with RDTb/Ck/for and RDT7/Ck/for respectively, the first step adds protokaryon ribosome bind site (SD sequence) at the N end.Second step made up T7 promotor, 5 '-loop-stem structure and 3 '-loop-stem structure respectively at the gene two ends.TaqDNA polymerase 5U, cumulative volume 50 μ l, 94 ℃ of 60s, 50 ℃ of 80s, 72 ℃ of 120s, after amplification 30 circulations, the gene fragment of structure is about 1000bp, sees Fig. 2, and constructed ribosomal display antibody gene Kuku capacity reaches 6.7 * 10
12
Required primer sequence is as follows:
Ck/for?5’ccg?cac?acc?agt?aag?gtg?tgc?ggt?gct?cta?gaa?cac?tct?cc?3’
RDTb?5’aga?cca?caa?cgg?ttt?ccc?tct?aga?aat?aat?ttt?gtt?taa?ctt?taagaa?gga?gat?ata?tcc?3’
RDT7?5’ata?cga?aat?taa?tac?gac?tca?cta?tag?gga?gac?cac?aac?gg3’
Embodiment 3: in-vitro transcription and translation
The expresswayTM Plus ExpressionSystem of invitrogen company is adopted in in-vitro transcription and translation, is undertaken by operational manual.After the termination reaction, in supernatant liquor, add the WBT that contains 10% (w/v) BSA of 1/5 volume of precooling.Reaction mixture, is transferred to supernatant liquor in the EP pipe of a new ice precooling behind the centrifugal 5min of 1400g at 4 ℃.
Embodiment 4: the affine screening of rrna mixture
Sealed with BSA-S.cerevisiae RNA-PBS by 96 orifice plates with reorganization Gp albumen (this chamber preparation) bag.The translation mixture is gone in the hole of bag quilt, and microwell plate is jog 1h in the cold house, inclines and falls translation reaction liquid, and microwell plate is washed 5 times with WBT.Add the elution buffer of precooling subsequently in the hole, jog 5min is with wash-out mRNA on ice.The mRNA that elutes carries out purifying with RNeasy kit.MRNA is dissolved in the RNase-free water behind the purifying, handles with RNase-free DNase, removes the dna molecular in the reaction solution.The mRNA of purifying carries out RT-PCR as template, and the PCR product that is: is transcribed-translate-screen as the next round circulation.
The clonal expression of embodiment 5:VH/k antibody gene
Nco I and Not I site are inserted in last RT-PCR gene product two ends of taking turns the mRNA of screening acquisition, and the expression vector pET22b (+) with the same double digestion of warp behind the double digestion is connected with the T4 ligase enzyme, then Transformed E .coli BL21 (DE3).Inoculate 200 positive recombinant list bacterium colonies and contain in the LB substratum of 100mg/LAmp in 10ml, 37 ℃ of shaking culture are spent the night.By 1: 50 overnight culture is inoculated in fresh 2 * YT-A (containing 100mg/L Amp) substratum, 37 ℃ of shaking culture are to OD
600=0.8, add IPTG and induce 1mmol/L, 37 ℃ of shaking culture 3.5h.The centrifugal 10min of 8000rpm collects thalline.Be suspended from lysis buffer, ultrasonication, centrifugal collection supernatant, product is used for ELISA.
Embodiment 6:ELISA identifies positive colony and carries out sequential analysis
By 96 orifice plates, 4 ℃ are spent the night in the wet box with Gp recombinant protein bag.3%BSA-PBS (pH 7.0) seals, 37 ℃ of 1h.Add above-mentioned preparation antibody, every hole 70 μ l, 37 ℃ of 1h.The anti-human IgG κ chain antibody that adds the HRP mark, every hole 50 μ l, 37 ℃ of 1h; 2M H is used in A, the colour developing of B liquid
2SO
4After the termination reaction, under the 450nm wavelength, measure the OD value, identify positive colony; Select 10 positive colonies, extract plasmid, send company's order-checking, itself and known sequence are compared, and use the related software analysis, wherein confirm the brand-new preferably human source anti-rabies virus VH/k antibody of a strain specificity, called after RD9, its heavy chain variable region gene, chain variable region gene have sequence table＜400〉sequence shown in 1 and＜400〉3, its heavy chain variable region gene, chain variable region gene encoded polypeptides sequence have sequence table＜400〉sequence shown in 2 and＜400〉4.
Embodiment 7: human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 efficiently expresses and purifying
7-1) the structure of antibody gene expression vector (embodiment 5): the RD9 antibody cloning is gone into expression vector pET22b (+), and successfully be transformed among the host bacterium E.coli BL21 (DE3);
7-2) in a suitable system or host antibody gene is expressed: the single bacterium colony of the positive RD9 that picking builds contains in the LB substratum of 100mg/L Amp in 10ml, and 37 ℃ of shaking culture are spent the night;
7-3) the antibody expression host that cultivates in a large number and increase: by 1: 50 overnight culture is inoculated in fresh 2 * YT-A (containing 100mg/L Amp) substratum, 37 ℃ of shaking culture are to OD
600=0.8, adding IPTG, to be induced to final concentration be 1mmol/L, 30 ℃ of shaking culture 5h, and the centrifugal 10min of 8000rpm collects thalline, is suspended from lysis buffer, ultrasonication, centrifugal collection supernatant;
7-4) antibody separation and purification to expressing: supernatant collected among the 7-3 is carried out purifying with the Ni-NTA post, with 5ml HiTrap Chelat ing HP (5ml) prepacked column and
FPLC protein purification instrument connects, and when washing pillar to baseline stability with buffer A, goes up sample at a slow speed.And then wash pillar with buffer A, consistent to baseline and the last sample, use buffer B (0.1M SODIUM PHOSPHATE, MONOBASIC then, 0.01MTris-HCI, pH8.0) and damping fluid C (0.1M SODIUM PHOSPHATE, MONOBASIC, 0.01M Tris-HCI, 250mmolimidazole, pH8.0) carry out gradient elution, collect the effluent liquid of elution peak, take a morsel and mix with isopyknic 2 * SDS sample buffer, boil behind the 5min centrifugally, get supernatant and carry out the 15%SDS-PAGE electrophoretic analysis, the result shows, realized people source RD9VH/K antibody efficiently expressing in prokaryotic cell prokaryocyte, expression amount reaches more than 25% of bacterial protein, has prepared the antibody purification of solubility, sees Fig. 3.
Embodiment 8: the specific antigens of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 detects in conjunction with activity Gp recombinant protein and rabies virus Vero vaccine is diluted to different concns with coating buffer, every hole adds 60 μ l on 96 orifice plates, repeat 3 holes, with TNFa as negative control, PBS makes blank, and 4 ℃ are spent the night in the wet box.3%BSA-PBS (pH 7.0) seals, 37 ℃ of 1h.The antibody purification that adding is diluted with PBS, every hole 70 μ l, 37 ℃ of 1h; The anti-human IgG κ chain antibody (sigma company) that adds the HRP mark, every hole 50 μ l, 37 ℃ of 1h; H is used in A, the colour developing of B liquid
2SO
4After the termination reaction, under the 450nm wavelength, measure the OD value, see Fig. 4.The result shows, people source VH/k antibody can be special combine with Gp and rabies virus, and can not combine with TNFa, show that this strain antibody is special human genetically engineered antibody at rabies virus glycoprotein, promptly its antibody gene, antibody gene encoded polypeptides are also special at rabies virus glycoprotein.
Embodiment 9: the relative affinity of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 is measured
Adopt the thiocyanate-elution method that the relative affinity of RD9 is detected.The Gp bag with the BSA-PBS sealing, is added above-mentioned purifying humanized antibody RD9, with containing different concns NH by 96 orifice plates
4The PBS solution of SCN is hatched, and washes plate.The anti-His-Tag antibody of HRP mark is anti-as two.With OD
45050% o'clock corresponding N H descends
4The concentration of SCN is seen Fig. 5 as the relative affinity index, and the result shows that the avidity of RD9VH/K is better, and its relative affinity index is 1.4mol/L.
Embodiment 10: the relatively stable Journal of Sex Research of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9
Stability is one of most important biological characteristics that influences antibody function.Hatch jointly at 37 ℃ with human serum, PBS and antibody purification, Gp recombinant protein wrapper sheet, the ELISA method detects the relative stability of antibody, sees Fig. 6.
The result shows, RD9 in PBS behind 37 ℃ of 2.5h activity reduction is arranged slightly, more stable always later on, still have activity during to 6d, and scFv generally it actively just sharply reduces behind 37 ℃ of 1h in PBS, do not had activity behind the 2h substantially.In serum, the activity of RD9 also can detect at 8d, and scFv in serum during about 2h its activity will reduce rapidly.
Embodiment 11: among the human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 and active research
The antibody fragment of preparation is adjusted into 400 μ g/ml with the MEM substratum, and makes gradient dilution with the MEM substratum.Getting a certain amount of each dilution antibody adds in the 24 porocyte culture plates, add in the equivalent again and use viral suspension, requiring the rabies virus plaque number in every hole is 10～60, makes positive control with rabies poison mouse McAb antibody, and anti-TNFa scFv makes negative control.Every extent of dilution is inoculated 4 holes, places 37 ℃ of water-bath 1h; Again with ready Vero cell suspension inoculation in above-mentioned 24 porocyte culture plates, every hole inoculation 1ml; Place 5%CO
2Leave standstill in 34 ℃ of incubators and cultivate after 8 days, go to use the 10% formaldehyde distilled water dyeing 5min that contains 0.1% Viola crystallina behind the nutrient solution, flowing water dashes the back and counts plaque down in the low power opticmicroscope, sees Fig. 7.
The result shows human antibody RD9 and the same live virus particle that can suppress to form plaque fully of rabies poison mouse McAb antibody, and the inhibition degree is directly proportional with the concentration of antibody fragment.And anti-TNFa scFv forms the unrestraint effect to the rabies virus plaque.The prepared anti-Gp human antibody RD9 of this explanation can be special in and rabies virus, stop the absorption of rabies virus to the Vero cell, suppressed the infection of rabies virus to target cell, reference source RD9 antibody is that a strain specificity is better, the neutralizing antibody of high-affinity.The acquisition of human source anti-rabies virus glycoprotein gene engineered antibody RD9 will be to the rabic prevention of China, and diagnosis and treatment provide new approach.
(6) sequence table:
＜110〉Inst. of Hygienics and Environmental Medical Science, Academy of Military Medici
＜120〉human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 and preparation thereof and application
<130>
<150>CN200710057213.6
<151>2007-04-26
<160>4
<170>PatentIn?version?3.3
<210>1
<211>378
<212>DNA
＜213〉people (human)
<220>
<221>V-region
<222>(1)..(378)
＜223〉nucleotide sequence of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 heavy chain variable region gene
<400>1
gaggtgcagc?tcgaggagtc?tgggggaggc?gtggtccagc?ctgggaggtc?cctgagactc 60
tcctgtgcag?cctctggatt?caccttcaat?agttatgctg?tccactgggt?ccgccaggct 120
ccaggcaagg?ggctggagtg?ggtggcagtt?gtctcaaatg?atggagatga?caaatactac 180
gcagactccg?tgaagggccg?attcaccatc?tccagagaca?attccaggaa?cacgctgtat 240
ctgcaaatga?acagcctgag?agctgaggac?acggctgtgt?attactgtgc?gagaggtttc 300
tatggttcgg?ggagtcttct?cgcggcctct?gatgcttttg?atatctgggg?ccaagggaca 360
gtggtcaccg?tctcttca 378
<210>2
<211>126
<212>PRT
＜213〉people (human)
<220>
<221>V-region
<222>(1)..(126)
＜223〉the proteinic aminoacid sequence of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 variable region of heavy chain
<400>2
Glu?Val?Gln?Leu?Glu?Glu?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?Arg
1 5 10 15
Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Thr?Phe?Asn?Ser?Tyr
20 25 30
Ala?Val?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val
35 40 45
Ala?Val?Val?Ser?Asn?Asp?Gly?Asp?Asp?Lys?Tyr?Tyr?Ala?Asp?Ser?Val
50 55 60
Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Asn?Ser?Arg?Asn?Thr?Leu?Tyr
65 70 75 80
Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys
85 90 95
Ala?Arg?Gly?Phe?Tyr?Gly?Ser?Gly?Ser?Leu?Leu?Ala?Ala?Ser?Asp?Ala
100 105 110
Phe?Asp?Ile?Trp?Gly?Gln?Gly?Thr?Val?Val?Thr?Val?Ser?Ser
115 120 125
<210>3
<211>324
<212>DNA
＜213〉people (human)
<220>
<221>V-region
<222>(1)..(324)
＜223〉nucleotide sequence of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 chain variable region gene
<400>3
gacattgtga?tgacgcagtc?tccaggcacc?ctgtctttgt?ctccagggga?gagagccacc 60
ctctcctgca?gggccagtca?gagtgttcac?ctcacctcct?tagcctggta?tcagcagaaa 120
cctggccagg?ctcccaggct?cctcgtctat?gctgcatcca?ccagggccac?tggcatccca 180
gacaggttca?gtggcggtgg?gtctgggaca?gacttcactc?tcaccatcag?cagactggag 240
cctgaggatt?ttgcaatgta?ttactgtcag?caatatggca?gctcaccaaa?gacgttcggc 300
caagggacca?aggtggatat?caaa 324
<210>4
<211>108
<212>PRT
＜213〉people (human)
<220>
<221>V-region
<222>(1)..(108)
＜223〉the proteinic aminoacid sequence of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 variable region of light chain
<400>4
Asp?Ile?Val?Met?Thr?Gln?Ser?Pro?Gly?Thr?Leu?Ser?Leu?Ser?Pro?Gly
1 5 10 15
Glu?Arg?Ala?Thr?Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?His?Leu?Thr
20 25 30
Ser?Leu?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu
35 40 45
Val?Tyr?Ala?Ala?Ser?Thr?Arg?Ala?Thr?Gly?Ile?Pro?Asp?Arg?Phe?Ser
50 55 60
Gly?Gly?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Leu?Glu
65 70 75 80
Pro?Glu?Asp?Phe?Ala?Met?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Ser?Ser?Pro
85 90 95
Lys?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Asp?Ile?Lys
100 105
Claims (10)
1, a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9, it is characterized in that: being human antibody, is by variable region of heavy chain VH, variable region of light chain VL with the three structural domain single-chain antibody VH-connection peptides-VKs of 12 amino acid whose sequences of CH1 (CH1) 5 ' end as connection peptides AKTTAPSVYPLA formation.
2, a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 according to claim 1, it is characterized in that: the heavy chain variable region gene of described antibody gene and chain variable region gene derive from the enrichment of human source anti-rabies virus rrna antibody library screening, and its heavy chain variable region gene, chain variable region gene have as sequence table＜400〉sequence shown in 1 and＜400〉3.
3, a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 according to claim 1 and 2, it is characterized in that: described antibody gene encoded polypeptides sequence is as sequence table＜400〉sequence shown in 2 and＜400〉4.
4, a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 according to claim 3, it is characterized in that: described aminoacid sequence is changed, lacks or adds one or several amino acid, and in still keeping and the ability of rabies virus.
5, the preparation method of a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9, it is characterized in that: this method utilizes the method for gene recombination technology to prepare, and concrete steps comprise:
(1) structure of ribosomal display single-chain antibody gene storehouse (VH/k):
1-1) extraction of cell total rna and cDNA's is synthetic;
1-2) the pcr amplification of human antibody VH and VL gene;
1-3) clone of PCR product and sequential analysis;
1-4) the splicing of antibody weight, light chain DNA;
(2) structure in ribosomal display single-chain antibody gene storehouse;
(3) in-vitro transcription and translation;
(4) the affine screening of rrna mixture;
(5) clonal expression of VH/k antibody gene;
(6) ELISA identifies positive colony and carries out sequential analysis;
(7) human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9's efficiently expresses and purifying;
(8) specific antigens of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 detects in conjunction with activity;
(9) relative affinity of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 is measured;
(10) the relatively stable Journal of Sex Research of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9;
(11) among the human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 and active research.
6, according to the preparation method of the said a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 of claim 5, it is characterized in that: the concrete steps that efficiently express with purifying of described step (7) human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 comprise:
(1) structure of antibody gene expression vector;
(2) in a suitable system or host, antibody gene is expressed;
(3) the antibody expression host that cultivates in a large number and increase;
(4) the antibody separation and purification to expressing.
7, the preparation method of a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 according to claim 6 is characterized in that: the antibody gene expression vector is plasmid vector or virus vector, the genetically modified carrier of animal or universal support in the above-mentioned steps (1); The expression vector of described antibody gene changes among a suitable system or the host and is expressed, and wherein system of Shi Heing or host comprise the cell of animal or plant, cell strain, bacterium or yeast; This animal is transgenic animal.
8, the application of a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 is characterized in that: described antibody is used for diagnosing or treating the application of rabies medicine in preparation, comprises antibody gene and antibody gene encoded polypeptides.
9, the application of a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 according to claim 8, it is characterized in that: described antibody gene is used for diagnosing or treating the application of rabies medicine in preparation.
10, the application of a kind of human source anti-rabies virus glycoprotein neutrality genetic engineering antibody RD9 according to claim 8, it is characterized in that: described antibody gene encoded polypeptides is used for diagnosing or treating the application of rabies medicine in preparation.
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| CN101812130A (en) * | 2010-05-06 | 2010-08-25 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody (RVFab5) against rabies virus glycoprotein |
| CN101812131A (en) * | 2010-05-06 | 2010-08-25 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody (RVFab8) against rabies virus glycoprotein |
| CN101812132A (en) * | 2010-05-06 | 2010-08-25 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody (RVFab3) against rabies virus glycoprotein |
| CN102643343A (en) * | 2011-02-17 | 2012-08-22 | 长春百克生物科技股份公司 | Human derived anti-rabies virus glycoprotein gene engineering antibody and preparation and application thereof |
| CN103130897A (en) * | 2011-12-05 | 2013-06-05 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Atrazine single-chain antibody screening method and purpose thereof |
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| CN104761639A (en) * | 2015-04-16 | 2015-07-08 | 哈尔滨博翱生物医药技术开发有限公司 | ScFv antibody, encoding gene thereof and application of scFv antibody to preparation of preparation for treating or preventing hepatitis B |
| CN104774844A (en) * | 2015-04-16 | 2015-07-15 | 哈尔滨博翱生物医药技术开发有限公司 | Method for sieving single-chain antibody by virtue of bacterial surface display system |
| CN111171145A (en) * | 2020-01-21 | 2020-05-19 | 兰州生物制品研究所有限责任公司 | Anti-rabies virus monoclonal antibody, preparation method and application |
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| CN1355253A (en) * | 2000-11-29 | 2002-06-26 | 中国预防医学科学院病毒学研究所 | Humanized genetically engineered neutralizing antibody of rabies virus |
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| CN101812130A (en) * | 2010-05-06 | 2010-08-25 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody (RVFab5) against rabies virus glycoprotein |
| CN101812131A (en) * | 2010-05-06 | 2010-08-25 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody (RVFab8) against rabies virus glycoprotein |
| CN101812132A (en) * | 2010-05-06 | 2010-08-25 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody (RVFab3) against rabies virus glycoprotein |
| WO2011137571A1 (en) * | 2010-05-06 | 2011-11-10 | 中国疾病预防控制中心病毒病预防控制所 | Human-derived neutralizing antibody rvfab5 against rabies virus glycoprotein |
| WO2011137570A1 (en) * | 2010-05-06 | 2011-11-10 | 中国疾病预防控制中心病毒病预防控制所 | Human-derived neutralizing antibody rvfab8 against rabies virus glycoprotein |
| CN101812132B (en) * | 2010-05-06 | 2012-07-04 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody (RVFab3) against rabies virus glycoprotein |
| CN101812131B (en) * | 2010-05-06 | 2012-07-04 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody (RVFab8) against rabies virus glycoprotein |
| CN101812130B (en) * | 2010-05-06 | 2012-07-04 | 中国疾病预防控制中心病毒病预防控制所 | Humanized neutralizing antibody (RVFab5) against rabies virus glycoprotein |
| CN102643343A (en) * | 2011-02-17 | 2012-08-22 | 长春百克生物科技股份公司 | Human derived anti-rabies virus glycoprotein gene engineering antibody and preparation and application thereof |
| CN102643343B (en) * | 2011-02-17 | 2015-03-25 | 长春百克生物科技股份公司 | Human derived anti-rabies virus glycoprotein gene engineering antibody and preparation and application thereof |
| CN103130897A (en) * | 2011-12-05 | 2013-06-05 | 中国人民解放军军事医学科学院卫生学环境医学研究所 | Atrazine single-chain antibody screening method and purpose thereof |
| CN104725501A (en) * | 2013-12-20 | 2015-06-24 | 深圳先进技术研究院 | Method for establishing HIV (human immunodeficiency virus) virus antibody yeast display library, method for screening virus broad-spectrum neutral antibody and application thereof |
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| CN104774844A (en) * | 2015-04-16 | 2015-07-15 | 哈尔滨博翱生物医药技术开发有限公司 | Method for sieving single-chain antibody by virtue of bacterial surface display system |
| CN111171145A (en) * | 2020-01-21 | 2020-05-19 | 兰州生物制品研究所有限责任公司 | Anti-rabies virus monoclonal antibody, preparation method and application |
| WO2023279803A1 (en) * | 2021-07-07 | 2023-01-12 | 高光 | Protein binding molecule of rbv and use thereof |
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