At above-mentioned shortcoming, the present invention has adopted molecular biology method structure, clone and has expressed anti-CEA phage single chain antibody.One of innovation is that the antibody molecule amount is little.It is the single-chain antibody variable region fragment (singlechain Fv fragment is called for short scFv) that is formed by connecting by a small-molecular peptides by antibody heavy chain variable region and variable region of light chain.The about 25kDa of molecular weight is the sixth of its parental generation monoclonal antibody molecule amount.Compare with the parental generation monoclonal antibody, the superiority of CL-3-scFv shows that mainly immunogenicity is less, is difficult for causing the human immunity rejection; . good penetrability, can penetrate fine and close tumor barrier effectively, help the diagnosis and the treatment of solid tumor; . because antiboy CL-3-scFv do not contain the Fc fragment, avoid and the cell generation non-specific responding that contains the Fc receptor, therefore in vivo during the imaging localization examination, background is low, clear picture; . antiboy CL-3-scFv does not need glycosylation, helps producing in enormous quantities with prokaryotic system; . antiboy CL-3-scFv and toxin gene reorganization, the recombinant immunotoxin of generation can overcome the unstability of traditional monoclonal antibody and toxin chemical coupling thing, for tumor biotherapy provides novel targeted medicine.
Two of innovation: its antibody production techniques and hybridoma technology relatively have following superiority:. saved the step of cell fusion and immune these time and effort consumings of humans and animals; . directly obtain and preserve antibody gene, avoided the frozen repeatedly back of hybridoma gene to lose problem; . by the antibody gene transformation, improve the affinity of antibody, improve the effector function of antibody; . can develop human antibody; . phage selection antibody method efficient height, easy and simple to handle, economical, be applicable to that rapid large-scale ground produces.
Three of innovation: the existing report of relevant CEA Study of Monoclonal Antibodies, and CEA strand genetic engineering antibody does not appear in the newspapers both at home and abroad.
Concrete technical scheme of the present invention is: at first separate from the mouse hybridoma of secreting anti-CEA monoclonal antibody, purified mRNA, reverse transcription becomes cDNA first chain.With cDNA first chain is template, amplifies heavy chain variable region gene (V respectively by round pcr
H) and chain variable region gene (V
L).DNA sequence by one section coding (Gly4Ser) 3 is V
HAnd V
LCouple together, constitute V
H-Linker-V
LFusion gene.Have in the expression vector of Sfi1 and Not1 restriction enzyme site for this gene is inserted, we add respectively at scFv genetic fragment two ends by PCR contains Sfi1 and Not1 restriction enzyme site.Through restriction endonuclease Sfi1 and Not1 digestion pcr amplification product and recovery, be connected transformed into escherichia coli TG1 competent cell with the expression vector pCANTAB5E that contains Sfi1 and Not1 double digestion then.
Because the single-chain antibody variable region gene inserts in the bacteriophage coat protein g3p gene, forms fusion gene with the g3p gene.Therefore single-chain antibody and coat protein g3p with the formal representation of fusion rotein in the surface of phage.With the affine screening method of immunity, be antigen with human colon carcinoma CEA, from the recombinant phages antibody library, screen.Those and the bonded recombinant antibodies phage of CEA are separated, be used to infect TG1.Make these CEA recombinant phages antibodies obtain enrichment by cultivating amplification.Through 3 take turns immunity affine-screening process of infection-amplification-again, finally obtain CEA specificity recombinant phages antibody.This antibody can be used for the external immunologic diagnosis of tumor.
In order to obtain can be used in the soluble single-chain antibody of oncotherapy, we select for use in the expression vector has an amber platinum (amber) termination codon between the single-chain antibody gene and g3p gene.In large intestine bar TG1 cell, contain the supE gene, can read over the amber codon, produce the scFv-g3p fusion rotein.And in the HB2151 cell, do not contain the supE gene, therefore translation stops at amber termination codon place, produces scFv albumen also constantly it to be transported in born of the same parents' pericentral siphon, leaks into gradually in the culture fluid, thereby obtains soluble single-chain antibody.
In order to detect single-chain antibody easily, the present invention has selected the pCANTAB5E expression vector for use.3 ' the genes of a coding small molecule peptide E-Tag of end connection at the scFv gene.Therefore the single-chain antibody of expressing can be with the antibody test of anti-E-Tag.Through the ELISA screening, obtain 6 strain CEA single-chain antibodies.Select two strains at random and carry out dna sequence analysis, the result shows that the two all is made up of 693 nucleotidess, and its sequence is in full accord.We are with its called after CL-3-scFv.Western Blot result shows that CL-3-scFv has the specificity identical with the parental generation monoclonal antibody.It can discern the tumor cell in colorectal cancer and the stomach organization, and does not organize reaction with normal gastrointestinal tract.
For the mass production antiboy CL-3-scFv, the present invention is cloned into the antiboy CL-3-scFv gene respectively on expression vector pJW2 and the pET5a, is built into antibody recombiant plasmid pJW2-CL-3-scFv and pET5a-CL-3-scFv efficient expression vector.Difference transformed into escherichia coli DH5 α and BL21 (DE3).Inducing with 42 ℃ (pJW2-CL-3-scFv) or 1mM IPTG (pET5a-CL-3-scFv) down, the expressing antibodies amount is 35% of a total protein concentration.Through the purification and the renaturation of inclusion body protein,, obtain purity at last greater than 90% single-chain antibody by MonoQ and Q Sepharose FF ion-exchange chromatography antibody purification.
Embodiment
(1) cell culture and evaluation: hybridoma (Yuan Mei, Liu Chenggui, Li Li etc., resistive connection intestinal cancer monoclonal antibody CL-3, the serodiagnostic application of CL-4 of secretion CL-3 antibody.The sick intestinal magazine of China's anus, 1988, the 4 phases: 3-6) be incubated in the complete RPMI1640 culture medium that contains 15% Ox blood serum.Incubator contains 5%CO
2Mist, humidity is 98%.Identify the specificity and the titre of monoclonal antibody in the culture supernatant with the ELISA method.
(2) isolation and purification of mRNA: with preparation fast, purified mRNA test kit (Promega) is from about 2 * 10
7Extract and purified mRNA in the individual hybridoma.
(3) preparation cDNA: the mRNA with purification is a template, and cDNA is synthesized in reverse transcription.
(4) amplification antibody variable gene: with PCR method is template with cDNA, in the PCR reaction system, add a cover light chain or a variable region of heavy chain primer respectively, 10 * PCR buffer, 5 μ l, the dNTP final concentration is 2.5Mm, mixing is after 100 ℃ of degeneration 5min add 2 Taq of unit archaeal dna polymerases.Total reaction volume is 50 μ l.Add mineral oil behind the mixing.Carry out 30 circular response, each circulation condition is: 94 ℃ of degeneration 30s, and 55 ℃ of annealing 90s, 72 ℃ are extended 90s, and reaction proceeds to last circulation back and be incubated 10min in 72 ℃.After reaction finishes, from heavy, variable region of light chain PCR reaction system, take out 3 μ l respectively and walk 1.5% agarose gel electrophoresis, remainder Sephagles
TMBandprep Kit reclaims.
(5) assembling of single-chain antibody gene and amplification: the V of recovery
HAnd V
LGenetic fragment be connected primer wait mole or near etc. under the molar concentration condition, add 2.5mM dNTP and 5 Taq of unit archaeal dna polymerases, 10 * PCR buffer, 2.5 μ l, 25mM MgCl
22.5 μ l, reaction volume are 25 μ l, with carrying out 7 anneal cycles after the saxol sealing, each circulation reaction condition is 94 ℃ of degeneration 30 seconds, anneals 4 minutes for 64 ℃.
With the scFv gene is template, in above-mentioned reaction volume, add a pair of 5 ' end and contain Sfi I, 3 ' end contains the primer of Not I restriction enzyme site, carry out 30 PCR circulations, each circulation condition is 94 ℃ of degeneration 1 minute, anneals 2 minutes for 55 ℃, 72 ℃ were extended 2 minutes, and last circulation back was 72 ℃ of insulations 10 minutes.Take out 3 μ l and walk 1.2% agarose gel electrophoresis from scFv gene PCR amplification reaction system, remainder reclaims with above-mentioned recovery test kit.
(6) foundation in single-chain antibody gene storehouse: the scFv gene after the recovery is connected transformed into escherichia coli TG1 competent cell through restriction endonuclease SfiI with Not I digestion back and with the plasmid pCAMTNB5E of Sfi I/Not I double digestion respectively.In culture fluid 2 * YT-G (1.7%Bacto-trypone, 1% Bacto-yeast extract, 0.5% NaCl, 2% glucose), cultivate transformed bacteria.Fraction (about 1/10 volume) transformed bacteria is stored in-70 ℃ with 13% glycerol.
(7) expression of recombinant phages antibody and immune affine screening: remaining transformed bacteria dilutes with 10ml2 * YT-G, and is cultured to OD at 37 ℃
600Value is 0.5.Adding ampicillin to final concentration then is 100 μ g/ml, M13K07 to final concentration be 2 * 10
9Pfu/ml cultivated 1 hour for 37 ℃, and is centrifugal.Bacterial sediment is resuspended in 10ml 2 * YT-AK, and (contain in 2 * YT) culture fluid of 100 μ g/ml ampicillin and 50 μ g/ml kanamycin, in 37 ℃ of overnight incubation, centrifugal collection contains the supernatant of recombinant phage.
The supernatant that will contain recombinant phage be coated on CEA antigen one on the culture dish and arise from 37 ℃ and hatched 1 hour.With PBST (PBS that contains 0.05%Tween20) wash dish 20 times, PBS wash dish 20 times.Add the recombinant phages antibody of 100mM triethylamine 1ml elution of bound on plate, use 1M Tris-HCl immediately, the pH9 neutralization is also collected the recombinant phage that elutes.Repeating above-mentioned infection-amplification-screening process 2 takes turns.
(8) preparation of recombinant phages antibody and evaluation: get the recombinant phages antibody 100 μ l that third round elutes and the TG1 cell 200 μ l of exponential phase, cultivated 30 minutes in 37 ℃ of shaking tables behind the mixing, infection cell was done doubling dilution (1: 10 with 2 * YT, 1: 100,1: 1000) after, be coated on SOBAG (2%Bacto-trypone, 0.5%Bacto-yeast extract, 0.05%NaCl, 10mMMgCl
2, 2% glucose, 100 μ g/ml ampicillin, 1.5%Bacto-Agar) on the solid medium in 30 ℃ of overnight incubation.From flat board, choose 72 single bacterium colonies at random, be inoculated into 100 μ l, 2 * YT-respectively
AG (contain 100 μ g/ml ampicillin, in 2 * YI) culture fluid of 2% glucose, 30 ℃ of overnight incubation.Get the 20 μ l bacterium that spends the night next day respectively and be transferred in the new 1.5mlEppendorf centrifuge tube, contain 200 μ l2 * YT-AG and 5 * 10
8Pfu/ml M13K07.Cultivated 2 hours for 37 ℃, centrifugal, with suspend respectively sedimentation cell in each centrifuge tube of 200 μ l2 * YT-AK (containing ampicillin and kanamycin) culture fluid, in 37 ℃ of overnight incubation.Centrifugal and collect supernatant.
By 96 hole elisa plates, coating buffer is 0.05MNa with antigens c EA bag
2CO
3(pH9.6), antigen concentration is 10 μ g/ml, and every hole adds coating buffer 100 μ l, and 4 ℃ are spent the night.Add confining liquid (1.5%BSA is dissolved among the PBS), in room temperature sealing 1 hour.100 μ l recombinant phages antibody supernatants are mixed with the confining liquid equal-volume, and room temperature is placed on the elisa plate that joins the bag quilt after 10 minutes, 37 ℃ of incubations 1 hour.With the negative contrast of M13 phage, 1%BSA, 2 * YT, PBS are blank, with the positive contrast of CL-3 monoclonal antibody, wash plate 3 times with PBST (PBS that contains 0.05%Tween20), and PBS washes plate 3 times.Add the anti-M13 phage of 100 μ l also with the IgG-HRP (1: 5000) of horseradish peroxidase-labeled, the positive control hole adds 100 μ l sheep anti-mouse igg-HRP, and 37 ℃ were reacted 1 hour, washed plate 3 times with PBST, and PBS washes plate 3 times.Add substrate OPD-H
2O
2100 μ l, room temperature effect 20 minutes adds 50 μ l2MH
2SO
4Cessation reaction, the absorbance value in the every hole of detection, 490nm place.Therefrom select the active higher clone of several strains, establish parallel hole and repeat The above results, determine the strongest positive colony of a strain activity, called after CL-3-scFv at last.
(9) evaluation of positive colony recombiant plasmid and dna sequence analysis thereof: extract the CL-3-scFv recombiant plasmid, use SfiI and NotI digestion with restriction enzyme respectively.The enzyme action product is through 1.2% agarose gel electrophoresis analysis.Use T7DNASequence
TMKit measures the DNA sequence of scFv gene on this recombiant plasmid.CL-3-scFv693,:ATG GCC CAG GTC CAG CTG CAG GAG TCA GGG TAT ACT TTCACA ACC TAT GGA ATG AGCTGG GTG AAA CAG GCT CCA GGA AAG GGT TTA AAG TGG ATGGGC TGG ATA AAC ACC TACTCT GGA GTG CCA ACA TAT GCT GAT GAC TTC AAG GGA CGGTTI GCC TTC TCT TTG GAAACC TCT GTC AGC ACT GCC TAT TTG CAG ATC AAC AAC CTCAAA AAT GAG GAC ACG TCAACA TAT TTC TGT GCA AGA TAT GCC TTC GGC TCT TGG TACTTC GAT GTC AGG GGC CAAGGC ACC ACG GTC ACC GTC TCC TCA GGT GGA GGC GGT TCAGGC GGA GGT GGC TCT GGCGGT GGC GGA TCG GAC ATC GAG CTC ACT CAG TCT CCA ATGGCT TCT TTG GCT GTG TCTCTA GGG CAG AGG GCC ACC ATC TCC TGC AGA GCC AGC GAAAGT GTT GAT ACT TAT GCCGTT AGT TTT ATG AAC TGG TTC CAA CAG AAA CCA GGA CAGCCA CCC AAA CTC CTC ATCTAT ACT GCA TCC AAG CAA GGG TCC GGG GTC CCT GCC AGGTTT AGT GGC AGT GGG TCTGGG ACA GAC TTC AGC CTC AAC ATC CAT CCT ATG GAG GAGGAT GAT GCT GCA ATG TATTTC TGT CAA CAA AGT AAG GAG GTT CCG TGG ACG TTC GGTGGA GGG ACC AAG CTG GAAATA AAA CGG
(10) preparation of soluble single-chain antibody and evaluation: with the recombinant plasmid transformed escherichia coli HB2151 that identifies.Choose 4 single bacterium colonies overnight incubation in 2ml2 * YT-AG culture fluid.Centrifugal, with containing 2 * YT culture fluid 2ml suspension sedimentation cell of 1mMIPTG, in 30 ℃ of inducing culture 20 hours.Centrifugal, collect supernatant respectively.Add equal-volume acetone, mixing left standstill 4 hours in 4 ℃ of refrigerators.Centrifugal, remove supernatant, in air drying 30 minutes.Add 50 μ lPBS dissolution precipitation things.
Get 15 μ l precipitate behind 12% SDS-PAGE electrophoresis, electricity consumption is changeed method and the protein substance on the gel is transferred on the nitrocellulose filter the anti-E-Tag antibody of reuse ( Phamacia ) detection soluble single-chain antibody.Westem Blot experimental results show that the molecular weight of this solubility scFv antibody fragment is about ( Fig. 6 ) about 26kDa.He is made up of 231 aminoacid.DNA:M A Q V Q L Q E S G Y T F T T Y G M S W V K Q A P G K G L K WM G W I N T YS G V P T Y A D D F K G R F A F S L E T S V S T A Y L Q I N N LK N E D T ST Y F C A R Y A F G S W Y F D V R G Q G T T V T V S S G G G GS G G G G S GG G G S D I E L T Q S P M A S L A V S L G Q R A T I S C R A S E SV D T Y A VS F M N W F Q Q K P G Q P P K L L I Y T A S K Q G S G V P A R FS G S G S G TD F S L N I H P M E E D D A A M Y F C Q Q S K E V P W T F G G GT K L E I K R
(11) immunocompetence of soluble single-chain antibody is identified: on ice, add 58.2 gram ammonium sulfate gradually in the 200ml supernatant and make its final concentration reach 50%, and with constantly stirring 4 hours of magnetic stirring apparatus.Centrifugal, abandon supernatant, precipitation is dissolved in the 2mlPBS buffer.
ELISA: by 40 hole elisa plates, coating buffer is 0.05MNa with antigens c EA bag
2CO
3(pH9.6), antigen concentration is 10 μ g/ml, and every hole adds coating buffer 100 μ l, and 4 ℃ are spent the night.Add confining liquid 1%BSA, be dissolved in the PBS buffer), in room temperature sealing 1 hour.If parallel hole, with confining liquid dilute spissated immune supernatant (1: 2,1: 4,1: 8,1: 16,1: 32 ...), every hole adds the immune supernatant of 100 μ l dilution.With the negative contrast of 1%BSA, 2 * YT, PBS is a blank, and with the positive contrast of CL-3 monoclonal antibody, 37 ℃ of incubations 1 hour are washed plate each 3 times with PBST and PBS.The anti-E-Tag antibody 100 μ l that add dilution in 1: 2000.Positive control adds 100 μ l diluents, and 37 ℃ of incubations 1 hour are washed plate each 3 times with PBST and PBS.Sheep anti-mouse igg-HRP100 μ the l that adds dilution in 1: 1000,37 ℃ of incubations 1 hour.Wash plate each 3 times with PBST and PBS.Add substrate OPD-H
2O
2100 μ l, room temperature effect 20 minutes adds 50 μ l2MH
2SO
4Cessation reaction, OD490 value and analysis result in the every hole of survey, A490 place.
Western Blot: with human colon carcinoma CEA is antigen, and on nitrocellulose filter, with the negative contrast of BSA, the PBS buffer is a blank with the antigenic dilution 2 μ l points of different concentration.After 20 minutes, add 5% defatted milk powder/PBS at air drying, spend the night in 4 ℃ of sealings.With film respectively with immune supernatant in CL-3-scFv or CL-3 monoclonal antibody reactive, shook gently 2 hours in room temperature.Wash film 3 times with the PBS buffer, each 10 minutes.Add the anti-E-Tag antibody 10ml of dilution in 1: 300 or the sheep anti-mouse igg-HRP10ml of dilution in 1: 500 respectively, shook gently 1 hour in room temperature.Reuse does not have phosphate buffer (150mM Tris-HCl pH7.4,50mM NaCl) and washed film 10 minutes.Add 10ml substrate (0.01M Tris-HCl pH7.4,0.03%CoCl
2, 6mg benzidine, 15 μ lH
2O
2), shook gently 2-3 minutes in room temperature, use the distilled water rinsing, add the 20mlPBS buffer preserving in 4 ℃.Dot Blot result shows the activity that CL-3-scFv can specific bond CEA, and approximate with the activity of positive control CL-3 monoclonal antibody.Illustrate that this soluble antibody fragment has affinity and the specificity identical with the parental generation monoclonal antibody.
(12) structure of CL-3-scFv efficient expression vector: for mass production CL-3-scFv, we are cloned into the antiboy CL-3-scFv gene respectively on expression vector pJW2 and the pET5a, are built into recombiant plasmid pJW2-CL-3-scFv and recombiant plasmid pET5a-CL-3-scFv efficient expression vector.Difference transformed into escherichia coli DH5a and BL21 (DE3).On the LB solid medium that contains 100 μ g/ml ampicillin, choose single bacterium colony at random, in the 1000mlLB culture medium 30 ℃ when being cultured to OD600=0.6, use 42 ℃ (pJW2-CL-3-scFv) or 1mM IPTG (pET5a-CL-3-scFv) inducing culture 5 hours respectively.Centrifugal, collecting cell.
(13) extraction of inclusion body protein [5] and renaturation [6]: thalline is resuspended in the 20ml distilled water, carrying out ultrasonic bacteria breaking.Centrifugal 12000 * g, 4 ℃, 30min.Abandon supernatant, precipitation both had been an inclusion body.After 50mM TE washing one time, in-20 ℃ of preservations.
With 8M carbamide (containing 10mM DTT) dissolving inclusion body, put room temperature after 2 hours, centrifugal 12000 * g, 4 ℃, 20min abandons precipitation.Supernatant dropwise add renaturation solution (0.1MTE, the 0.5ML-arginine, 1mMGSSG, 0.2mM GSH pH8.4) makes proteic final concentration be about 30 μ g/ml.Leave standstill 10 ℃, more than 48 hours.
(14) purification of antiboy CL-3-scFv: the antibody that concentrates renaturation with PEG.With MonoQ and Q SepharoseFF ion-exchange chromatography antibody purification.With 20mMTE balance pillar (2.6 * 20cm).Last sample 20mMTE eluting, flow velocity 60ml/h.After waiting to pass the peak, with 20mMTE, 0.3M NaCl eluting is collected the purpose peak.Identify antibody with SDS-PAGE and Western Blot, the purity that obtains single-chain antibody is greater than 90%.