CN1786016B - Artificial constructed bioactive molecule and its preparation method - Google Patents

Abstract

The invention belongs to the field of bioengineering, and relates to an artificially constructed bioactive molecule and a preparation method thereof.

Description

A kind of artificial constructed bioactive molecules and preparation method thereof

Technical field

The invention belongs to bioengineering field, relate to a kind of artificial constructed bioactive molecules and preparation method thereof.

Background technology

In theory, the antibody variable region structural domain can be used as an ideal skeleton structure (scaffold) of showing the exogenous peptide section.At first, V _HStructural domain can keep certain rigid, and the loss that combines entropy when forming mixture like this with target protein is less, and bonding force is stronger.At antibody variable region (heavy chain: V _H, light chain: V _L) in the structure, three loop support that by two antiparallel βZhe Die lamellas these β lamellas are more stable on conformation, can support short peptide sequence, form a definite three-dimensional structure, the space that keeps correct folds.Secondly, the antibody variable plot structure spatially can concentrate on the small peptide of several displayings in certain zone, forms the interface that can combine closely with target protein.In addition, the exogenous peptide segment length that the antibody variable region frame is showed can change, because the skeleton of antibody variable region is when showing CDR loop district, length can change.Report was once arranged, heavy chain CDR2 was prolonged to strengthen the binding ability of antiestrogenic antibody.Report is also arranged, insert 4 residues, can keep not changing with haptenic binding ability at the contiguous combining site of framework region FR3 and near the site of CDR.Therefore the antibody variable region structural domain can be illustrated in the exogenous peptide section in the certain-length scope.Simultaneously, utilize people's antibody variable region skeleton to support, greatly reduce the allos reaction as the displaying of external source small peptide.

Tumor necrosis factor alpha (TNF-α) is a kind of active cytokine of various biological that has, be mainly derived from monocyte and scavenger cell, T lymphocyte, neutrophil leucocyte, mastocyte and endotheliocyte also can produce (Old L (1985) Science 230:630-632) under certain condition.Mature T NF-alpha molecule amount is 17kD, combines mediation various biological activity with the TNF-α acceptor (TNFR) of cell surface with trimeric form.The TNF-α of normal level can participate in resisting bacterium, viral and parasitic infection, promote tissue repair, cause apoptosis of tumor cells, but TNF-α in vivo a large amount of generations and discharge the immunologic balance then can destroy body, produce multiple pathology damage with other inflammatory factors, for example the multiple organ dysfunction syndrome due to emaciation and the septic shock, rheumatoid arthritis (RA), multiple sclerosis (MS), inflammatory bowel (IBD), disorderly syndromes multiple disease (Moeller A et al. (1990) the Cytokine 2:162-169 such as (MDS) of graft versus host disease (GVH disease) (GVHD) and marrow hemopoiesis; No. 5231024, the United States Patent (USP) of Moeller etc.; VasilliP (1992) Annu Rev Immunol 10:411-452; Tracey KJ, Cerami A (1994) Annu Rev Med 45:491-503).Therefore, the antagonist of use TNF-α reduces the TNF-alpha levels in these diseases, for these treatment of diseases provide a kind of new way.

Up to the present, the antagonist of the TNF-α that has developed mainly contains two big classes, and a class is the fusion rotein that forms of the Fc section of TNFR and IgG and the monoclonal antibody of anti-TNF-α, and compares sufficient clinical study.Research through nearly ten years, developed commercial TNFR II-Fc fusion rotein by American I mmunex company, be called Etanercept, commodity are called Enbrel, by FDA approval listing, be used for the treatment of rheumatoid arthritis, ankylosing spondylitis and inhibition psoriatic patient's bone and joint injury.Another the big class effectively macromole of antagonism TNF-α effect is an antibody, especially monoclonal antibody, and carried out humanization, U.S. Centocor company has developed the antibody of the anti-TNF-α of a kind of mosaic type, called after Infliximab, commodity are called Remicade, and the FDA approved is used for the treatment of RA and colon C rohn disease.

Use immunological diseases such as TNF-alpha-2 antagonists treatment rheumatoid arthritis and colon C rohn disease and obtained definite results, this also illustrates and reduces the effective measure that the TNF-alpha levels is these diseases of treatment really.But the antagonist of this two class TNF-α exists a lot of side effects.The using dosage of Etanercept and Infliximab is all very big, and the course of treatment is long, needs the hundreds of milligram course of treatment.Side effect is big, and mouse source antibody can produce human antimouse antibody (HAMA) reaction, and human mouse chimeric antibody also can cause the anti-chimeric antibody of people (HACA) reaction (ElliottMJ etc. (1994) Lancet 344:1125-1127) in the process of long term administration.

The unsurmountable shortcoming that these TNF-alpha-2 antagonists itself exist will impel people to remove to develop the more effectively novel molecular of antagonism TNF-α.Other and the closely-related some diseases of high TNF-alpha levels also provide wide space for the application of the novel antagonistic molecule of TNF-α.

A series of researchs were carried out over past ten years in this laboratory aspect the TNF-Alpha antibodies, in having obtained and the monoclonal antibody (called after Z12) of TNF-α, made up the mixture model of Z12 and hTNF-α effect by computer-implemented method, determined the epi-position (141-146 position) of antibody Z12 identification TNF-α, according to this pattern layout some suppress the small peptide molecule (Shen doubly put forth energy to wait (2003) Chinese patent application number 03104870.6) of TNF-α.After the biological experiment checking, find that these small peptide molecules have activity, but the Cytotoxic inhibition effect of TNF-α is not reached theoretical expected value, and large usage quantity, infer that reason may be because these small peptide molecule space volumes are less, after the TNF-alpha molecule combines, still be not enough to block the effect between TNF tripolymer and these two macromole of TNF acceptor.

Summary of the invention

Based on above-mentioned prior art, the present invention proposes the novel method that makes up new bioactive molecules, promptly utilize human antibody heavy chain variable region (V _H) skeleton construction, show to have the active peptide section of particular organisms, thereby show the biological activity of bioactive peptide with the macromole form.And this bioactive macromolecule can not excite rejection in human body.Therefore, have a good application prospect.

Based on aforesaid method of the present invention, the present invention utilizes three to design and verified its active small peptide molecule: the framework with selected people's antibody variable region is support, the small peptide that will have the antagonism function substitutes the CDR of people's antibody variable region, made up to TNF-α have antagonistic action novel molecular (antigen antagonism peptide antibody).

The present invention relates to a kind of artificial constructed bioactive molecules, wherein contain people's antibody variable region skeleton and bioactive peptide, described people's antibody variable region skeleton has stable three-dimensional structure, and in its native sequences one section or several sections sequences are replaced by bioactive peptide, thereby bioactive peptide is showed in molecular surface, shows the biological activity of bioactive peptide.

In people's antibody variable region, substituted sequence preference is the CDR district.

Various people's antibody variable regions all can be used for realizing the present invention, but preferably adopt people's IgG antibody heavy chain or variable region of light chain, and particularly preferred people's antibody variable region is IgG V _H5.

Bioactive peptide can be any active peptide section of particular organisms that has, as long as this bioactive peptide can show the activity of itself in people's antibody variable region skeleton.In specific embodiments of the present invention, described bioactive peptide is small peptide, particularly PT2, PT3 and the PT4 that TNF-α is had antagonistic activity.

The invention still further relates to the preparation method of artificial constructed bioactive molecules of the present invention, may further comprise the steps:

1) provides a kind of people's antibody variable region with stable three-dimensional structure;

2) provide one or more bioactive peptides;

3) with step 2) bioactive peptide step of replacing 1) in people's antibody variable region in one section or several sections sequences, make bioactive peptide be showed in molecular surface, show the biological activity of bioactive peptide.

In people's antibody variable region, substituted sequence preference is the CDR district.

Various people's antibody variable regions all can be used for realizing the present invention, but preferably adopt people's IgG antibody heavy chain or variable region of light chain, and particularly preferred people's antibody variable region is IgG V _H5.

Bioactive peptide can be any active peptide section of particular organisms that has, as long as this bioactive peptide can show the activity of itself in people's antibody variable region skeleton.In specific embodiments of the present invention, described bioactive peptide is small peptide, particularly PT2, PT3 and the PT4 that TNF-α is had antagonistic activity.

Description of drawings

The mixture space conformation model that Fig. 1 .1.1PTVH1 and TNF form.Wherein, green line chart is the TNF tripolymer, and pink colour is the VH skeleton construction, and cyan mallet structure is PT1, and yellow is PT2, and redness is PT3, down together.

The mixture space conformation model that Fig. 1 .1.2PTVH2 and TNF form.

The mixture space conformation model that Fig. 1 .1.3PTVH3 and TNF form.

The mixture space conformation model that Fig. 1 .1.4PTVH4 and TNF form.

The mixture space conformation model that Fig. 1 .1.5PTVH5 and TNF form.

The mixture space conformation model that Fig. 1 .1.6PTVH6 and TNF form.

The mixture space conformation model that Fig. 1 .1.7PTVH7 and TNF form.

The secondary structure analysis of 6 sections sequences of Fig. 1 .2.1PTVH5 normal chain.Among the figure, from top to bottom, be respectively the RNA secondary structure of sequence 1-6 from left to right.

The secondary structure analysis of 6 sections sequences of Fig. 1 .2.2PTVH5 minus strand.Among the figure, from top to bottom, be respectively the RNA secondary structure of sequence F1-F6 from left to right.

Fig. 1 .3.1 complete synthesis PTVH5 DNA of PCR method, wherein 1 is dna molecular amount marker, 2 is PTVH5 DNA.

The enzyme of Fig. 1 .3.2 recombinant plasmid PTVH5/pET22 is cut evaluation, among the figure

1. molecular weight marker thing, 2.PTVH5/pET22 is after Xba I and EcoRI enzyme are cut.

The sequencing result of Fig. 1 .3.3 recombinant plasmid PTVH5/pET22.

MRNA secondary structure before and after Fig. 1 .4.1PTVH5/pET223 ' end is revised.The left side is the RNA secondary structure of the 70bp of terminator codon before PTVH5/pET223 ' end is revised near among the figure; The right side is amended secondary structure.

Sequencing result before and after Fig. 1 .4.2PTVH5/pET223 ' distal process becomes.The left side is the sequence before PTVH5/pET223 ' distal process becomes among the figure; The right side is amended sequence, and red line part is the sudden change part.

Abduction delivering analysis before and after Fig. 1 .4.3PTVH5/pET223 ' distal process becomes.Among the figure, the left side is that PTVH5/pET223 ' distal process becomes the pre-induction result, and the right side is sudden change back result.

1.PTVH5/pET22 do not induce; 2.0.5mMIPTG after inducing; 3.DNA molecular weight marker thing.

The SDS-PAGE that Fig. 1 .5.1PTVH5 expresses analyzes.Among the figure:

1. do not induce; 2. the precipitation after ultrasonic; 3. after inducing; 4. the supernatant after ultrasonic.

The Western trace that Fig. 1 .5.2PTVH5/pET22 expresses is identified.Among the figure:

1. do not induce; 2.PTVH5/pET22 after inducing; 3.PTVH5/pET22 after inducing; 4. the precipitation after ultrasonic; 5. the supernatant after ultrasonic.

The Ni of Fig. 1 .5.3PTVH5 ²⁺The post affinity chromatography.

Fig. 1 .5.4PTVH5 is through Ni ²⁺SDS-PAGE after the enrichment of post affinity chromatography analyzes.

Fig. 1 .6.1PTVH5 is to the competition inhibition test of Z12-HRP.Among the figure, triangle curve is represented the PTVH5 albumen of purifying, the supernatant after diamond curve represents pET22/BL21 ultrasonic.

Fig. 1 .6.2PTVH5 is to the cytotoxic retarding effect of TNF-α.Among the figure, triangle curve is represented the PTVH5 albumen of purifying, the supernatant after diamond curve represents pET22/BL21 ultrasonic.

Fig. 1 .6.3PTVH5 is to the specificity analyses of TNF-α cell toxicant retarding effect.Among the figure, diamond curve is represented TNF-α+PTVH5 (200 μ g/ml), the supernatant after square curve represents TNF-α+pET22/BL21 (200 μ g/ml) ultrasonic, and triangle curve is represented TNF-α.

Embodiment

Below specifying with TNF-α antagonism peptide (PT2, PT3 and PT4) is the process of the new bioactive molecules of fundamental construction, simultaneously, has verified the biological activity of this new biological activity molecule by experiment.It must be noted that method of the present invention is not limited to TNF-α antagonism peptide, the peptide section of other biologically active can be according to method of the present invention, and preparation makes new advances has the active molecule of particular organisms.

The design of PTVH5 and Function Identification

Mixture model according to crystalline structure, TNF-α and the antibody Z12 effect of mouse source of TNF-α, with people's IgG antibody variable region is framework, select three bioactive peptides (Shen doubly put forth energy to wait (2003) Chinese patent application number 03104870.6), replace the CDR district of antibody variable region, thereby design the novel molecular (antigen antagonism peptide antibody) that suppresses TNF-α.

Utilize the Octane2 graphics workstation, by InsightII (2000) routine package, to 7 classes with people's IgG antibody heavy chain (V _H) investigate theoretically as the novel molecular of frame design and the interaction pattern between the TNF-α, by differentiation, select the 5th class (V in conjunction with the zone of free energy, recognition function territory (Domain), participation effect and the intermolecular hydrogen bonding that forms etc. _H5) skeleton of antibody variable region is as support, and the bioactive peptide of selecting to have antagonistic action is designed to novel molecular PTVH5 as the CDR alternative.Utilize the DNA of the complete synthesis coding of PCR method PTVH5, and obtain PTVH5 albumen through prokaryotic expression and purifying.Utilize the competition inhibition test of PTVH5 and Z12 and to the cytotoxic inhibition experimental verification of TNF-α its biologic activity.

Particularly, said process can be divided into following steps:

(1) computer aided molecular design PTVH5

(2) dna sequence dna of complete synthesis coding PTVH5

(3) PTVH5/pET22 construction of recombinant plasmid

(4) revise the structure that 3 of PTVH5 ' holds, improve expression level

(5) purifying of PTVH5 and evaluation

(6) PTVH5 is to the competition inhibition test of Z12-HRP

(7) PTVH5 is to the cytotoxic retarding effect of TNF-α

(8) PTVH5 is to the specificity analyses of the cytotoxic retarding effect of TNF-α

1.1 material

The pET22 plasmid is preserved by this chamber

Chelating Ni ²⁺The sepharose chromatography column available from this yuan Zhenyang, Beijing Genentech, Inc. (US) 460 Point San Bruno Blvd, South San Francisco, CA, 94080

Anti-his antibody is available from Invitrogen company

The sheep anti-mouse igg of HRP mark is preserved by this chamber preparation

All the other material therefors are with 1.1 material parts

1.2 method

1.2.1 computer aided design (CAD) new function molecule PTVH5

According to mouse source antibody Z12 and the interactional mixture model of antigen TNF-α, by people's IgG antibody variable region structure type, by the reasonable triage techniques of computer, replace the CDR of people's antibody variable gene with the functional peptides PT2 with antagonistic action, PT3, PT4 (Shen (2003) Chinese patent application such as doubly put forth energy number 03104870.6), making up with people's IgG antibody variable region skeleton is supports, antagonism peptide PT2, PT3, PT4 carry out the structure displaying at corresponding C DR antigen antagonism peptide antibody.

On graphics workstation Octane2, the homology mould that the antigen antagonism peptide antibody that makes up carries out space conformation is built by the Homology software of InsightII (2000) routine package.Under CVFF, the Amber field of force, by mouse source antibody Z12 and TNF-α interaction pattern, (PDB number: 1tnf) combination obtains the space conformation of antigen antagonism peptide antibody and the interactional mixture of TNF-α with antigen antagonism peptide antibody that makes up and the TNF-α with crystalline structure by the molecular docking method.Determine the stable conformation that it is final by molecular mechanics, molecular dynamics dynamic simulation.

With interaction pattern, identified region, interaction free energy, intermolecular hydrogen bonding as criterion, utilize distance geometry, computer graphics techniques, to fixed serve as that the biological effect that supports the antigen antagonism peptide antibody obtain carries out theoretical prediction with human IgG antibody's variable region of heavy chain 7 class skeletons, determine best design.

1.2.2 the DNA of complete synthesis coding PTVH5

Select the framework of VH5 as displayed polypeptide section PT2, PT3 and PT4, called after PTVH5, its aminoacid sequence is:

QVQLQESGPGLVKPSETLSLTCTVS INYTGEPTYSQSVSNVVWYWIRQP

PGKGLEWIG? YTYYPTVNENRSQSVSNVFRVTISVDTSKNQFSLKLSSVTAAD

TAVYYCAR? YINTGYDGLYYNSMD?WGQGTLVTVSS(SEQ?ID?NO：1)

Line portion is the sequence after CDR-H1, CDR-H2 and CDR-H3 are replaced by PT2, PT3 and PT4 respectively.

Serves as that the aminoacid sequence that supports the antigen antagonism peptide antibody PTVH5 that makes up carries out reverse translation to what obtain with people's antibody variable region VH5 skeleton.Consider genetic expression, in translation process, the bias codon is revised.

With the anti-dna sequence dna (SEQ ID NO:2) of translating into of aminoacid sequence:

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

16 E T L S L T C T V S I N Y T G

31 E P T Y S Q S V S N V V W Y W

46 I R Q P P G K G L E W I G Y T

61 Y Y P T V N E N R S Q S V S N

76 V F R V T I S V D T S K N Q F

91 S L K L S S V T A A D T A V Y

106 Y C A R Y I N T G Y D G L Y Y

121 N S M D W G Q G T L V T V S S

For the PTVH5 gene order that obtains, attempt obtaining by the method for pcr amplification.So, with above sequence segmentation, with the free energy of each section of Goldkey computed in software sequence, keeping adjusting base under the constant prerequisite of aminoacid sequence, to eliminate hair clip (hairpin) structure.Like this, obtained following sequence (SEQ ID NO:3)

Dash area is for desiring each section of synthetic sequence.

1：5′-GCGACTTC CATATGCAAGTT?CAACTTCAGG?AATCTGGTCC-3′(40bp)(SEQ?ID?NO：4)

2：5′-TGAAACCCTG?TCTCTGACTT?GTACAGTGTC?TATCAACTAT?ACCGGCGAGC?CGACCTAC-3′(58bp)(SEQ?IDNO：5)

3：5′-GTAGTTTGGT?ACTGGATTCG?CCAACCTCCG?GGTAAAGGTC?TGGAGTGGAT?CGGTTACAC-3′(59bp)(SEQ?IDNO：6)

4：5′-GAAAACCGTT?CTCAATCCGT?CTCCAACGTT?TTCCGTGTTA?CTATCTCTGT?TGATACCAG-3′(59bp)(SEQ?IDNO：7)

5：5′-TGAAGCTGTC?TTCCGTAACA?GCAGCTGATA?CAGCCGTGTA?TTACTGCGC?GCGTTAC-3′(56bp)(SEQ?IDNO：8)

6：5′-CGGCTTATAT?TATAACTCTA?TGGATTGGGG?TCAAGGCACT?TTAGTGACGG?TGAGCTCC-3′(58bp)(SEQ?IDNO：9)

F1：5′-AAGTCAGAGA?CAGGGTTTCA?GATGGTTTTA?CCAGACCCGG?ACCAGATTCC?TGAAGTTG-3′(58bp)(SEQ?IDNO：10)

F2：5′-GAATCCAGTA?CCAAACTACG?TTAGACACAG?ACTGGGAGTA?GGTCGGCTCG?CCGGTATAG-3′(59bp)(SEQ?IDNO：11)

F3：5′-GGATTGAGAA?CGGTTTTCGT?TGACGGTCGG?ATAATACGTG?TAACCGATCC?ACTCCAG-3′(57bp)(SEQ?IDNO：12)

F4：5′-TTACGGAAGA?CAGCTTCAGG?GAAAACTGGT?TTTTGCTGGT?ATCAACAGAG?ATAGTAAC-3′(58bp)(SEQ?IDNO：13)

F5：5′-CCATAGAGTT?ATAATATAAG?CCGTCATAGC?CCGTGTTAAT?GTAACGCGCG?CAGTAATAC-3′(59bp)(SEQ?IDNO：14)

F6：5′-TGCCGA? CTC?GAG?GGAGCTCACC?GTCACTAAAG?TGCCTTGAC-3′(41bp)(SEQ?ID?NO：15)

The every section long 56-59 of sequence bp, first section sequence line part of normal chain is the NcoI restriction enzyme site, the F6 sequence line part of minus strand is the XhoI restriction enzyme site.

Obtain complete PTVH5DNA sequence with the PCR method amplification: the sequence 1-6 and the F1-F6 that get 1 OD unit, add deionized water 108,74.4,72,73.6,76.8,73.6,73.6,72.4,74.8,73.2,72.8 and 105.2 μ l respectively, make final concentration be 50pmol/ μ l, dilute 5 times, be 10pmol/ μ l, respectively get 2 μ l to 26 μ l H ₂Among the O, mix, the concentration of every section sequence is 20pmol/50 μ l, gets 10 μ l mixtures and makes template, adds each 1 μ l of upstream primer (sequence 1) and downstream primer (sequence F6), dNTP2 μ l, 10 * pyrobest buffer, 2.5 μ l, H ₂O 8 μ l, high-fidelity enzyme pyrobest 0.5 μ l.

PCR reaction conditions: 94 ℃ of sex change 4min; 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s, 30 circulations; 72 ℃ are extended 5min, reduce to 4 ℃ then.Amplified production is got the agarose electrophoresis detection of 3 μ l with 1-5%.

1.2.3 the structure of PTVH5/pET22 recombinant expression plasmid:

The enzyme of dna fragmentation and plasmid is cut: pET22 plasmid or dna fragmentation 5 μ l, 10 * buffer II, 2 μ l, BSA (100 *) 0.2 μ l, NcoI and XhoI are 0.5 μ l, adding deionized water to cumulative volume is 20 μ l, 37 ℃ 2 hours.Use the dna fragmentation fast purifying of QIAGEN company to reclaim test kit, reclaim the purpose fragment.

Carry out pET22 and dna fragmentation that ligation: NcoI and XhoI enzyme cut by following condition and respectively get 7 μ l, 5 * ligase enzyme damping fluid, 4 μ l, T4 dna ligase 2 μ l.Room temperature is placed 6 hours postposition and is spent the night for 4 ℃.

Transform the BL21 competence by 1.2 described methods, the picking mono-clonal with colony polymerase chain reaction (PCR) method primary dcreening operation positive colony, is identified with the enzyme blanking method.

Enzyme is cut system: recombinant plasmid 5 μ l, 10 * EcoR I buffer, 2 μ l, NcoI and XhoI are 0.5 μ l, adding deionized water to cumulative volume is 20 μ l, 37 ℃ 2 hours.Agarose electrophoresis with 1.5% detects enzyme and cuts effect.The recombinant plasmid PTVH5/pET22 that selection has the big or small fragment of expection to downcut send Bo Ya Bioisystech Co., Ltd, checks order with universal primer.

1.2.4 the proteic expression and purification of PTVH5

(1) abduction delivering of PTVH5/pET22 plasmid

The recombinant plasmid that above-mentioned evaluation is correct, Transformed E .coli BL21 (DE3) host bacterium, comparing with BL21 (DE3) bacterial strain that transforms the pET22b empty carrier, is that the inducing of 0.1-1mmol/L carries out a small amount of down and express and test at the IPTG final concentration, and with 12% SDS-PAGE detection expression.

(2) 3 ' terminal sequence of modification PTVH5 DNA improves expression level

Design following primer:

The line part is the base after suddenling change, the codon TCC of Ser residue of 135 of codings is sported AGC, near the original low excessively free energy of the secondary structure of terminator codon is increased, help translating and transcribing, still keep XhoI restriction enzyme site (drawing the sequence of wavy line).

With the PTVH5/pET22 plasmid that checks order correct is template, is upstream primer with sequence 1, is downstream primer with Lower3, pcr amplification, again be connected into pET22 after amplified production cut with NcoI and XhoI enzyme, cut through enzyme and identify and order-checking is identified and induced after correct, analyze expression with SDS-PAGE.

Expression product mainly existed with the inclusion body form when concentration of inductor IPTG was 0.1-1mM, reduced IPTG concentration to 0.01mmol/L, part was then arranged for soluble-expression, selected the highest bacterial strain of expression amount and carried out great expression.

(3) purifying of PTVH5:

Recovery PTVH5/pET22/BL21, by 1: 500 transferred species to 400ml LB substratum, 37 ℃ when being cultured to A600 ≈ 0.5, add IPTG, making final concentration is 0.01mmol/L, induces 145rpm, 20 hours in 20 ℃.Coinduction 4000ml, centrifugal, 10mM PBS (pH7.2) washing three times suspends with 400ml 10mM PBS (pH7.2), and after the ultrasonication, centrifugal 15 minutes of 12000 * g collects supernatant, transfers pH to 8.0, adds NaCl to 500mM, uses chelating Ni ²⁺The agarose affinity chromatography column purification.Add 500mMNaCl and the 20mM imidazoles washs to baseline with the 20mM PB of pH8.0, with 50,100,150 with different imidazole concentration wash-out such as 250mM, collect elution peak, the purity of SDS-PAGE electrophoresis detection target protein with 12%.

(4) Western-blot identifies:

The 12%SDS-PAGE electrophoresis, 40v transfer printing 2 hours, 4 ℃ of sealings of 5% skim-milk are spent the night.One anti-is Anti-His (1: 4000), 37 ℃ 1 hour, PBST (pH7.4) washing is three times for 0.2%Tween20,10mM PBS, and the sheep anti-mouse antibody that adds the HRP mark is as two anti-(1: 500), 37 ℃ 1 hour, PBST washing three times develops the color with ECL.

1.2.5PTVH5 the mensuration of function:

(1) PTVH5 is to the competition inhibition test of Z12-HRP:

With humanTNF-'s coated elisa plate, TNF-α bag is 0.2 μ g/ml by concentration, every hole 50 μ l, 4 ℃ are spent the night, PBST washing one time, 37 ℃ of sealings of 10% foetal calf serum 1 hour, PBST washing three times, every hole adds Z12-HRP50 μ l (1: 500), the PTVH5 albumen (200,100,50,10 μ g/ml) that adds different concns respectively, every hole 50 μ l, the supernatant after ultrasonic compares with PBS and pET22/BL21 respectively, hatched 1 hour for 37 ℃, PBST washing three times adds the OPD substrate, every hole 50 μ l, color development at room temperature 10 minutes, every hole add 50 μ l 2N H ₂SO ₄Termination reaction, 492nm wavelength are surveyed the OD value down.

(2) PTVH5 is to the cytotoxic retarding effect of TNF-α

L929 cell suspension furnishing 3.2 * 10 ⁵/ ml is inoculated into 96 orifice plates, and every hole 80 μ l add dactinomycin, and making final concentration is 1 μ g/ml.The hTNF-α that adds degerming, every hole 10 μ l, the PTVH5 albumen of adding different concns, every hole 10 μ l, mixing, the supernatant of the filtration sterilization after control wells adding pET22/BL21 is ultrasonic, 37 ℃ of 5%CO ₂Incubator was cultivated 24 hours.The supernatant that inclines adds 0.5% Viola crystallina, every hole 15 μ l, and room temperature 10 minutes, the distilled water thorough washing adds 33% acetate, and every hole 100 μ l survey 570nm OD value.Calculate inhibiting rate as follows.

In addition, other establishes one group of experiment, the fixing concentration of PTVH5 and the pET22/BL21 supernatant after ultrasonic, be 200 μ g/ml, the amount that adds hTNF-α is a different set of concentration, be respectively 10,100,500,1000 and 5000pg/ml, all the other methods are the same, investigate PTVH5 to the inhibiting specificity of TNF-α cytotoxicity.

1.3 result

1.3.1 the design of novel molecular PTVH5 and biological function theoretical prediction

According to human IgG antibody variable region heavy chain three-dimensional conformation, be divided into 7 kinds of main types according to Kabat classification, skeleton and CDR district conformation with crystalline structure.With above-mentioned V with crystalline structure _H1～V _H7 seven class V _HBe template, use PT2, PT3 and PT4 (Shen (2003) Chinese patent application such as doubly put forth energy number 03104870.6) to replace respectively in three CDR districts of VH, make up novel antigens antagonism peptide antibody.With the TNF crystalline structure is template, by mouse source antibody Z12 and TNF interaction pattern, makes up antigen antagonism peptide antibody molecule and the interactional space three-dimensional model of TNF (seeing Fig. 2 .1.1-Fig. 2 .1.7).By theoretical analysis, calculate interaction free energy, the intermolecular hydrogen bonding of novel molecular and TNF-α, determine mutual identified region and binding mode.

The result who builds from computer mould in this seven class VH, is that the PTVH5 and the effect between the TNF (3) of stencil design is the strongest with VH5.The Van der Waals of the effect between PTVH5 and the TNF (3) can be-76.43794kJ for-142.95993kJ, electrostatic energy, and is minimum in conjunction with free energy (Van der Waals energy+electrostatic energy), for-219.39787kJ, in conjunction with the effect of TNF the strongest (seeing Table 1).

Table 1 is the interaction energy of the PTVH recruit and the TNF (3) of frame design with seven class VH

The zone of action of PTVH5 and TNF-α is wider, can with two monomer effects in the tripolymer, wherein main recognition site zone is the reactive site 141-146 position (seeing Table 2) of TNF-α, the PTVH5 that obtains so finally may in and the toxic action of TNF-α, and three CDR districts of alternate all participate in identification TNF-α (seeing Table 3), can form 15 pairs of hydrogen bonds (seeing Table 4) with TNF (3), the effect between PTVH5 and the TNF-α is played an important role.

Table 2 seven class PTVH recruits discern the zone of TNF (3)

The classification of novel molecular	The zone (TNF epitiope) of identification
		Z12	A141-A146，B141-B146，C141-C146
PTVH1	A103(R)，B23-B24(EG)，B65-B72(KGQGCPST)，B103-B110(RETPEGAE)，B114(W)， B138-B142(RPDYL)，B144(F)，C107-C110(EGAE)，C112(K)
		PTVH2	A92-A95(NLLS)，B20-B24(PQAEG)，B32(R)，B34(N)，B65-B68(KGQG)，B70(P)， B104(E)，B106-B113(PEGAEAKP)，B138-B149(RPDYLLFAESGQ)
PTVH3	A20-A24(PQAEG)，A27(Q)，A65(K)，A67-A71(QGCPS)，A107(E)， A110-A111(EA)，A113(P)，A115(Y)，A138-A145(RPDYLLFA)
		PTVH4	C20-C25(PQAEGQ)，C65(K)，C67-C71(QGCPS)，C73-C74(HV)，C105-C113(TPEG AEAKP)，C137-C142(NRPDYL)，C144(F)
PTVH5	B71-B75(STHVL)，C20-C24(PQAEG)，C65-C75(KGOGCPSTHVL)， C105-C115(TPEGAEAKPWY)，C138(R)，C140-C145(DYLLFA)
		PTVH6	A20-A25(PQAEGQ)，A65-A74(KGQGCPSTHV)，A106-A112(PEGAEAK)， A114(W)，A138-A145(RPDYLLFA)
PTVH7	C20-C21(PQ)，C23-C24(EG)，C65-C70(KGQGCP)，C105-C111(TPEGAEA)，C138(R)， C140-C146(DYLLFAE)

Table 3 seven class PTVH recruits participate in discerning the zone of TNF (3)

The novel molecular classification	The zone of action of identification TNF
		PTVH1	12-14(KKP)，48(Q)，50(P)，55-57(EWM)，106(Y)，108(A)，[PT3]110(Y)， 113-123(TGYDGLYYNSM)
PTVH2	[PT1]37-44(SVSNVVWY)，[PT2]63(P)，65-71(VNENRSQ)；85(K)，109(R)， [PT3]111-116(INTGYD)，118(L)，120(Y)
		PTVH3	34-35(YS)，37-41(SVSNV)，62-64(YPT)，85(T)，110-112(YIN)， 114-118(GYDGL)，121-123(NSM)
PTVH4	38-39(VS)，41-44(VVWY)，58-59(YT)，61(Y)，63-66(PTVN)， 68-76(NRSQSVSNV)，85(N)，112-116(NTGYD)
		PTVH5	11-13(LVK)，34-45(YSQSVSNVVWYW)，64-66(TVN)，86-88(SKN)， 109-116(RYINTGYD)
PTVH6	1-2(EV)，5-8(VQSG)，21-23(SCK)，27-36(NYTGEPTYSQ)，38(V)，100(T)，138(Q)
		PTVH7	43-44(WY)，54-56(LEW)，61(Y)，63(P)，65(V)，73(V)，110-121(YINTGYDGLYYN)

Table 4 VH5 and the hydrogen bond that (TNF) forms between 3

1.3.2 the DNA of complete synthesis coding PTVH5:

Select the codon of E.coli preference, with the anti-dna sequence dna of translating into of the aminoacid sequence of PTVH5, long 405bp, CAI=0.5217 (CAI:codon adaptation index, bias codon index), this is also similar to the CAI value that the intracytoplasmic high-abundance proteins matter of most of E.coli is adopted.

Normal chain and the minus-strand dna of PTVH5 respectively are divided into 6 sections, and corresponding two sections (as sequence 1 and F1) overlapping 18-22bp in normal chain and the minus strand help the extension of PCR process.Analyze every section secondary structure, revise base, eliminate too much hairpin structure, be convenient to pcr amplification (seeing Fig. 1 .2.1 and Fig. 1 .2.2).12 sections sequences are mixed the back as template, obtained the product consistent (seeing Fig. 1 .3.1) with the purpose clip size through pcr amplification.

1.3.3PTVH5/pET22 the structure of recombinant expression plasmid:

After the total length PTVH5 dna sequence dna that pcr amplification is obtained was connected to the pET22 carrier, the recombinant plasmid PTVH5/pET22 of structure cut through XbaI and EcoRI enzyme and identifies correct (seeing Fig. 1 .3.2), and whole sequence is measured entirely true (seeing Fig. 1 .3.3).

1.3.4PTVH5 the raising of protein expression level and purifying are identified

After the PTVH5/pET22 recombinant plasmid that has made up is induced expression is arranged, but expression level is very low, this brings very big difficulty can for follow-up purifying.Factors such as the secondary structure of the initiator secondary structure of Recombinant Protein Expression level and mRNA, near the secondary structure the terminator codon and full length mRNA and half life are relevant.In PTVH5/pET22, a pelB signal peptide was arranged before target protein PTVH5, long 21 amino-acid residues (63bp) therefore can be got rid of the influence of the initiator secondary structure of mRNA substantially.For mRNA one-piece construction and half life, up to the present also there are not very accurate calculating and Forecasting Methodology.Near the terminator codon TAA RNA secondary structure also has a significant impact expression level, structure is tight, free energy is relatively low, help keeping the stability of RNA in translation process, be difficult for degraded, help proteic efficiently expressing, but free energy can not be too low, and free energy crosses that RNA often is folding imporosity when low, can give to transcribe and translation causes very big difficulty.

Attempted revising the structure of 3 ' end among the present invention, calculated near the free energy of the RNA secondary structure of the 70bp of terminator codon TAA, compared the influence of structural changes then expression level.The design mutant primer sports AGC with the codon TCC of Ser residue of 135 of codings, and the free energy of 3 ' end is increased to 8.782kJ/mol from-49.348kJ/mol, and the expression level of PTVH5 is increased to 12% (seeing Fig. 1 .4.1-Fig. 1 .4.3).

On the other hand, among the PTVH5 two halfcystines must form disulfide linkage could be correct folding, PTVH5 also just has activity.Because the pelB signal peptide sequence of pET22 can carry PTVH5 (periplasm) to the colibacillary periphery chamber, redox potential helps the formation of disulfide linkage than cytoplasm height herein, thereby can impel PTVH5 correctly folding.The target protein that does not enter the periphery chamber then in endochylema the form with inclusion body have (seeing Fig. 1 .5.1, Fig. 1 .5.2).After ultrasonic, correct folding PTVH5 albumen is owing to having 6 * His label, through chelating Ni in the supernatant ²⁺The agarose affinity chromatography post can obtain enrichment.

Utilizing chelating Ni ²⁺Agarose affinity chromatography column purification PTVH5 the time, the Tween 20 that adds 20mM imidazoles and 0.2% in the supernatant before upper prop, can reduce non-specific adsorption effectively, help the enrichment of PTVH5, with 5-10 column volume of imidazoles washing of 50mM, can further increase the purity of target protein during washing.The target protein purity that finally obtains reaches more than 80% (sees Fig. 1 .5.3).

1.3.5PTVH5 the mensuration of function:

(1) PTVH5 is to the competition inhibition test of Z12-HRP:

If the activated words of PTVH5 can combine the TNF-α that is coated on the enzyme plate with the Z12-HRP competition so, the colour developing of Z12-HRP will reduce.Experimental result explanation, PTVH5 can compete combine (the seeing Fig. 1 .6.1) that suppresses Z12-HRP and TNF-α.When the Z12-HRP extent of dilution was 1: 500, the inhibiting rate of the PTVH5 of 200 μ g/ml was 52%.

(2) PTVH5 is to the cytotoxic retarding effect of TNF-α

Experimental principle and PT4FC are similar to the cytotoxic retarding effect of TNF-α.Under TNF-α effect, apoptosis can take place in the L929 cell, suppresses the fissional while at the adding dactinomycin, and the apoptotic quantity of the L929 just activity with TNF-α is relevant, adds the cytotoxicity that PTVH5 can partly suppress TNF-α.When TNF-α concentration was 0.2 μ g/ml, the PTVH5 of 200 μ g/ml can suppress 45% cytotoxicity (seeing Fig. 1 .6.2).

When the concentration of PTVH5 is 200 μ g/ml, TNF-α for different concns, resulting OD570 absorbance is obviously different when not adding PTVH5, also obviously different with the contrast of the supernatant of the PTVH5/pET22 that adds same concentration, illustrate that PTVH5 is special (seeing Fig. 1 .6.3) to the cytotoxic retarding effect of TNF-α.

Reference

1.Smyth?MJ?and?Johnstone?RW.Microscopy?Research?and?Technique，2000，50：196-208.

2.Michael?E，Weinblatt，Edward?C，et?al.Arthritis?and?Rheumatism，2003，48(1)：35-45.

3.Present?DH，Rutgeerts?P，Targan?S，et?al.N?Engl?J?Med，1999，340(18)：1398-1405.

4.Moreland?L，Schiff?M，Baumgartner?S.Ann?Intern?Med，1999，130：478-486.

5.Wajdula?J.Ann?Rheum?Dis，2000，59(Suppl?1)：163-170.

6.Genovese?M，Martin?R，Fleischmann?R..Ann?Rheum?Dis，2000，43(suppl)：S269-S276.

7.Weinblatt?ME，Kremer?JM，and?Bankhurst?AD，et?al.N?Engl?J?Med，1999，340：253-259.

8.Abraham?E，Laterre?PF，Garbino?J，et?al.Crit?Care?Med，2001，29(3)：503-10.

9.Rau?R，Sander?O，van?Riel?P，et?al..J?Rheumatol，2003，30(4)：680-690.

10.Maini?RN，Feldmann?M.Arthritis?Research，2002，4(Suppl?2)：S22-S28.

11Hanauer?SB，Feagan?BG，Lichtenstein?GR，et?al.Lancet，2002，359(9317)：1541-1549.

12.Maini?R，Breedveld?F，Kalden?J?et?al.Arthritis?and?Rheumatism，1998，41：1552-1563.

13.Maini?R，St?Clair?E，Breedveld?F?et?al.Lancet，1999，359：1932-1939.

14.Lipsky?P，van?der?Heijde?D，St?Clair?E，et?al.N?Engl?J?Med，2000，343：1594-1602.

15.Raza?A.Microscopy?Research?and?Technique，2000，50：229-235.

16.Targan?SR，Hanauer?SB，van?Deventer?SJ，et?al.N?Engl?J?Med，1997，337(15)：

1029-1135.

17.De?Smit?MH?and?van?Duin?J.J.Mol.Biol，1994，244：144-150.

Sequence table

＜110〉Qin Weisong

Feng Jiannan

Li Yan

Put forth energy fully in Shen

＜120〉a kind of artificial constructed bioactive molecules and preparation method thereof

<130>CP1040125

<160>16

<170>PatentIn?version?3.3

<210>1

<211>135

<212>PRT

＜213〉artificial sequence

<220>

<223>PTVH5pro

<400>1

Gln?Val?Gln?Leu?Gln?Glu?Ser?Gly?Pro?Gly?Leu?Val?Lys?Pro?Ser?Glu

1 5 10 15

Thr?Leu?Ser?Leu?Thr?Cys?Thr?Val?Ser?Ile?Asn?Tyr?Thr?Gly?Glu?Pro

20 25 30

Thr?Tyr?Ser?Gln?Ser?Val?Ser?Asn?Val?Val?Trp?Tyr?Trp?Ile?Arg?Gln

35 40 45

Pro?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Tyr?Thr?Tyr?Tyr?Pro?Thr

50 55 60

Val?Asn?Glu?Asn?Arg?Ser?Gln?Ser?Val?Ser?Asn?Val?Phe?Arg?Val?Thr

65 70 75 80

Ile?Ser?Val?Asp?Thr?Ser?Lys?Asn?Gln?Phe?Ser?Leu?Lys?Leu?Ser?Ser

85 90 95

Val?Thr?Ala?Ala?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Tyr?Ile?Asn

100 105 110

Thr?Gly?Tyr?Asp?Gly?Leu?Tyr?Tyr?Asn?Ser?Met?Asp?Trp?Gly?Gln?Gly

115 120 125

Thr?Leu?Val?Thr?Val?Ser?Ser

130 135

<210>2

<211>405

<212>DNA

＜213〉artificial sequence

<220>

<223>PTVH5

<400>2

caggttcaac?ttcaggaatc?tggtccgggt?ctggtaaaac?catctgaaac?cctgtctctg 60

acttgtacag?tgtctatcaa?ctataccggc?gagccgacct?actcccagtc?tgtgtctaac 120

gtagtttggt?actggattcg?ccaacctccg?ggtaaaggtc?tggagtggat?cggttacacg 180

tattatccga?ccgtcaacga?aaaccgttct?caatccgtct?ccaacgtttt?ccgtgttact 240

atctctgttg?ataccagcaa?aaaccagttt?tccctgaagc?tgtcttccgt?aacagcagct 300

gatacagccg?tgtattactg?cgcgcgttac?attaacacgg?gctatgacgg?cttatattat 360

aactctatgg?attggggtca?aggcacttta?gtgacggtga?gctcc 405

<210>3

<211>405

<212>DNA

＜213〉artificial sequence

<220>

＜223〉PTVH5 of Xiu Shiing

<400>3

caagttcaac?tgcaagaatc?tggtccgggt?ctggtaaaac?catctgaaac?cctgtctctg 60

acttgtacag?tgtctatcaa?ctataccggc?gagccgacct?actcccagtc?tgtgtctaac 120

gtagtctggt?actggattcg?ccaacctccg?ggtaaaggtc?tggagtggat?cggttacacg 180

tattatccga?ccgtcaacga?aaaccgttcc?cagtctgtct?ccaacgtttt?ccgtgttact 240

atctctgttg?ataccagcaa?aaaccagttt?tccctgaagc?tgagctccgt?aacagcagct 300

gatacagccg?tgtattactg?cgcgcgttac?attaacacgg?gctatgacgg?cttatattat 360

aactccatgg?attggggtca?gggcacttta?gtgacggtga?gctcc 405

<210>4

<211>40

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>4

gcgacttcca?tatgcaagtt?caacttcagg?aatctggtcc 40

<210>5

<211>58

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>5

tgaaaccctg?tctctgactt?gtacagtgtc?tatcaactat?accggcgagc?cgacctac 58

<210>6

<211>59

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>6

gtagtttggt?actggattcg?ccaacctccg?ggtaaaggtc?tggagtggat?cggttacac 59

<210>7

<211>59

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>7

gaaaaccgtt?ctcaatccgt?ctccaacgtt?ttccgtgtta?ctatctctgt?tgataccag 59

<210>8

<211>56

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>8

tgaagctgtc?ttccgtaaca?gcagctgata?cagccgtgta?ttactgcgcg?cgttac 56

<210>9

<211>58

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>9

cggcttatat?tataactcta?tggattgggg?tcaaggcact?ttagtgacgg?tgagctcc 58

<210>10

<211>58

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>10

aagtcagaga?cagggtttca?gatggtttta?ccagacccgg?accagattcc?tgaagttg 58

<210>11

<211>59

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>11

gaatccagta?ccaaactacg?ttagacacag?actgggagta?ggtcggctcg?ccggtatag 59

<210>12

<211>57

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>12

ggattgagaa?cggttttcgt?tgacggtcgg?ataatacgtg?taaccgatcc?actccag 57

<210>13

<211>58

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>13

ttacggaaga?cagcttcagg?gaaaactggt?ttttgctggt?atcaacagag?atagtaac 58

<210>14

<211>59

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>14

ccatagagtt?ataatataag?ccgtcatagc?ccgtgttaat?gtaacgcgcg?cagtaatac 59

<210>15

<211>41

<212>DNA

＜213〉artificial sequence

<220>

＜223〉the PTVH5 fragment of Xiu Shiing

<400>15

tgccgactcg?agggagctca?ccgtcactaa?agtgccttga?c 41

<210>16

<211>34

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>16

tgccgactcg?aggctgctca?ccgtcactaa?agtg 34

Claims (1)

1. An artificially constructed TNF-α antagonist PTVH5, the amino acid sequence of which is shown in SEQ ID NO: 1.

Images