CN1523350A

CN1523350A - Process for three-dimensional structural information design new pattern pharmaceutical molecule based on antigen-antibody action

Info

Publication number: CN1523350A
Application number: CNA031048706A
Authority: CN
Inventors: 沈倍奋; 冯建男; 黎燕; 张巍
Original assignee: Beijing Sihuan Medicine Science & Technology Co ltd; Institute of Basic Medical Sciences of AMMS
Current assignee: Beijing Tianguangshi Bio-Tech Co., Ltd.; Institute of Basic Medical Sciences of CAMS
Priority date: 2003-02-21
Filing date: 2003-02-21
Publication date: 2004-08-25

Abstract

The invention relates to a new-drug molecule designing method based on stereochemical structure information by the action of antigen and antibody. Concretely speaking, it designs theoretical conformation of changeable region of antigen and antibody on the computer, on this basis builds the theoretical conformation of antigen-antibody complex, identifies epitope in antigen combined with antibody and verifies the epitope by experiment, and designs a new-drug molecule according to the sequence of the epitope.

Description

Design the method for newtype drug molecule based on the spatial structure information of antigen-antibody effect

Technical field

The present invention relates to carry out the newtype drug molecule design method based on the spatial structure information of antigen-antibody effect.Particularly, the present invention is by making up the theoretical conformation of antigen and antibody variable region on computers, set up the theoretical conformation of antigen-antibody complex on this basis, in the identification antigen with the epi-position of antibodies, and verify this epi-position, the drug molecule new by experiment according to the sequences Design of this epi-position.The examples of antigens of using among the present invention is TNF (TNF-α), the antibody example is the anti-TNF monoclonal antibody Z12 that screening obtains from hybridoma, utilize this conformation and structural information to the antigen-antibody interaction zone, by CAD method, obtain to substitute the small peptide molecule of antibody Z12.

Background technology

Because it is big that protein drug prepares difficulty, need more Protocols in Molecular Biology, means, owing to be difficult to discharge, absorb, in the medication process, can only be undertaken simultaneously by the mode of injection.The protein drug molecular weight is big, pliability is big, exists a plurality of differently from epi-position, may activate other Disease-causing gene and produce beyond thought spinoff in treatment.

The antibody class medicine has caused people's extensive concern as the swift and violent protein drug of development in recent years.Yet " human antimouse antibody (HAMA) " reaction that mouse source antibody causes has limited its clinical practice; Human mouse chimeric antibody can cause the anti-chimeric antibody of people (HACA) reaction in the process of long term administration; Although utilize the monoclonal antibody of people-people's hybridoma technology preparation can overcome the shortcoming of above-mentioned mouse source antibody and chimeric antibody, the preparation of people's monoclonal antibody exist yield poorly, unstable, problem such as affinity is low so that can not reach the corresponding treatment purpose.What is more important, when treatment rheumatoid arthritis diseases such as (RA), dose needs 1-10mg/kg anti-TNF antibodies or 16mg/m ²Soluble recepter need continue some months the course of treatment to more than half a year.Because dosage is big, the spinoff of protein drug is just more obvious.

E Fintan Walton (being proficient in the expert of extensive biological technical field) once pointed out, " chance that has the medicine of sensation property from the recombinant protein acquisition is very limited ".Protein is difficult to preparation, be difficult to discharge, and be easy to generate spinoff, and small-molecule drug has selectivity, can exist with tablet, capsule form, make small-molecule drug substitute the focus that protein molecular becomes current MOLECULE DESIGN, rational drug design, Moffat A S promptly proposed " getting back to the synthetic epoch (Going back to the future with small syntheticcompounds) of micromolecular compound " in 1993.

The area of computer aided drug design that development in recent years is swift and violent, no matter be theoretical, technology, still relevant method has all possessed quite rich basis, and serves experiment effectively, provide foundation, theoretical thinking for new drug designs and synthesizes, also shortened the R﹠D cycle of new drug simultaneously.In conjunction with computing machine medicine auxiliary design method, will speed up undoubtedly have particular target to the research and development of lead compound, improve the success ratio of new drug development greatly.

Utilizing the area of computer aided drug design method to obtain candidate compound, to enter the successful small-molecule drug of clinical research a lot, utilize the crystal structure model of fibrin ferment as Hoffmann La Roche company, optimize the hydrophobic region structure of candidate compound at the enzymatic center, design, acquisition thrombin inhibitor Ro46-6240 have now entered anticoagulant clinical trial; The crystal structure of Merck research department integrated use carbonic anhydrase (CA) and quantum chemistry conformational analysis, determine the activity conformation that known CA inhibitor combines with CA, carry out structure of modification on this basis and obtain Trusopt (MK-507), now as the medicine listing for the treatment of the glaucoma disease.

At present, obtain bigger progress based on protein conformation design micromolecular compound in the world, yet utilize antigen-antibody complex modelling antibody analog just just to begin.Takahashi M etc. utilize computer-aided design (CAD) to obtain the porphyrin binding peptide by site, antigen binding antibody hypervariable region (CDR), have biological function through experimental verification; Utilize two-dimentional 1H nuclear magnetic resonance technique to disclose the conformational characteristic that 12 peptides combine with porphyrin, utilize computer-aided design (CAD), make up more small peptide section-9 peptide, and final the achieving success.Domestic also the development in recent years based on macromolecular structure design small-molecule drug, and obtained certain progress on theoretical model, computing method and integrates with in the research aspect this in the world.Yet, but do not carry out as yet at antigen epitope-antibody hypervariable region CDR binding mode design small-molecule drug based on Ag-Ab effect compound.

HTNF-α is by the multi-functional cell factor that has of monocyte and macrophage generation, can kill and wound or suppress tumour cell, has important biologic activity such as the neutrophil leucocyte of raising phagocytic activity.But, in recent years along with going deep into to hTNF-α biological activity research, find infectious shock, inflammation that a variety of causes causes, that autoimmune disease all shows as the TNF expression is too high, great deal of research results shows that TNF-α participates in such as multiple human diseasess such as pyemia, infection, autoimmune disease, graft rejection, graft versus host disease(GVH disease)s.Therefore, progressively go deep into about the research of hTNF-α antagonist and mechanism thereof, especially anti-TNF antibodies is in the ascendant especially to the effect study and the clinical research of animal model.

Because the illeffects of hTNF-α in the human immunity disorder set about designing therapeutic strategy at present to suppress or to offset the activity of hTNF-α.Wherein, in the searching of carrying out both at home and abroad and the antibody of hTNF-α, soluble TNF acceptor have development prospect widely as the means that suppress the hTNF-alpha active.

In recent years, anti-hTNF-Alpha antibodies, soluble TNF acceptor have obtained the FDA approval, and are used for the treatment of RA, regional ileitis.With respect to early stage medicine (as DMARD), the anti-hTNF-Alpha antibodies, the soluble TNF acceptor that obtain the FDA approval have more excellent result of treatment, and tolerance, clinical remission effect are better.A large amount of molecular biology experiments show, no matter be neutralizing antibody, or solubility hTNF-α acceptor, as long as can block the TNF activity, above-mentioned disease just there is certain therapeutic action.

This invention selects TNF-α as research object, in conjunction with this laboratory over past ten years in the molecular biology research achievement aspect the TNF antibody, make up the compound model of antibody and hTNF-α effect targetedly by computer-implemented method; By molecular biology experiment, the structured data correction research that binding isotherm simulation is determined, and then utilize that the homology mould is built, the molecular docking Design Theory is modified the compound model; The epitope space conformation feature of antibodies is determined in the variation of binding molecule dynamic method dynamic similation compound effect conformation; Method appropriate design TNF antibody simulating peptide by brand-new drug design.

Summary of the invention

The method of the spatial structure information design newtype drug molecule based on the Ag-Ab effect of the present invention may further comprise the steps:

(1), makes up the theoretical conformation of antigen and antibody variable region based on the primary structure sequence of antigen and antibody;

(2), make up the theoretical conformation of antigen-antibody complex according to the theoretical conformation of antigen and antibody variable region;

(3) epi-position of binding antibody in the identification antigen;

(4) epi-position of binding antibody in the experimental verification antigen;

(5) according to the sequence of above-mentioned epi-position, design can influence the molecule of itself and antibodies.

The present invention adopts the method that combines with structure biology, molecular biology, bioinformatics, is example with TNF-α, and theoretical combining with experiment determined the TNF-α epitope of antibody Z12 specific recognition.

At the antibody Z12 that obtains from filtering hybridoma,, utilize the compound theoretical model that the homology mould is built, the method for molecular docking obtains the Ag-Ab effect in conjunction with TNF-alpha-crystal structure with neutralization activity.

By computer graphics techniques, form the space structure that theory, free energy of reaction theory, the effect of general moral China and electrostatic interaction are tentatively determined antibody Z12 specific recognition TNF-α epitope theoretically by intermolecular ydrogen bonding.

Utilize Protocols in Molecular Biology, design TNF-α deletion mutant detects by biological means such as ELISA, Western-Blot, thereby determines theoretical prediction result's reliability from experiment.

The present invention is by the correctness of said method confirmation theoretical prediction, and the position of definite Z12 antibody recognition TNF-α epitope.

The present invention utilizes the computer aided molecular design principle by epi-position structure and the physicochemical property feature of the Z12 specific recognition TNF-α that determines, theoretically the small peptide that design can analog antibody Z12.Utilize biological means, small peptide is carried out biological assessment.

The present invention has obtained that by above-mentioned strategy the specificity combination of antigen (TNF-α)-antibody (Z12) is had remarkable inhibiting Z12 antagonism peptide.

Method of the present invention can be referring to appended process flow diagram (Fig. 7).

Description of drawings

The RT-PCR amplified production of Fig. 1 .1 antibody Z12.M:DL2000 wherein; 1:Fd segment genome 2: κ chain gene.Fig. 1 .2 contains the enzyme of the recon Z12 of Fd and κ chain gene and cuts evaluation.M:DL2000 wherein; 1:pGEM-T Easy (Fd)/XhoI+SpeI; 2:pGEM-T Easy (κ)/Sac I+Xba I.

Fig. 1 .3Fd section sequencing result.

The κ chain order-checking result of Fig. 1 .4 antibody Z12.

Fig. 2 .1 utilizes ψ, ω and main chain C atom optical activity ζ antagonist Z12 secondary structure heavy, light chain to predict the outcome.

Fig. 2 .2 utilizes Ramachandran figure that weight, the variable region of light chain space structure that makes up carried out rationality and detects.

Fig. 2 .3 utilizes molecular docking, heavy, the interactional stable composite space conformation of light chain of the antibody Z12 that obtains by molecular mechanics, molecular dynamics simulation.Green portion is represented heavy chain of antibody framework region (FR) among the figure, red part is represented light chain of antibody framework region (FR), purple, Chinese red, pale blue are represented heavy chain CDR1, CDR2, CDR3 respectively, and yellow, avy blue, grey are represented light chain CDR1, CDR2, CDR3 respectively.

TNF-α three-dimensional conformation under Fig. 3 .1 molecular force field.Red, green, purple ribbon is represented every strand of TNF-α triplet respectively, and spheroid is represented the TNF-α catastrophe point that obtains in the molecular biology experiment.

The apparent static in Fig. 3 .2 antibody Z12 variable region gene CDR district distributes.The apparent static distribution in antibody Z12 variable region gene CDR district (the blue positive electricity district that represents, the red negative electricity district that represents, the non-electric district of white representative)

The TNF-α that Fig. 3 .3 theoretical modeling obtains and the stable space conformation of Z12 effect.The TNF-α that theoretical modeling obtains and the stable space conformation of Z12 effect (the triplet pattern yellow, red, that grey is represented TNF respectively; The FR green, that purple is represented antibody Z12 variable region gene, wherein green is represented light chain, and purple is represented heavy chain; The CDR of tangerine, light blue representative antibody Z12 variable region gene, wherein Chinese red is represented light chain, light blue representative heavy chain)

Determining of Fig. 4 .1TNF-α and Z12 effect compound key position.TNF-α and Z12 effect compound key position determine that (yellow line chart is represented antibody Z12, and green line chart is represented TNF-α; Sphere role of delegate zone, the purple ball is represented antibody CDR, green ball is represented the 141-146 of TNF-α).

Fig. 4 .2 participates in the TNF-α identified region mode chart in conjunction with Z12.

Escherichia coli bias password table and the corresponding amino acid sequence thereof of Fig. 4 .3hTNF-α.

Fig. 5 .1pUTNF92-157 construction of recombinant plasmid synoptic diagram.

Fig. 5 .2pUTNFD53-91 construction of recombinant plasmid synoptic diagram.

Fig. 5 .3pGEX-2T and different hTNF-α deletant genetic recombination synoptic diagram.1. the foreign gene of Cha Ruing is the encoding gene of hTNF-α or hTNF92-157 or hTNFD53-91 or hTNFD29-88; 2. the foreign gene of Cha Ruing is the hTNF1-91 encoding gene.

The enzyme of Fig. 5 .4 recombinant plasmid is cut evaluation.(A) pUTNF92-157; (B) pUTNFD53-91; (C) pUTNFD29-88; 1: recombinant plasmid pUTNF92-157; 2:pUTNF92-157/EcoN I; 3:pUTNF-α/EcoN I; 4:pUTNF92-157/HincII; 5:pUTNF-α/Hinc II; 6:pUTNF92-157/Hind III; 7:pUTNF-α/Hind III; 8:pUTNFD53-91/Nco I+Hind III 9:pUTNFD29-88/NcoI+Hind III; 10:pUTNF-α/Nco I+Hind III.

Fig. 5 .5PCR amplification hTNF-α and deletant gene thereof.

M：DL2000

1：PCR?products?of?hTNF92-157(254bp)

2：PCR?products?of?hTNFD29-88(347bp)

3：PCR?products?of?hTNFD53-91(410bp)

4：PCR?products?of?hTNF-α(527bp)。The enzyme of Fig. 5 .6 recombinant plasmid is cut evaluation.

M：DL2000

1：pGTNF92-157/EcoR?I+BamH?I

2：pGTNFD29-88/EcoR?I+BamH?I

3：pGTNFD53-91/EcoR?I+BamH?I

4：pGTNF-α/EcoR?I+BamH?I

Fig. 5 .7pGTNF1-91 construction of recombinant plasmid and evaluation.

M1：DL2000；M2：λDNA/Hind?III

1:hTNF-α/BamH I+Hinc II PCR product; 2:hTNF-α PCR product; 3:pGEX-2T/BamH I+Sma I; 4,5:pGTNF1-91/BamH I+EcoR I.

Fig. 5 .8hTNF-α deletant gene sequencing figure.Fig. 5 .9 is through the electrophoretic analysis of the fusion of IPTG abduction delivering.

(A) M: molecular weight protein marker; 1:GST (26kDa);

2：GST/hTNF92-157(33kDa)； 3：GST/hTNFD29-88(37kDa)；

4：GST/hTNFD53-91(39kD)； 5：GST/hTNF-α(43kDa)

(B) M: molecular weight protein marker; 1:GST (26kDa);

2：GST/hTNF92-157(33kDa)； 3：GST/hTNF?1-91(37kDa)；

4：GST/hTNF-α(43kDa)。

The affinitive layer purification of Fig. 5 .10Z12 antibody.(A) wash-out collection of illustrative plates (B) protein electrophoresis analysis.

The electrotransfer of Fig. 5 .11 fusion reaches the Western-blot analysis with the effect of Z12 antibody.

(A): protein electrophoresis and transfer (B): Western-blot

M: molecular weight protein marker; 1:GST (26kDa); 2:GST/hTNF92-157 (33kDa); 3:GST/hTNFD29-88 (37kDa); 4:GST/hTNFD53-91 (39kD); 5:GST/hTNF-α (43kD).

The electrotransfer of Fig. 5 .12 fusion reaches the Western-blot analysis with the effect of Z12 antibody.

(A): protein electrophoresis and transfer (B): Western-blot

M: molecular weight protein marker; 1:GST (26kDa); 2:GST/hTNF92-157 (33kDa); 3:GST/hTNF1-91 (37kDa); 4:GST/hTNF-α (43kDa).The ELISA of Fig. 5 .13Z12 antibody and hTNF-α N end and C end fragment interactively detects.Fig. 5 .14 one step inverse PCR method makes up hTNF-α deletant synoptic diagram.The agarose gel electrophoresis of Fig. 5 .15PCR amplified production.M: λ DNA-HindIII fragment; 1: primer 1PCR amplification; 2: the primer 2 pcr amplification.

The enzyme of Fig. 5 .16 recombinant plasmid is cut evaluation.1：pUTNF-α/Hinc?II+Hind?III；2：pUTNFD141-146/Hinc?II+Hind?III；3：pUTNF-α/Nco?I+Hinc?II；4：pUTNFD29-36/Nco?I+Hinc?II。

The protein electrophoresis analysis of Fig. 5 .17hTNF-α disappearance protein expression.

1，2：hTNF-α(17kDa)；3，4：hTNFD141-146(16kDa)；5，6：hTNFD29-36(16kDa)；7，8：pUC18。

Fig. 5 .18hTNF-α and deletant thereof detect the toxicity of L929 cell.

Fig. 5 .19Z12 antibody and the interactional elisa assay of hTNF-α deletant.

Fig. 6 .1 computer-aided design (CAD) small peptide with (TNF) ₃Interactional compound model.

Fig. 7 carries out the process flow diagram of newtype drug MOLECULE DESIGN based on antigen-antibody action space structural information.

Embodiment

Comprise following 7 parts

1, TNF-α function antibody Z12 variable region gene obtains

2, the simulation of antibody Z12 variable region gene sequence space conformation

3, the acquisition of antibody Z12 variable region gene and TNF-α effect compound

4, antibody Z12 variable region gene identification TNF-α epitope determines

5, utilize the checking of molecular biology experiment to notional result

6, computer-aided design (CAD) newtype drug molecule

7, competition is in conjunction with experiment and other function test

1, anti-TNF-α function antibody Z12 variable region gene obtains

1.1 material

PCR product cloning carrier pGEM-T Easy is available from Promega company, and clone strain JM109 is frozen by this chamber.

1.2 the extraction of the total RNA of hybridoma and cDNA's is synthetic

Recovery hybridoma (Z12) cell, grow up to individual layer after, it is blown down gently, place the 15ml test tube, the centrifugal 10min of 1000r/min uses the physiological saline washed twice on low speed centrifuge.Add 4ml physiological saline re-suspended cell, be sub-packed in the sterilization Ep pipe.The centrifugal supernatant of abandoning adds 1mlTRIzol Reagent solution (Gibco company), makes the lysis mixing, and room temperature leaves standstill 15-20min.Every pipe adds the 0.2ml chloroform, the vibration mixing, and room temperature leaves standstill 10min.4 ℃ of centrifugal 10min of 12000r/min carefully draw upper strata water (containing cell total rna) and place another sterilization Ep pipe, and room temperature leaves standstill 10min behind the adding 0.5ml isopropyl alcohol mixing.4 ℃ of centrifugal 10min of 12000r/min abandon supernatant, and precipitation is washed twice with 1ml 75% absolute ethyl alcohol.Allow be deposited in natural drying at room temperature (being sure not to drain), the resuspended RNA precipitation of sterilization deionized water with 20 μ l do not have RNAase places ice bath to be used for the synthetic of cDNA immediately.

Get about 1-5 μ g cell total rna, add random primer 0.5 μ g, 200 μ M dNTP, 5 * reverse transcriptase reaction buffer, 4 μ l, water supply and make volume reach 18 μ l.Above mixed liquor is boiled 5min in water-bath, and place ice bath to cool off immediately, add 5U AMV reverse transcriptase (Promega company).For preventing that the RNA enzyme from polluting the RNA degraded that causes, adds RNA enzyme inhibitor Rnasin 10-20U in reaction system.With above reaction system mixing, behind 42 ℃ of extension 1h, in water-bath, boil 10min, centrifugal stand-by.

1.3PCR amplification antibody Fd and κ chain gene

Get above-mentioned synthetic cDNA product 4 μ l as template, take each 1uM of upstream and downstream primer of murine antibody Fd and κ chain gene, dNTP 200uM, 10 * Taq buffer, 5 μ l, 1U Taq enzyme, water complement to 50 μ l.The pcr amplification condition is: 95 ℃ of 5min; 94 ℃ of 1min, 53 ℃ of 1min, 30 circulations of 72 ℃ of 1min; 72 ℃ of 10min.

Amplification is as follows with 5 ' end mix primer (totally 8) sequence of murine antibody heavy chain Fd fragment gene:

5′--AC?TCC?CAG? CTC?GAG?CTG(T)GTG?C(G)AG?TCA(T)GG--3′

The recognition sequence (line part) that wherein contains restriction endonuclease XhoI.

Amplification is as follows with 3 ' end primer sequence of murine antibody heavy chain Fd fragment gene:

5′--AGG?CTT? ACT?AGT?ACA?ATC?CCT?GGG?CAC?AAT--3′

The recognition sequence (line part) that wherein contains restriction endonuclease SpeI.

Amplification is as follows with 5 ' end mix primer (totally 7) sequence of murine antibody κ chain gene:

5′--CC?AGT?TCC? GAG?CTC?GTT?GTG?ACT?CAG?GAA?TCT--3′

5′--CC?AGT?TCC? GAG?CTC?GTG?TTG?ACG?CAG?CCG?CCC--3′

5′--CC?AGT?TCC? GAG?CTC?GTG?CTC?ACC?CAG?TCT?CCA--3′

5′--CC?AGT?TCC? GAG?CTC?CAG?ATG?ACC?CAG?TCT?CCA--3′

5′--CC?AGA?TGT? GAG?CTC?GTG?ATG?ACC?CAG?ACT?CCA--3′

5′--CC?AGA?TGT? GAG?CTC?GTC?ATG?ACC?CAG?TCT?CCA--3′

5′--CC?AGT?TCC? GAG?CTC?GTG?ATG?ACA?CAG?TCT?CCA--3′

The recognition sequence (line part) that wherein contains restriction endonuclease SacI.

Amplification is as follows with 3 ' end primer sequence of murine antibody κ chain gene:

5′--GCG?CCG? TCT?AGA?ATT?AAC?ACT?CAT?TCC?TGT?TGAA--3′

The recognition sequence (line part) that wherein contains restriction endonuclease XbaI.

1.4PCR the recovery/purifying of product

Use the dna fragmentation fast purifying/recovery kit of Beijing ancient cooking vessel state biotech firm, undertaken by its product description.

1.5Fd and the clone and the sequencing of κ chain gene

Fd and κ chain gene are connected into pGEM-T Easy carrier respectively, and the coupled reaction system is as follows: reclaim the pcr amplification product 3 μ l of purifying, pGEM-T Easy carrier 1 μ l, T4 dna ligase 1 μ l, 2 * T4 dna ligase buffer, 5 μ l.With the reaction solution mixing, instantaneous centrifugal, room temperature is placed 2h, just can be used for transforming.With coupled reaction product 10 μ l, transform in e. coli jm109, coated plate is cultivated 16-20h for 37 ℃.Picking monoclonal bacterium colony is put 37 ℃ of overnight incubation in the 4ml Amp+LB nutrient culture media.Extract bacterium liquid plasmid.Use XhoI+SpeI and SacI+XbaI double digestion recombinant plasmid respectively, after the clip size that evaluation cuts out is correct, bacterium liquid is served the order-checking of Hai Boya biotech firm.

1.6 determining of antibody weight, variable region of light chain and CDR region sequence

The BLAST at the login U.S. state-run biological information center (NCBI) (basic local contrast research tool, Basic Local Alignment Search Tool) sequence retrieval website http://www.ncbi.nlm.nih.gov/BLAST/, in text box, paste Fd and κ chain order-checking gene respectively by clipbook, select the nr database (to comprise all nonredundancy sequences that GenBank, EMBL and DDBJ include, got rid of EST, STS, GSS part), use the blastn program to carry out the sequence similarity retrieval.The dna sequence dna document of the gene (Query) that the search sequence similarity is higher, according to the relative conservative characteristic of CH1 with the CL gene, determine genes of interest (Sbjct) CH1 or CL code area, search genes of interest head 5 ' end primer sequence position then, base sequence between the two is exactly the gene order of variable region amino acid of encoding substantially.Can determine initial amino acid by CDS (Coding Sequence, the coded sequence) note in the known homogenic dna sequence dna record.

The variable region gene sequence of will encoding is translated into amino acid sequence, with this sequence is probe, in the PDB database, retrieve, the architectural feature of the albumen (Query) that the search sequence similarity is higher, be combined in the manual information retrieval result in the Kabat immunoglobulin amino acid sequence library simultaneously, determine CDR region sequence and other architectural feature such as the disulfide bond of antibody.

1.7RT-PCR amplification

Extract total RNA from the Z12 hybridoma of secreting anti-hTNF-Alpha antibodies,, can obtain the band (seeing Fig. 1 .1) of size about 700bp, big or small consistent with Fd and κ chain gene through RT-PCR amplification.

Cut evaluation 1.8 contain the enzyme of the recon of Fd and κ chain gene

Under the Taq enzymatic, carry out the product that pcr amplification obtains and have poly (A) tail, utilize these characteristics that antibody Fd and the κ chain gene product that amplification obtains directly is connected in the pGEM-T Easy carrier.Connexon Transformed E coli JM109, amplification cultivation, and extract plasmid.5 of the upstream and downstream primer of Fd and κ chain gene ' end has XhoI and SpeI (Fd), SacI and XbaI (κ chain) restriction enzyme site respectively owing to be used to increase, utilize these two groups of enzymes to cut corresponding recombinant plasmid respectively, all cut out the band (seeing Fig. 1 .2) of about 700bp size, big or small consistent with pcr amplification product proves correct recon.

1.9 sequencing result

To cut through enzyme and identify that correct recombinant plasmid send company's order-checking, sequencer address is seen Fig. 1 .3,1.4.Concrete nucleotide sequence is face 1.10 joints as follows.

1.10 determining of antibody heavy and light chain variable region gene

With the BLAST command search nr database of Fd sequencing sequence by NCBI, and carry out homology analysis, search program is blastn, and promptly nucleotide sequence is to the retrieval of nucleic acid database.Preceding 100 genes that retrieval is come out are mouse (Mus Musculus) antibody (IgG1/antibody) variable region of heavy chain (heavy chain variable region) or Fd district (VH+CH1) gene (mRNA), and sequence similarity is all more than 93%.

The disclosed dna sequence dna document of inquiry some of them, be to be for AB048527, gi number that 13359422 nucleotides sequence is classified example as with the searching number, the nucleotide sequence of " C_region " among this CDS (murineantibody heavy chain CH1 region) indication is corresponded to (tilted letter in the Fd sequenced genes that vides infra) in the genes of interest, find that this position sequence is in full accord between the two.In view of the CH1 district more conservative, thereby determined the CH1 code area of genes of interest, sequence before this promptly is a VH district coded sequence.

Search out 5 ' end primer sequence (primer directly is connected in the pGEM-T Easy carrier by pcr amplification) at the sequenced genes head and use shadow representation, the restriction enzyme recognition sequence in 5 ' end primer represented in letter in the black surround.In conjunction with the VH coded message in the homologous gene CDS record, determine VH nuclei originis thuja acid, thereby determine VH code area (representing) with unrestrained line.

Z12 antibody Fd (VH+CH1) sequenced genes (594bp):

GGGCCCGACGTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATT

TCAGCCAA

AACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACT

CCATGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGT

GAGCTGGAACTCTGGATCCCTGTCCAGCGGTGTGCACAGCTTCCCAGCTTGTCTG

GCN

Similarly, determine κ chain CL code area earlier, determine the coding variable region gene then.

Z12 antibody κ chain (VL+CL) sequenced genes (593bp):

GTCGCATGCTCCCGGCCGCCATGGCGGCCGCGGGAATTCGATT

CGGGCTGATGCTGCACCAACTGTATCCATCTTCC

CACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAAC

AACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACA

AAAATGGCGTCCTGAACAGTTGGGACTGGTCANGGACAGGCAAAAGACAGCACCTA

CC

The nucleotide sequence of coding variable region is translated into amino acid sequence corresponding.

Anti-hTNF-alpha monoclonal antibodies (Z12) heavy and light chain variable region sequence:

VH?Seqyence Lebgth：363bp?121aa

1 Q L E L V E S G G G L V K S G 15

16 G S L K L S C A A S G F A F N 30

31 N Y D M S W V R Q T P E R R L 45

46 E W V A Y I N T G G G Y T Y Y 60

61 P D T V K G R F T I S R D N A 75

76 K N T L Y L Q M S S L R S E D 90

91 T A M Y Y C A S E R Y D G L Y 105

10 6 Y A M D Y W G Q G T S V T V S 120

(VH Sequce3-9 bit base is XhoI recognition sequence: CAG C ^ TC GAGCTT)

VL?Sequence Length：339bp?113aa

1 E L Q M T Q S P S S L A V S A 15

16 G E K V T M S C K S S Q S L L 30

31 N S R T R K N Y L A W Y Q Q K 45

46 P G Q S P K L L I Y W A S T R 60

61 E S G V P D R F T G S G S G T 75

76 D F T L T I S G V Q A E D L A 90

91 V Y Y C K Q S Y N L P W T F G 105

106 G G T K L E I K 113

(VL sequence 1-6 bit base is the SacI recognition sequence: ¹GAG CT ^C)

1.11 antibody weight, light chain CDR district determine

According to the Kabat classification schemes, to angling the Z12 antibody variable gene of getting CDR district light, heavy chain to analyze.

2, antibody Z12 variable region gene space conformation simulation

Utilize Www.ncbi.nlm.nih.gov/BLAST and www.nih.gov/FASTAThe sequence comparative approach, retrieve by the protein structure proximity database (pdb) proximity, obtain same From Template albumen (PDB number of registration: the 1AR1 (83%) that sequence similarity is higher than 75% antibody Z12 variable region gene sequence of heavy chain, 1IGT (81%), 1HFN (78%), 1KFA (77%), 1OPG (76%), 1QKZ (76%), the percentage representative in the bracket and the sequence similarity degree of Z12 heavy chain) and sequence similarity be higher than same From Template albumen (PDB number of registration: the 1H3P (88%) of 80% antibody Z12 variable region gene sequence of light chain, 1A3R (84%), 1A5F (83%), 1AP2 (83%), 1SRS (82%), 1MCP (82%), 1IFH (81%)).

Utilize The sequencing results, carry out forecast analysis by statistical study antagonist Z12 weight, the light chain secondary structure of dihedral angle (ψ, ω).Fig. 2 .1 has provided and has utilized ψ, ω and main chain C atom optical activity ζ antagonist Z12 secondary structure heavy, light chain to carry out prediction result.

By graphics workstation Octane2 (R12000), utilize the Homology module of InsightII (2000) routine package, be template with above-mentioned antibody molecule with crystal structure, structure Z12 antibody variable region is heavy, the three-dimensional conformation of light chain.

Under CVFF, the Gromos field of force, utilize steepest descent (convergence step 10 successively, 000 step, convergence criterion 0.01KJ/mol), conjugate gradient (convergence step 20,000 step, convergence criterion 0.01KJ/mol), three kinds of molecular mechanics methods of Newtonian mechanics (convergence step 25,000 step, convergence criterion 0.001KJ/mol) are optimized the Z12 that obtains initial conformation heavy, light chain.

The antibody Z12 that obtains is heavy to optimizing, the light chain space conformation carries out rationality and detects to utilize Ramachandran figure, and Fig. 2 .2 has provided testing result.Mark the rationality degree of residue conformation among the figure with color distortion: because glycocoll (Gly) pliability is big, do not have side chain, proline (Pro) is a corner amino acid, does not consider the rationality of above-mentioned two seed amino acid conformations in evaluation procedure; Red area is represented conformation coupling optimal zone among the figure, and buff Regional Representative residue conformation allows the district, khaki Regional Representative's residue conformation still can, it is the bigger zone of possible error that white portion is represented the residue conformation.As can be seen from the figure, the predict of acquisition is rational: it is suitable that all residue conformations of variable region of heavy chain are all simulated, and the light chain conformation is defectiveness slightly, and 7 residue conformations wherein are uncertain, yet that whole conformation still is tending towards is rational.

Utilize antibody variable gene weight, light chain binding mode, by CDR district, surface texture featur, in conjunction with single-chain antibody ScFv architectural feature, method by molecular docking has obtained antibody weight, the interactional compound model of light chain, under the relevant field of force (CVFF, Gromos), by molecular mechanics, heavy, the interactional stable composite space conformation of light chain of molecular dynamics normal temperature simulation acquisition antibody Z12.Fig. 2 .3 has provided antibody Z12 weight, the interactional composite structure of light chain.

3, the acquisition of antibody Z12 and TNF effect compound space conformation

With TNF-alpha-crystal structure (PDB number: 1tnf) be template, select force field parameter identical when simulate, the TNF-α structure of structure theory with antibody.Binding molecule biology mutating experiment carries out space orientation to the experiment catastrophe point of TNF-α.Fig. 3 .1 has provided the TNF-α space conformation that theoretical modeling obtains.

TNF-α three-dimensional conformation, Z12 antibody variable region space structure with simulation are template, utilize the method for molecular docking to simulate TNF-α and the interactional compound model of Z12 antibody theoretically, in the process of molecular docking, consider the influence of solvent effect.

Parameters such as the space orientation by analyzing TNF-α catastrophe point, the distribution of apparent static, accessibility surface area, investigate antibody Z12 variable region gene CDR district's correlation parameter (Fig. 3 .2), pass through dynamic similation, the intermolecular ydrogen bonding number is many to form, interaction energy is low is evaluation criterion, makes up the interactional composite structure of TNF-α and Z12 theoretically.

The initial composite object model that obtains, select the CVFF/Gromos field of force, pass through steepest descent (convergence criterion 0.05KJ/mol successively, convergence step 40,000 step), conjugate gradient (convergence criterion 0.01KJ/mol, convergence step 60,000 step), Newtonian mechanics (convergence criterion 0.005KJ/mol, convergence step 80,000 step) carry out configuration energy minimization; Under the situation of considering solvent effect, the method for utilizing the normal temperature molecular dynamics to optimize is carried out simulative optimization to it and is handled.Fig. 3 .3 has provided the compound space conformation of TNF-α and the effect of Z12 antibody.

4, antibody Z12 variable region gene identification TNF-α epitope determines

Utilize the intermolecular ydrogen bonding theory TNF-α and the interactional compound model of antibody Z12 are analyzed, table 4.1 listed participate in forming hydrogen bond between antigen-antibody effect complex molecule give body (Donor), acceptor (Acceptor), distance () and angulation.Background colors different in the table are classified the CDR district of antibody.Antibody variable gene heavy chain CDR2, light chain CDR3 participate in the effect of synantigen TNF-α; The 135-148 position residue of TNF-α mainly participates in binding antibody heavy chain CDR2, and the 84-92 position residue of TNF-α mainly participates in binding antibody light chain CDR3.

In order further to inquire into the interaction of TNF-α and antibody Z12, by the free energy of reaction theory its interaction energy is calculated, the results are shown in the table 4.2.Wherein Van der Waals energy can be made up of repulsive energy and chromatic dispersion, electrostatic energy only is half of Van der Waals energy, the work of antigen TNF-α and antibody Z12 act as the master in order to hydrophobic effect, polarity effect and salt bridge, associative list 4.1 as can be seen, the 135-148 position residue of heavy chain of antibody CDR2 and TNF-α just in the above described manner the effect.

Further utilize computer graphics techniques, distance geometry method, the main region that can obtain TNF-α and Z12 effect from TNF-α and Z12 effect compound model is positioned at 141-146.Fig. 4 .1 has provided the mode chart of TNF-α and Z12 effect, and the spheroidite among the figure is the key position of TNF-α and Z12 effect.

At TNF-α and Z12 binding mode, by hydrophilic and hydrophobic the zone of TNF-α participation effect is analyzed, Fig. 4 .2 has provided the distribution pattern of the antigen TNF-α participation zone of action.Can determine further that the 141-146 position of TNF-α is the important area of Z12 antibody specificity identification.

5, utilize the checking of molecular biology experiment to notional result

At first by deletion method with hTNF-α partitioned representation, determine the hTNF-α fragment of Z12 antibody recognition according to Western-blot and ELISA experimental result.Though the epitope zone of Que Dinging is bigger in this way, it can be further epi-position research work favourable reference is provided.

5.1 materials and methods

5.1.1 material

Plasmid pUTNF-α ^{| 2.1|}Be by amino acid and the nucleotide sequence of this laboratory, with Escherichia coli bias password synthetic its total length CDNA (seeing Table 5.1), be cloned into again on the pUC18 plasmid vector, this recon called after pUTNF-α according to the hTNF-α that has delivered.Clone and expressive host bacterium are E.coli JM109 or DH5 α.Above plasmid, bacterial strain and carrier are frozen by this chamber.

Fusion expression vector pGEX-2T and bacterial strain E.coli BL21 (DE3) are frozen by this chamber, and the medicine IPTG of abduction delivering is available from Sigma company.

The toolenzyme restriction enzyme is all available from BioLab company.E.coli Klenow pol is available from TaKaRa company.T4 DNA Ligase is available from Gibco company.

ELISA agents useful for same coating buffer: 0.015mol/L PH9.6 carbonate buffer solution (NaCO ₃: 1.59g, NaHCO ₃: 2.93g, H ₂O is settled to 1L).Confining liquid: 10% bovine serum albumin.The rabbit anti-mouse antibody (HRP-RAM) of horseradish peroxidase-labeled is by this prepared in laboratory and purifying.Chromogenic substrate o-phenylenediamine (OPD) is available from Sigma company.Stop buffer: 2mol/L H ₂SO ₄

5.1.2 basic experiment operation

Plasmid extracts (1) alkaline lysis, with reference to " molecular cloning experiment guide " second edition (chapter 1 the 3rd joint).(2) plasmid rapid extraction kit, vast company product, by specification carries out.

The enzyme of plasmid is cut (1) single endonuclease digestion: plasmid DNA 2～5 μ g, and 10 * endonuclease reaction damping fluid, 2 μ l, restriction enzyme 10U, the sterilization deionized water complements to 20 μ l.37 ℃ of incubation 1～3h.(2) double digestion: plasmid DNA 2～5 μ g, 10 * endonuclease reaction damping fluid, 2 μ l, two kinds of each 10U of restriction enzyme, the sterilization deionized water complements to 20 μ l.37 ℃ of incubation 1～3h.Should select two kinds of damping fluids that enzyme is all suitable.Before carrying out the double digestion experiment, single endonuclease digestion reaction in advance, the agarose gel electrophoresis observations guarantees that two enzymes separately can be with plasmid digestion fully in same damping fluid.

The Li PCR that falls does before enzyme cuts evaluation single bacterium colony being increased, extracts plasmid, bacterium colony PCR in advance, and the picking positive colony carries out enzyme again and cuts evaluation, can reduce the blindness that enzyme is tested conscientiously.Use the finely disseminated monoclonal bacterium colony of the aseptic suction nozzle picking of 200 μ l, be placed in the 1.5ml microcentrifugal tube, add the aseptic deionized water of 50 μ l, with the abundant mixing of vortex oscillator.Get 5 μ l mixed liquors and be used for 25 μ l PCR reaction, all the other place 4 ℃ of refrigerators to preserve.

Reaction system:

dNTP(2.5mM) 2μl

Each 0.5 μ l of primer (50uM)

10×Taq?buffer 2.5μl

Taq (5U/ μ l) 0.2/ μ l cumulative volume 25uL

In case electrophoresis result is shown as suspicious positive bacterium colony, remaining bacterium liquid is contained in the LB nutrient culture media of Amp shaken cultivation in 3ml spend the night, extract plasmid DNA then and carry out enzyme and cut evaluation.

The recovery of dna fragmentation/purifying uses the dna fragmentation fast purifying/recovery kit of Beijing ancient cooking vessel state biotech firm, is undertaken by its product description.

Filling-in 3 ' recessed end product (the big fragment of carrier) 0.5～1 μ g of 3 ' recessed end, 10 * Klenow buffer, 2 μ l, E.coli Klenow pol 1 μ l (5U), dNTP (2.5mmol/L) 1 μ l, the sterilization deionized water complements to 20 μ l.37 ℃, reaction 30min.70 ℃ of 5min or add the EDTA solution of 1 μ l 0.5mol/L in reactant liquor are with deactivation Klenow, cessation reaction.

Connect in coupled reaction (1) molecule: have the big molecular dna 6 μ l (about 50-100ng) of two smooth end, 5 * T4 ligase buffer, 3 μ l, T4 ligase 1 μ l (10U), aqua sterilisa 5 μ l.Room temperature connects 2h or longer time.(2) intermolecular connection: carrier 3 μ l (about 30-60ng), dna fragmentation 5 μ l (about 60-120ng), 5 * T4 ligase buffer, 3 μ l, T4 ligase 1 μ l (10U), aqua sterilisa 3 μ l.Room temperature connects 2h or longer time.

The preparation of competent escherichia coli cell and conversion CaCl ₂Legal system is equipped with competent escherichia coli cell, with reference to " molecular cloning experiment guide " second edition (chapter 1 the 5th joint).The frozen pipe that fills competent cell is put into ice earlier, put 4 ℃ of refrigerator overnight, put again afterwards in-70 ℃ of liquid nitrogen and preserve, can produce the high frequency transformation efficiency.During conversion, add plasmid 0.5 μ l in the 100 μ l competent cells or add DNA connection product 8 μ l.

Protein electrophoresis, transfer and Western-blot are with reference to " molecular cloning experiment guide " (second edition) related Sections.The used instrument of electrotransfer is ' Bio-Rad PowerPAC 200 '.

5.1.3hTNF-the clone of α deletant and C end fragment gene

Being cloned in the pUTNF-α plasmid of hTNF92-157 (only keeping hTNF-α C end 92-157 amino acids) gene, there is Nco I restriction enzyme site the codon ATG position, and the recognition site of flush end nickase Hinc II is just before the codon of coding hTNF-α the 92nd amino acids (seeing Fig. 5 .1).Digest pUTNF-α plasmid jointly with above-mentioned two enzymes, the big fragment of recovery has only kept the encoding gene of hTNF-α 92-157 amino acids.3 ' the recessed end that utilizes E.coli Klenow pol that Nco I cutting is produced is mended flat, obtains initial code ATG just.Under the catalysis of T4 DNA Ligase, two flush ends connect from beginning to end, make the ATG back connect the encoding gene of hTNF-α 92-157 amino acids, with its recombinant plasmid called after pUTNF92-157 (seeing Fig. 5 .1).

Its principle of clone and the operating process and the above-mentioned pUTNF92-157 recon basically identical of hTNFD53-91 (53-91 amino acids in the middle of the disappearance hTNF-α) gene.With EcoN I and Hinc II double digestion pUTNF-α plasmid, E.coli Klenow pol mends 3 ' the recessed end that flat EcoN I digestion back produces, and carries out connection in the molecule, thereby removes the encoding gene of hTNF-α 53-91 amino acids.With this recombinant plasmid called after pUTNFD53-91 (seeing Fig. 5 .2).

The clone of hTNF D29-88 (29-88 amino acids in the middle of the disappearance hTNF-α) gene uses the recombinant plasmid that a step inverse PCR method makes up hTNF D29-88 gene and pUC18, with its called after pUTNFD29-88.Operation steps and principle schematic are referring to second portion two, 1.8 joints.

Above recombinant plasmid is carried out enzyme cut evaluation and order-checking.

5.1.4pGEX-2T structure with hTNF-α, hTNF92-157, hTNFD53-91 and hTNFD29-88 fusion recon

HTNF-α need not to induce just on the pUC18 carrier and can obtain high expressed, even and above-mentioned deletant can not be expressed under the condition of inducing.Therefore, the deletant genetic recombination to the pGEX-2T fusion expression vector, is obtained the expression of disappearance albumen.In the plasmid of hTNF-α and above-mentioned deletant and pUC18 reorganization, sequence before the initial code of encoding gene, after the stop code is in full accord, therefore with reference near the sequence initial code and the stop code, design a pair of primer, hTNF-α and deletant gene simultaneously thereof increase.Primer sequence is as follows:

Upstream primer: 5 '-CGC GGA TCCGAG GAA AAA ACC ATG-3 '

(the line part is the BamHI restriction enzyme site).

Downstream primer: 5 '-CCG GAA TTCAAG CTT GGC TGC AGT-3 '

(the line part is the EcoRI restriction enzyme site).

Get plasmid 0.2 μ l, each 1 μ M of upstream and downstream primer, dNTP 200 μ M, 10 * Pyrobest buffer5 μ l, 1U Pyrobest, water complement to 50 μ l.The pcr amplification condition is: 95 ℃ of 5min; 94 ℃ of 45s, 52 ℃ of 45s, 72 ℃ of 45s, 30 circulations; 72 ℃ of 5min.

The PCR product that reclaims is through BamH I and EcoR I double digestion, and is connected through the same pGEX-2T carrier of handling, thereby foreign gene is connected in the afterbody (see among Fig. 5 .3 1.) of GST encoding gene.Recombinant plasmid is called after pGTNF-α, pGTNF92-157, pGTNFD53-91 and pGTNFD29-88 respectively.

5.1.5pGEX-2T structure with hTNF1-91 (only keeping hTNF-α N end 1-91 amino acids) fusion recon

The recognition site of flush end nickase Hinc II is just at the base end of hTNF-α the 91st amino acids of encoding, and the recognition site of another flush end nickase SmaI is arranged also in the multiple clone site of pGEX-2T carrier, cut the PCR product of amplification hTNF-α full-length gene simultaneously with BamH I and HincII, with put down with the carrier segments that Sma I handled through BamH I-cohesive end is connected, thereby make up pGEX-2T/hTNF1-91 recombinant plasmid (see among Fig. 5 .3 2.), with its called after pGTNF1-91.

5.1.6 the abduction delivering of fusion

With above-mentioned recon Transformed E .coli DH5 α host bacterium, the plasmid that extracts is after enzyme is cut evaluation and order-checking correctly, Transformed E .coli BL21 (DE3) host bacterium, compare with the bacterial strain that transforms the pGEX-2T empty carrier, under the IPTG of 1mmol/L induces, carry out a small amount of and express experiment, and detect expression with 12% SDS-PAGE.Select the highest bacterial strain of expression and carry out great expression.With yeast culture to A ₆₀₀=0.3～0.6 o'clock, with final concentration is that 1mmol/L IPTG induces 4h in 37 ℃, the centrifugal 10min results of 8000r/min thalline, after the ultrasonication, the centrifugal 30min of 8000r/min, collect supernatant, adopt the ultraviolet absorption method quantitative protein, calculate protein content (mg/ml) as follows: (1.45 * A ₂₈₀-0.74 * A ₂₆₀) * extension rate.

5.1.7 preparation and the purifying of anti-hTNF-α monoclonal antibody Z12

After the anti-hTNF-α of the stably excreting monoclonal antibody Z12 hybridoma enlarged culture of (belonging to the IgG1 subclass), be inoculated in the BLAB/c mouse peritoneal, week back collection ascites was treated to extract once more after ascites is gathered at interval in 2-3 days.With the micro-pore-film filtration ascites in 0.45 μ m aperture, remove bigger solid particle, fibrin clot and fat drop.Then, the centrifugal 15min of 12000r/min removes cell residue and finely ground particle substance, collects supernatant, and is standby.

The above-mentioned pretreated mouse ascites Xml that learns from else's experience adds the dilution of equivalent physiological saline, under the ice bath, drips saturated ammonium sulfate 2Xml and makes and reach 50% saturation degree, the stirring while dripping.Solution is put 4 ℃ more than the 2h.Behind the equilibrium at room temperature, the centrifugal 30min of 3000r/min abandons supernatant, and precipitation is used the 2Xml physiological saline solution, dropwise adds saturated ammonium sulfate Xml, stirs while dripping, and makes to reach 33% saturation degree, puts 4 ℃ more than the 2h.Behind the equilibrium at room temperature, centrifugal treating, precipitation is dissolved with a small amount of PBS, and the gained material was the IgG crude extract after this kind saltoutd.

After the IgG crude extract is dialysed to 0.1mol/L PB (PH8.0), be splined on Protein A-Sephorase CL-4B affinity column, behind identical PB buffer solution elution foreign protein, 0.1mol/L citrate buffer solution wash-out destination protein (IgG1) with PH6.0, collect the effluent of this eluting peak, and immediately with 10mmol/L PB PH8.0 damping fluid neutralization and to its dialysis.Concentrated and purified sample.

5.1.8Z12 the Western-blot of antibody recognition hTNF-α deletant analyzes

HTNF-α deletant albumen is through SDS-PAGE, and electrotransfer is cut Marker to nitrocellulose filter, put in the amino black solution and develop the color.Transfer printing is had the filter membrane of albumen seal with 5% skimmed milk power, 4 ℃ are spent the night.Outwell confining liquid, fully wash filter membrane 3 times, each 10min with TBS-T.Filter membrane is put into than its bigger slightly polybag, added the Z12 antibody-solutions of 5 μ g/mL, sack is sealed, shake 1h gently in room temperature with plastic packaging machine by the amount of every square centimeter of 0.1ml.Take out film,, add two anti-(HRP-RAM), shake 40min gently in room temperature with the TBS-T washing.Use clear water flush away Ponceaux at once, make the color before film returns to dyeing, and seal.After the TBS-T washing, develop the color with DAB.

5.1.9Z12 the elisa assay of antibody recognition hTNF-α deletant

(1) antigenic solution is diluted to variable concentrations with coating buffer, every hole adds 50 μ l on 96 orifice plates, repeats 3 holes, does blank with coating buffer, and 4 ℃ are spent the night or 37 ℃ of 2h in the wet box.Outwell the reactant liquor in the plank, before adding next step experiment agents useful for same each time, fully wash, remove the antigen or the antibody of not absorption, reduce nonspecific reaction as far as possible with PBS-T.(2) every hole adds 200 μ l, 10% calf serum (FCS dilutes with PBS), seals, and 37 ℃ of 1h or 4 ℃ spend the night.(3) add the Z12 antibody (5 μ g/mL) of purifying, every hole 50 μ l, 37 ℃ of 1h or 4 ℃ spend the night.(4) add HRP-RAM two and resist every hole 50 μ l, 37 ℃ or room temperature 40min.(5) do substrate colour developing with OPD, lucifuge 5-10min becomes yellow to reacting hole, and is as the criterion with the nondiscolouring of blank hole, uses 50 μ l 2N H immediately ₂SO ₄Cessation reaction, this moment, reacting hole became orange red.Under the 490nm wavelength, measure the A value.

5.2 result

5.2.1hTNF-cutting, identifies and expression the enzyme of α deletant gene (pUC18)

Make up correct pUTNF92-157 plasmid, Nco I and Hinc II restriction enzyme site disappear; In the base sequence of removed coding hTNF-α 1-91 amino acids, contain EcoN I recognition sequence, so the also corresponding disappearance of EcoN I restriction enzyme site.With EcoN I and Hinc II cutting pUTNF92-157, the product that obtains will become the band shape of plasmid respectively.Because Hind III restriction enzyme site still exists, therefore by after its digestion, pUTNF92-157 equally presents a linear band (seeing Fig. 5 .4A) with pUTNF-α.

In pUTNF-α plasmid, before the initial code of encoding gene and after the stop code Nco I and Hind III restriction enzyme site are arranged respectively, digest the small fragment that can obtain 494bp simultaneously with them.PUTNFD53-91 and pUTNFD29-88 recon lack 39aa (117bp) and 60aa (180bp) respectively, therefore obtain the small fragment of 377bp (seeing Fig. 5 .4B) and 314bp (seeing Fig. 5 .4C) respectively with Nco I and Hind III double digestion.

Enzyme cut the result all with the conforming to of expection, confirm to have obtained correct recon.Sequencing result shows, deletant gene entirely true (figure slightly).With recombinant plasmid difference Transformed E .coli JM109 and DH5 α competent cell, under the condition that nothing is induced or IPTG induces, above hTNF-α missing gene does not all have expression (figure slightly) in the pUC18 carrier.

5.2.2 merge the structure and the evaluation of recon

Can't obtain to express owing to be structured in hTNF-α missing gene on the pUC18 carrier, therefore with above transgenosis in the pGEX-2T carrier, expectation obtains to express with amalgamation mode.Design a pair of universal primer, amplification simultaneously is kept at hTNF-α, hTNF92-157, hTNFD53-91 and the hTNFD29-88 gene (seeing Fig. 5 .5) on the pUC18 carrier.Utilize restriction enzyme BamHI and EcoR I, above gene is inserted in the pGEX-2T carrier, and identify that with these two enzymes 1% agarose electrophoresis result shows: all cut out the fragment (seeing Fig. 5 .6) of expection size, obtained correct recon.

Save described method by 5.1.5 and make up the pGTNF1-91 recombinant plasmid: the amplified production that at first cuts hTNF-α full-length gene with BamH I and Hinc II, cut the fragment of glue recovery 294bp, with put down-the cohesive end coupled reaction through the big fragment of carrier of BamH I and Sma I cutting and purifying, Transformed E .coli JM109, picking list bacterium colony, amplification cultivation, extract plasmid, identify with BamH I and EcoR I double digestion, cut out the small fragment (Fig. 5 .7) of 297bp, confirm to have obtained correct recon.

Above all recons are checked order, and the result shows gene entirely true (Fig. 5 .8).

5.2.3 the abduction delivering of fusion

Fusion recon Transformed E .coli BL21 (DE3) with in 5.1.3 and the 5.1.4 joint with the IPTG abduction delivering, with the bacterial sediment behind an amount of PBS suspended centrifugal, carries out ultrasonication behind the multigelation.12%SDS-PAGE result shows that several fusions have all obtained the high expressed of solubility, and after IPTG induces 4h, the Expression of Fusion Protein amount reaches maximal value, further prolongs induction time and can not improve expression efficiency.Empty carrier is through inducing, the oneself expression molecular weight is at the glutathione S of 26kDa transferase (GST), and the molecular weight that hTNF-α, hTNFD53-91, hTNFD29-88, hTNF92-157 and hTNF1-91 and GST merge is respectively 43,39,37,33 and 37kDa (seeing Fig. 5 .9).

5.2.4Z12 purifying antibody

Protem A-Sepharose CL-4B dry powder swelling 30min in 0.1mol/L PB (PH8.0) is in the chromatographic column of packing into then, with same damping fluid balance.PH is 8.0 o'clock, and A albumen only combines with mouse IgG, thereby separates with the Ig and the impurity of other classification.After treating that the foreign protein peak is returned to baseline, use the citric acid buffer wash-out of PH6.0 instead, and collect the effluent of this eluting peak.When PH6.0, IgG1 is at first eluted, thereby separates with other subclass (IgG2a, IgG2b and IgG3).After this use the PH3.0 elution requirement, all IgG class materials that are adsorbed on the A albumen are eluted, make affinity column obtain regeneration, be used for purifying (seeing Fig. 5 .10A) next time.Collect 0.01mol/L PB (PH8.0) dialysis 12～24h of liquid, change liquid therebetween 2～3 times 50 times of its volumes.With the sample freeze-drying, the dry powder that takes a morsel is dissolved among an amount of PBS (PH7.0), measures OD on ultraviolet spectrophotometer ₂₈₀Value is pressed following formula calculating antibody solution concentration (mg/ml): (OD ₂₈₀÷ 1.4) * extension rate.The capable 10%SDS-PAGE of the sample that takes a morsel, electrophoresis result shows that the Z12 antibody molecule amount of purifying is at 120～150kDa, purity (is being seen Fig. 5 .10B) more than 90%.After this all use in the experiment of Z12 antibody, and used antibody reagent is the sample of same batch of purifying.

5.2.5 the Western-blot of Z12 antibody recognition hTNF92-157, hTNFD53-91 and hTNFD29-88 analyzes

Fusion is through 12% SDS-PAGE, and (40V, constant voltage is 65min) to nitrocellulose filter for electrotransfer.Dye film with Ponceaux, the colour developing result shows that the albumen transfer ratio is more complete.Cut Marker, put in the amino black solution and develop the color.Use clear water flush away Ponceaux at once, make the color before film returns to dyeing, and seal.According to the Western-blot operation steps, carry out immunoblotting assay, finally develop the color with DAB.The result shows: Z12 antibody specificity identification contains the fusion of hTNF-α, hTNF92-157, hTNFD53-91 and hTNFD29-88, and with the GST no cross reaction (seeing Fig. 5 .11) of empty carrier oneself expression.The common sites of these several fusions is hTNF-α 92-157 districts, and the antigenic determinant of prompting Z12 antibody recognition is arranged in hTNF-α 92-157 district.

5.2.6Z12 the Western-blot of antibody nonrecognition hTNF1-91 analyzes

By the work of front, we think that the epitope of Z12 antibody recognition is positioned at hTNF-α C end 92-157 district, and conversely, if with hTNF-α N end 1-91 district single expression, antibody should be nonrecognition.Western-blot result (Fig. 5 .12) shows: the fusion that Z12 antibody nonrecognition GST and hTNF1-91 form, the conclusion above having confirmed from the negative, i.e. Z12 antibody specificity identification hTNF-α 92-157 district.

5.2.7Z12 the ELISA of antibody and hTNF-α N end and C end fragment interactively detects

ELISA result (Fig. 5 .13) is consistent with front Western-blot result, confirms Z12 antibody recognition hTNF-α C end 92-157 district, and nonrecognition N end 1-91 district.

5.3Z12 determining of the epitope of antibody recognition

The experimental study of front makes us determine that the antigen zone of Z12 antibody recognition is positioned at hTNF-α C end 92-157 district.The antibody identification meter position (hTNF-α 141-146 amino acids) that utilizes computer simulation method prediction just in this zone, preliminary identification the reliability that predicts the outcome.Utilize reduction to absurdity, be about to hTNF-α 141-146 amino acids residue and lack, detect the variation of interaction relationship between disappearance albumen and the Z12 antibody, thereby determine whether hTNF-α 141-146 position is the epitope of Z12 antibody recognition.

5.3.1 materials and methods

5.3.1.1 material

Clone, the expression vector of disappearance albumen are pUC18, and the host bacterium is E.coli JM109.

Toolenzyme: restriction enzyme is all available from BioLab company.T4 DNA Ligase is available from Gibco company.High-fidelity DNA amplification enzyme Pyrobest ^TMAnd related reagent is available from TaKaRa company.T4 polynueleotide kinase (T4 PNK) and ATP are available from TaKaRa company.

The preparation and the purifying of Z12 antibody save referring to 5.1.7.HRP-RAM two is anti-by this prepared in laboratory and preservation.

The L929 cell is frozen by this chamber.Radiating streptozotocin D is available from Fluka company.

Other reagent is homemade or import is analyzed pure.

5.3.1.2 the analysis of Oligonucleolide primers is with synthetic

The primer that utilizes the Goldkey software analysis to design is guaranteed no hairpin structure in the primer, and no dimer forms between primer, and other position of primer and template does not have specificity and combines.Afterwards, primer sequence being served sea living worker's bioengineering company limited synthesizes.

Primer 1P5:5 '-TCTGGTCAGGTTTACTTC-3 ';

Primer 1P3:5 '-GTCAGGACGGTTGATTTC-3 '

Primer 2 P5:5 '-CTGGCTAACGGTGTTGAA-3 '

Primer 2 P3:5 '-CCACTGCAGCTGACCTTC-3 '

Primer 1 is used for making hTNF-α 141-146 position deletion mutant (to call hTNFD141-146 in the following text, its recombinant plasmid is represented with pUTNFD141-146) the about 2660bp of amplified production length; Primer 2 is used for making hTNF 29-36 position deletion mutant (to call hTNFD29-36 in the following text, its recombinant plasmid is represented with pUTNFD29-36), the about 2650bp of amplified production length.

5.3.1.3PCR amplification

Reaction system: template 0.1ug, each 1 μ mol/L of upstream and downstream primer, dNTPs 200 μ mol/L, 10 * Pyrobest buffer, 5 μ l, Pyrobest 1U, the sterilization deionized water complements to 50 μ l, adds the 50ul paraffin oil in the reactant liquor surface.Reaction conditions: 95 ℃ of sex change 5min; 94 ℃ of 1min, 56 ℃ of 1min, 72 ℃ of 3min (press 72 ℃ of 1kb DNA and extend 1min calculating), 35 circulations of amplification; 72 ℃ are extended 10min.

5.3.1.4PCR the recovery/purifying of amplified production

Use the DNA fast purifying/recovery kit of Beijing ancient cooking vessel state biotech firm, undertaken by product description.

5.3.1.5PCR product 5 ' terminal phosphateization

Reclaim the PCR product 0.5 μ g of purifying, 200mmol/L ATP 0.5 μ l, 10 * T4 PNKbuffer, 2.5 μ l, T4 PNK 5U, the sterilization deionized water complements to 25 μ l.With the reactant liquor mixing, put 30min in 37 ℃ of water-baths, afterwards 68 ℃ of 10min deactivation T4 PNK.

5.3.1.6 the recirculation of linear DNA

The PCR product 5 μ L of 1.4 step process of learning from else's experience (about 0.1 μ g), T4 DNA Ligase 5 * Buffer 3 μ l, T4 DNA Ligase 1 μ L (5U), sterilization deionized water 6 μ L, making the reaction cumulative volume is 15 μ L.Room temperature or 4 ℃ of connections are spent the night.

5.3.1.7DNA conversion, extraction and enzyme cut evaluation

With reference to " molecular cloning experiment guide " related Sections.

5.3.1.8hTNF-α deletant construction of recombinant plasmid and expression

Primer

1,2 is a template with pUTNF-α circular plasmids respectively, carries out pcr amplification under Pyrobest catalysis in opposite direction.Reclaim purified pcr product, achieve 5 ' terminal phosphateization, utilize T4 DNA Ligase to make the linear molecule of the capable phosphorylation of N end realize connecting in the molecule (seeing Fig. 5 .14) afterwards.Transformed E .coli JM109 competent cell is done the suspicious bacterium of bacterium colony PCR preliminary screening.Amplification cultivation, enzyme is cut evaluation behind the extraction plasmid, order-checking.

Enzyme cut and check order that correct plasmid transforms again, coated plate, choose monoclonal 37 ℃ of overnight incubation in the LB of Amp resistance nutrient culture media, make positive control, make negative control with the bacterial strain of conversion pUC18 empty carrier with the bacterial strain that transforms pUTNF-α plasmid.The bacterial sediment of centrifugal results suspends with an amount of PBS solution, ultrasonication, and centrifugal results supernatant adopts protein content (mg/ml) in the uv absorption standard measure supernatant: (1.45 * A ₂₈₀-0.74 * A ₂₆₀) * extension rate.

5.3.1.9hTNF-the bioactive mensuration of α and deletant thereof

Recovery L929 cell, treat that it covers with individual layer after, use 0.025% trypsinization, re-suspended cell is in the RPMI-1640 nutrient solution, transferring cell concentration is 3 * 10 ⁴/ hole is inoculated in the Tissue Culture Plate, every hole 80 μ l.In each hole, add 10 μ l radiating streptozotocin Ds (final concentration is 2 μ g/ml) and 10 μ l testing samples (the ultrasonic supernatant that contains hTNF-α, hTNFD141-146 and hTNFD29-36) respectively, if 4 sample concentrations: 1,0.1,0.01 and 0.001mg/ml, and establish 3 multiple holes.Establish simultaneously and only add radiating streptozotocin D and do not add the positive control of sample and only add the L929 cell and do not add the negative control of radiating streptozotocin D and sample.Culture plate is put 5%CO ₂Cultivate 24h for 37 ℃ in the incubator, outwell mixed-culture medium, and wash with PBS-T.Add 10 μ l, 0.5% crystal violet in every hole, clean extracellular crystal violet gently with distilled water behind the room temperature dyeing 10min.Behind the air drying, every hole adds 100 μ l, 33% acetic acid, treat that cell melts fully after, measure absorbance A value under the 570nm with automatic reading enzyme connection instrument.Represent the sample activity with the cell toxicant percent, computing formula: (1-b/a) * 100%, ab is respectively the optical density value of control group and experimental group.

5.3.1.10Z12 antibody detects with the ELISA of disappearance protein-interacting

The ELISA operation steps saves referring to 5.2.1.9.

5.3.2 result

5.3.2.1hTNF-α deletant construction of recombinant plasmid

At first, be that template is carried out pcr amplification with the primer solution of diluting with pUTNF-α circular plasmids, described in reaction system and reaction conditions such as material and the method.0.8% agarose gel electrophoresis is observed, the size of amplified production conform to 2650bp with the 2660bp of expection (seeing Fig. 5 .15).Cut glue and reclaim the PCR product, row N end phosphorylation connects, thereby finishes construction of recombinant plasmid.

5.3.2.2hTNF-the enzyme of α deletant recombinant plasmid is cut evaluation

The restriction enzyme site of restriction enzyme Nco I, Hind III, Hinc II is 15 and the 91st amino acids residue end behind the last position of the initiation codon of the hTNF-α that encodes, termination codon respectively.PUTNF-α plasmid can downcut the small fragment of a 216bp through the digestion of uniting of Hind III and Hinc II; HTNFD141-146 lacks 6 amino acid (18bp), so the small fragment size that its recombinant is cut generation through this two enzyme should be 198bp.Equally, pUTNF-α and pUTNFD29-36 plasmid be through the digestion of uniting of Nco I and Hinc II, and respectively can cut size be respectively 277 and the small fragment of 253bp.The result of endonuclease reaction conforms to expection, sees Fig. 5 .16.Enzyme is cut the correct recombinant plasmid of checking serve the order-checking of Hai Boya company, result entirely true (figure slightly).

5.3.2.3hTNF-the expression of α deletant recon in Escherichia coli

The recombinant plasmid that order-checking is correct is Transformed E .coli JM109 again, the 37 ℃ of joltings in the LB nutrient culture media of single bacterium colony of picking are spent the night, the precipitation of centrifugal results suspends with PBS (bacteria culture fluid 1/10 volume), after the ultrasonication, centrifugal results supernatant, the capable 18%SDS-PAGE of the sample that takes a morsel, Coomassie brilliant blue dyeing (Fig. 5 .17).

5.3.2.4hTNF-the biologic activity analysis of α deletant

L929 cell suspension furnishing 3 * 10 ⁴/ ml is inoculated into 96 orifice plates, every hole 80ul.HTNF-α and deletant thereof dilute every hole 10ul in back and radiating streptozotocin D (0.1ug/3 * 10 by a certain percentage ⁴) the common adding.37 ℃ of 5%CO ₂Incubator was cultivated 24 hours, and 0.5% violet staining is surveyed 570nm OD value.By formula (1-b/a) * 100% calculates cytotoxicity, and a, b are respectively the optical density value of control group and experimental group.With the sensitivity and the accuracy of hTNF-α standard items checking system, the drawing standard curve shows reliable results.Make radix (100%) with the cytotoxicity of hTNF-α supernatant, the relative cytotoxicity of hTNFD141-146, hTNFD29-36 is respectively 23.47% and 17.80% (mean value, see Fig. 5 .18), the biologically active that these two deletants are described all obviously reduces, show that 141-146 and 29-36 district are the active sites of hTNF-α, by its performance biological action necessary.

5.3.2.5Z12 antibody detects with the ELISA of disappearance protein-interacting

The ultrasonic supernatant that will contain hTNF-α, hTNFD141-146 and hTNFD29-36 is diluted to 4 variable concentrations such as 100,50,10,1 μ g/ml and wraps quilt with coating buffer.The saturation plot of the antibody recognition antigen of doing according to preliminary experiment, when the ultrasonic supernatant concentration of hTNF-α during at 100～10 μ g/ml, the Z12 antibody of purifying and antigen reactive optium concentration are 2～10 μ g/ml, therefore carry out ELISA with the antibody concentration of 5 μ g/ml and detect.The result shows: the ability of complete hTNF-α of Z12 antibody recognition and 29-36 position deletant thereof is basic identical, and the ability of identification 141-146 position deletant is compared almost forfeiture with the above two.Proof hTNF-α 141-146 position is the epitope (seeing Fig. 5 .19) of Z12 antibody recognition.

6, computer-aided design (CAD) newtype drug molecule

According to antibody (Z12) that obtains and the interactional compound conformation of antigen (TNF-α), in conjunction with the conclusion identical that experiment is determined, utilize the CAD method design to obtain following three small peptide (PT3:INYTGEPTYSQSVSNVVWY with theoretical modeling; PT4:YTYYPTVNENRSQSVSNVF; PT6:YINTGYDGLYYNSMD).Simulating peptide design process: the TNF-α epi-position 141-146 of analog antibody Z12 identification (it is the important structure territory that antigen participates in binding antibody that experiment and theoretical model are all pointed out this zone), the polypeptide of design satisfies corresponding pliability and easily folding feature, for satisfying its hydrophobic property, introduce corresponding hydrophobic residue simultaneously; For guaranteeing its static behaviour and corresponding polarity, introduce relevant water wettability residue.

For comparing, at the irrelevant peptide PT1 (GGY TYYTHSNV) of binding mode design.

According to the method for from the beginning building, four small peptides are carried out structural simulation.Select the CVFF/Gromos field of force, successively through steepest descent (convergence criterion 0.01KJ/mol, convergence step 5000 step), conjugate gradient (convergence criterion 0.005KJ/mol, convergence step 5000 step), Newtonian mechanics (convergence criterion 0.001KJ/mol, convergence step 8000 step), small peptide is carried out the conformation optimization of molecular mechanics, obtain to stablize conformation.And then utilize the molecular dynamics method to carry out dynamic similation, determine the ambient stable conformation.

The TNF-α theoretical model that selection makes up previously utilizes the method for molecular docking to build the interactional compound model of small peptide PT3, PT4, PT6 and TNF-α.Results suggest, wherein preceding two PT3, PT4 have the conformational characteristic shown in Fig. 6 .1 (a), and the 3rd PT6 has the architectural feature shown in Fig. 6 .1 (b).

7, competition is in conjunction with experiment

7.1 peptide is synthetic

PT1, PT3,4 peptide molecules such as PT4, PT6 that design is obtained synthesize, all by U.S. Genenmed Synthesis, and the Inc synthesizing and purifying, the purity of four peptides is respectively 99.9%, 99.9%, 81.4%, 92.8%.

7.2ELISA competition is in conjunction with experiment

This patent is used the EILSA competition in conjunction with experimental technique, whether can combine the TNF-alpha molecule with anti-TNF-Alpha antibodies competition to above designed with synthetic small peptide and verify.Method: 100 μ l TNF-α/holes (final concentration 2 μ g/ml) wrap by 96 microwell plates, 4 ℃ spend the night or 37 ℃ hatched 2 hours.PBS-T washes plate 3-4 time, and the sealing of 200 μ l/ holes, 4% casein was hatched 30 minutes for 37 ℃; PBS-T washes plate 3-4 time, behind formamide dissolving peptide, dilute peptide and Z12-HRP (the mouse anti human TNF-alpha monoclonal antibodies of horseradish enzyme labeling) respectively with PBS again, the peptide (final concentration be respectively 0.047,0.236,1.180,4.720mmol/L) and the Z12-HRP (final concentration is 1: 600) that get 100 μ l add every hole, 4 ℃ of overnight incubation.PBS-T washes plate 3-4 time, the colour developing of OPD substrate, 2N H ₂SO ₄Cessation reaction, 490nm wavelength are measured the OD value down.

The combination rate computing formula:

Experimental group: peptide+Z12-HRP; Control group: Z12-HRP

PT3, PT4, PT6 are with the increase of dosage, the effect that combines TNF-α with the Z12 antibody competition obviously strengthens (table 3), do not combine TNF-α effect and demonstrate with the Z12 antibody competition with the dosage increase as the PT1 of irrelevant control peptide, above three the designed small peptides of results suggest all have with the Z12 antibody specificity competes the effect that combines TNF-α.

7.3 in and TNF-α biologically active analyze

With cytotoxicity experiment methods analyst PT3, PT4, PT6 hTNF-α is mediated bioactive effect in the application.Get L929 cell (3 * 10 ⁴/ hole) is inoculated into 96 well culture plates, adds or do not add TNF-α 10 simultaneously ^-2μ g/3 * 10 ⁴/ hole and radiating streptozotocin D 0.1 μ g/3 * 10 ⁴/ hole does not add or adds different dilution small peptides respectively, 37 ℃, 5%CO ₂Cultivated 24 hours, 0.5% violet staining is surveyed 570nm OD value.

Among the TNF-α and active computing formula:

(1) inhibiting rate of TNF-α on cell proliferation calculates:

Experimental group: cell+TNF-α+actinomycin D;

Control group: cell+actinomycin D

(2) peptide to TNF-α on cell proliferation inhibiting in and efficiency calculation:

Experimental group inhibiting rate: cell+TNF-α+actinomycin D+peptide;

Control group inhibiting rate: cell+TNF-α+actinomycin D;

Table 4 results suggest PT3, PT4, PT6 demonstrate different dosage neutralizing effects respectively to the alpha mediated cytotoxicity of TNF-, among PT3, the PT4 and the alpha mediated Cytotoxic efficient of TNF-obviously be better than PT6.Among EILSA competitive assay and the TNF-α and result of experiment confirmed the designed small peptide biologically active of TNF-α epi-position discerned according to antibody in this patent.

Hydrogen-bond donor (Donor) between table 1 TNF-α and Z12 complex molecule, acceptor (Acceptor), distance () and angulation (°)

Table 2 TNF-α and Z12 form the interaction energy (Kcal/mol) of compound

The ELISA competition combination of the synthetic small peptide of table 3

The combination rate of hTNF-α and Z12 antibody (%)

Small peptide small peptide concentration (mM)

0.00 0.14 0.68 1.36 2.72

PT1 100±0 118.6±20.3 119.1±14.2 112.5±8.5 111.7±13.9

PT3 100±0 109.0±17.3 96.1±13.2 81.4±12.4 ^* 55.2±12.1 ^**

PT4 100±0 90.8±8.9 88.7±9.9 79.0±3.5 ^* 54.7±3.2 ^**

PT6 100±0 70.1±17.3 ^* 59.5±15.3 ^** 54.3±20.5 ^** 37.8±9.1 ^**

^**：P＜0.01； ^*：P＜0.05

The synthetic small peptide of table 4 is to the alpha mediated Cytotoxic neutralization of TNF-

In and efficient (%)

Small peptide small peptide concentration (mM)

0 0.62 1.25 2.50 5.00 10.00

PT1 0 7.9 9.1 9.1 12.0 42.4

PT3 0 12.9 7.4 16.7 ^* 17.3 87.9 ^*

PT4 0 14.8 14.7 20.6 ^* 33.9 ^* 66.0 ^*

PT6 0 17.3 10.8 7.1 33.2 ^* 28.3

^*：P＜0.05

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Claims

1. design the method for newtype drug molecule based on the spatial structure information of Ag-Ab effect, may further comprise the steps:

(3) epi-position of binding antibody in the identification antigen;

(4) epi-position of binding antibody in the experimental verification antigen;

2. the method for claim 1, wherein the building process of antibody variable region conformation is as follows: according to the analysis of antibody gene variable region sequences, determine its conserved region, utilize the homology mould to set up antibody variable region initial space conformation, obtain antibody variable region through conformation optimization and stablize conformation.

3. the process of claim 1 wherein that resisting the mother crystal structure carries out the conformation that theoretical treatment obtains antigen.

4. the method for claim 1, wherein according to the space conformation feature of antigen and antibody variable region conformation, obtain the antigen-antibody complex space conformation through molecular docking and molecular dynamics simulation, described space conformation feature is that geometry, accessibility surface area and apparent static distribute.

5. the process of claim 1 wherein by making up the deletion mutant of the epi-position region of binding antibody in the disappearance antigen, and analyze itself and the interaction of antibody, the epi-position of binding antibody in the affirmation antigen.

6. the process of claim 1 wherein that described antigen is TNF α, described antibody is Z12.

7. the epitope that contains TNF alpha amino acid sequence 141-146.

8. the epi-position of claim 7, wherein said epi-position can specificity binding antibody Z12.

9. the epi-position of claim 8, described epi-position is made up of TNF alpha amino acid sequence 141-146.