CN1781936A - B lymphocyte stimulus factor inhibiting peptide and its screening and preparing method - Google Patents

Abstract

The present invention discloses a kind of small peptide with the amino acid sequence of P-M-K-M-R-T-M or Y-E-P-K-I-R-G, and its preparation. The preparation process includes screening bacteriophage peptide library with B lymphocyte stimulus factor as target molecule to obtain B lymphocyte stimulus factor specifically combined small peptide and determining its sequence, and subsequent artificial synthesis of corresponding small peptide. The small peptide may be used in preparing medicine for treating B lymphocyte stimulus factor related diseases.

Description

Bone-marrow-derived lymphocyte stimulating factor inhibiting peptide and screening thereof, preparation method

Technical field

The present invention relates to a kind of little peptide and screening thereof, preparation method, the invention particularly relates to little peptide of a kind of bone-marrow-derived lymphocyte stimulating factor (BLyS) inhibition and screening thereof, preparation method.

Background technology

Systemic lupus erythematous (SLE), rheumatoid arthritis (RA) and sjogren syndrome (Sj gren ' s syndrom, SS) be that three kinds of morbiditys are higher, (with China is example to the autoimmune disorder of serious threat human health, the SLE morbidity is 0.07%, and the women is about 0.1%; The RA morbidity is 0.32%～0.36%; The SS morbidity is 0.3%～0.7%, but morbidity can be up to 3%～4% in elderly population).Up to now, mainly be to use anti-inflammatory drug and immunosuppressor about three kinds of treatment of diseases measures, the while is in conjunction with the general treatment of anti symptom treatment.Also can take surgical operation therapies such as synovectomy or joint replacement according to circumstances for RA.But generally speaking, still there is not specific short at present.Therefore, seek the specific treatment methods for several diseases is about focus in these disease researches and difficult point problem always.The discovery of BLyS and the research of effect thereof are that the solution of above-mentioned focus and difficult point problem has brought new hope.

BLyS is called BAFF (B cell activating factor belonging to the TNF family again, the B cell activation factor of TNF family), TALL-1 (TNF-and apoptosis ligand-related leukocyte expressedligand 1, the white corpuscle expression ligand 1 that TNF is relevant with apoptosis ligand), THANK (TNF homologue thatactivates apoptosis, NF-κ B and JNK, the TNF homologue of apoptosis-induced and activation NF-κ B and JNK), zTNF4 etc.This molecule was found and cloned in 1999, and it is a newcomer of TNF superfamily, and it belongs to II type transmembrane protein, and N holds in born of the same parents, and C holds outside born of the same parents.BLyS has film mating type and two kinds of forms of solubility, the biologic activity basically identical of the two.People BLyS total length is 285 amino acid, and wherein 47-73 amino acid is for striding the film district, and 74-285 amino acid is extracellular region, and 133-285 amino acid is the main region of its performance function.BLyS can exist with the solvable pieces of extracellular region, its biologic activity and total length embrane-associated protein basically identical.

BLyS mainly is expressed in marrow sample monocytic series, its not only wide participation regulate B cell development and differentiation and production of antibodies, and recent findings its also activation and answering of wide participation T cell.In addition, people's BLyS not only can activate human B cell, and can make the B cell activation of mouse.

The BLyS monomer is one to contain the structure of 9 conservative β-lamellas, and is the same with other many TNF superfamily members, and oligomerization forms homotrimer form show activity.BLyS is as a kind of ligandin, by bringing into play its biological action with its receptors bind.The BLyS acceptor of having found at present that is expressed on the B cytolemma has three at least, they are TACI (transmembrane activator and cyclophilin ligand interactor, stride membrane activator and ciclosporin ligand interaction thing), BCMA (B cell maturation antigen, B cell maturation antigen) and BR3 (BLyS receptor3, BLyS acceptor 3).And, only found TACI at present about being expressed in the BLyS acceptor on the T cytolemma, and whether also have the existence of other acceptor, still imperfectly understand.

In recent years many studies show that, BLyS crosses and plays a part uniqueness in the morbidity that is expressed in some autoimmune disorder.The evidence of having found at present mainly contains:

1. patient SLE, the sBLyS level obviously raises in the serum, and its titre with anti--double-stranded-DNA antibody is proportionate, and the BLyS in the serum can be at stimulated in vitro B cell activation.The BLyS transgenic mouse serious B cytosis can occur and typical SLE sample changes.

2. patient RA, the sBLyS level obviously raises in the serum, and is proportionate with patient RA factor titre.SBLyS level in patient's RA synovium of joint liquid also raises simultaneously, and its pathology damage with local joint is relevant.

3. SS (it is feature that this syndrome is destroyed with severe sialadenitis, salivation minimizing, submaxillary gland) patient, the sBLyS level obviously raises in the serum, and in the sialisterium of inflammation, the expression level of BLyS obviously raises.At the BLyS transgenic mouse, the SS sample can occur and change.

4. the interior experiment confirm of external and animal body, the soluble receptors of BLyS (Fc section common and antibody merges) both can suppress the activation of human B cell NF-κ B and the generation of immunoglobulin (Ig), T cell activation also capable of blocking.At the SLE model mouse, give the soluble receptors of BLyS after, animal proteinum urine degree is obviously alleviated, can prolong the survival time of animal simultaneously.At the RA model mouse, give behind the soluble receptors of BLyS inflammation is alleviated greatly, can obviously suppress the destruction and the advancing of disease of bone and cartilage simultaneously.

5. the functional site with sBLyS or sealing (or sterically hindered) film mating type BLyS during anti--BLyS antibody can pass through suppresses B cell activation and multiple production of antibodies.

Because SLE, RA and SS etc. are to relate to B, the overactive autoimmune disorder of T lymphocyte simultaneously, and BLyS has participated in the lymphocytic excessive activation of B, T simultaneously, thereby manage to block the biologic activity of BLyS, then can make two kinds of lymphocytic activation be subjected to press down, provide new (and even specific) method and medicine thereby might and recur prevention for above-mentioned various autoimmune treatment of diseases.

As previously mentioned, confirm that through external or experimentation on animals such strategy is effective.The preparation of blocking-up BLyS biological action mainly contains two classes at present, i.e. the soluble receptors of BLyS (Fc section common and antibody merges) and anti--BLyS antibody.In addition, can only be in conjunction with the BLyS acceptor can not activated lymphocyte the BLyS mutant also might be used as the competitive inhibitor (just still lacking strong experimental evidence at present) of BLyS.The mankind, anti--BLyS antibody enters preclinical test in calendar year 2001, and in order to the relevant autoimmune disorder of treatment BLyS hyperfunction, it is clinical to have entered the second phase at present.It is bigger that yet the BLyS that above this three class is possible suppresses molecule, prepare relative inconvenience with purifying, even when using side effect might appear, as the soluble receptors that merges with antibody Fc section, because the existence of Fc section, might cause unnecessary biological effect, thereby the functioning efficiency of preparation is reduced.For anti--BLyS antibody, when being used for human body, need to use humanized antibody, otherwise will cause the anti-heterogenetic antibody reaction of people, even anaphylaxis.Yet the preparation expense height of corresponding humanized antibody, difficulty are big.Therefore, if can make its preparation and purifying convenient simultaneously the molecule miniaturization of BLyS inhibitor, the practicality of corresponding preparations in medical research and clinical practice be improved greatly.

Phage display techniques was at first invented by Smith in 1985, the imagination of phage random peptide library was proposed to make up by Parmley and Smith in 1988, then also successfully screened phage peptide library in nineteen ninety, thereby indicated the birth of phage peptide library technology by three research groups' structures.The ultimate principle of phage peptide library is that the dna sequence dna of peptide section is at random inserted the genome of people to the phage capsid protein, the DNA that inserts is after phage expression, be illustrated on the phage capsid protein with linear peptides, peptide section and phage capsid protein form fusion rotein and are presented in phage surface, and utilize the characteristics of phagocytosis physical efficiency massive duplication, obtain a plurality of copies of different recombinant phages, and do not influence infecting and the amplification ability of phage.This technology makes genotype be combined dexterously with phenotype, thereby provides favourable instrument for different research.

Interaction between the biomacromolecule comes down to the interaction between functional site, does not need the contact fully of whole molecule, and the interaction between interaction between BLyS and its acceptor and BLyS and anti--BLyS antibody is also all unexceptional.Therefore, as long as can find on acceptor molecule or the blocking antibody molecule and BLyS bonded peptide sequence (or simulating peptide sequence, be that function is same or similar, but amino acid is formed different peptide sequences), just can utilize corresponding peptides (or simulating peptide) to come competitiveness or closure to suppress BLyS and combine, and then reach inhibition BLyS activated b, the lymphocytic purpose of T with acceptor on the lymphocyte film.

In phage random peptide library, exactly comprised huge amount, amino acid form or sequence on discrepant little peptide mutually, these little peptides are presented in phage surface, they can serve as or simulate the functional site of biomacromolecule interphase interaction on sequence.The advantage of phage random peptide library technology is that its screening flux is big, easy and simple to handle, both can screen true peptide sequence or the simulating peptide sequence of representing the protein function site from the storehouse, also can screen the peptide sequence of simulation nonprotein functional site.

What commercial phage random peptide library was the most frequently used at present is the phage library of being developed on M13 phage basis by U.S. New England Biolabs company (being called for short NEB), the M13 phage is a kind of lysogenic phage, it only infects and not cracking host bacterium, finally discharge with the form of secretory protein, the host bacterium keeps its original activity, and constantly secretes the phage of amplification.Be long filament shape structure under the M13 phage Electronic Speculum, be about 1 μ m, diameter 600-700nm, its genome are a strand cyclic DNA, are made up of 6407 Nucleotide, the 10 kinds of albumen of encoding altogether, and wherein 5 kinds is the structural protein of virion.NEB company inserts the external source fragment to express foreign protein by molecular biological means in the gene of the less important capsid protein p3 of M13 phage, be presented in the proteic N end of phage P3 behind the exogenous gene expression of insertion.The product of NEB company comprises 7 peptide storehouses, and three kinds of libraries, 7 peptide storehouses are encircled in 12 peptide storehouses, and wherein the front belongs to non-conformation type peptide library for two kinds, and the third is a conformation type peptide library.The difference in two class peptide storehouses mainly is: conformation type peptide library is that the nucleotide sequence with two halfcystines is added on the two ends of peptide dna sequence dna at random, thereby disulfide linkage can combine into ring makes the peptide section that presents form the space structure with functionally active, and then the interaction of simulation natural molecule; But not the peptide Duan Zewei that conformation restricted type peptide storehouse is presented is linear, does not then make the peptide at random that is presented form disulfide linkage.Based on the fundamental principle of peptide storehouse screening, conformation type peptide library is selected in our research for use, and this will help filtering out the little peptide that has high-affinity with target molecule more.

Studies show that phage presents the performance of the little peptide effect of type, related to the amino-acid residue in the phage capsid protein that aim sequence closes on sometimes.Therefore, the corresponding free little peptide that phage presents little peptide of type and synthetic can not fit like a glove on function sometimes, so must carry out Function Identification once more to the free little peptide of synthetic.In addition, phage presents the little peptide of type because of the existence of phage composition is arranged, and can not be used for that is to say in body research, has only the phage that is screened is presented the little peptide of type to carry out just having real development and application value behind the synthetic.

Based on above existing situation, weak point for the inhibitor that overcomes present BLyS, if can screen conformation type peptide library with BLyS by phage random peptide library technology, artificial synthesis peptide's technology and immunologic function test, screening, evaluation and synthetic aforementioned little peptide with inhibition people BLyS function, to lay the foundation for the new inhibitor of further researching and developing, thereby the treatment of the relevant autoimmune disorder of bone-marrow-derived lymphocyte is had suitable help at BLyS.

Summary of the invention

The object of the present invention is to provide a kind of little peptide molecule: P-M-K-M-R-T-M, PRO-MET-LYS-MET-ARG-THR-MET with following aminoacid sequence; Or Y-E-P-K-I-R-G, TYR-GLU-PRO-LYS-ILE-ARG-GLY.

A kind of little peptide molecule provided by the present invention has high-affinity with combining of BlyS, can and suppress its activity with the BLyS specific combination, also can antagonism reorganization BLyS peptide section stimulate lymphopoietic activity simultaneously.

Another object of the present invention is to provide a kind of preparation method of little peptide molecule of the present invention, adopt the synthetic little peptide molecule of the present invention of Fmoc solid-phase synthesis.

A further object of the present invention is to provide a kind of screening method of bone-marrow-derived lymphocyte stimulating factor inhibiting peptide.

For realizing purpose of the present invention, a kind of the present invention is provided the preparation method of little peptide molecule, may further comprise the steps:

1. use Fmoc (9-fluorenylmethyloxycarbonyl) group that amino acid whose alpha-amino group is protected, by a support arm it is attached on the insoluble macromolecule resin upholder carrier, use the piperidines base with the alpha-amino group deprotection subsequently, remove the protectiveness of Fmoc, washing amino acid-support arm-resin;

2. the amino acid of activatory alpha-amino group protection in advance is connected to amino acid-support arm-resin by coupled reaction, repeats deprotection, washing, coupling connection then, up to obtaining the purpose peptide;

3. at last with peptide-support arm-resin cracking, behind resin cutting and deprotection, synthetic little peptide is carried out purifying.

The preparation method of the little peptide of tool P-M-K-M-R-T-M sequence of the present invention is characterized in that, may further comprise the steps:

1. with the 9-fluorenylmethyloxycarbonyl group alpha-amino group of proline(Pro) is protected, by a support arm it is attached on the insoluble macromolecule resin upholder carrier, use the piperidines base with the alpha-amino group deprotection subsequently, remove the protectiveness of Fmoc, wash amino acid-support arm-resin with dichloromethane solution;

2. the methionine(Met) of activatory alpha-amino group protection in advance is connected to amino acid-support arm-resin by coupled reaction, carries out deprotection, washing then;

3. repeat 2., Methionin, methionine(Met), arginine, Threonine and the methionine(Met) of the protection of activatory alpha-amino group in advance carried out the coupling connection successively, up to the little peptide that obtains tool P-M-K-M-R-T-M sequence;

With peptide-support arm-resin cracking, behind resin cutting and deprotection, synthetic little peptide is carried out purifying at last.Little peptide of the present invention also can prepare with other synthetic method.

The preparation method of the little peptide of tool Y-E-P-K-I-R-G sequence of the present invention is characterized in that, may further comprise the steps:

1. with the 9-fluorenylmethyloxycarbonyl group alpha-amino group of tyrosine is protected, by a support arm it is attached on the insoluble macromolecule resin upholder carrier, use the piperidines base with the alpha-amino group deprotection subsequently, remove the protectiveness of Fmoc, wash amino acid-support arm-resin with dichloromethane solution;

2. the L-glutamic acid of activatory alpha-amino group protection in advance is connected to amino acid-support arm-resin by coupled reaction, carries out deprotection, washing then;

3. repeat 2., proline(Pro), Methionin, Isoleucine, arginine and the glycine of the protection of activatory alpha-amino group in advance carried out the coupling connection successively, up to the little peptide that obtains tool Y-E-P-K-I-R-G sequence;

With peptide-support arm-resin cracking, behind resin cutting and deprotection, synthetic little peptide is carried out purifying at last.

Behind the synthetic little peptide of the present invention it is suppressed lymphopoietic detection and show that they have high-affinity with combining of BlyS, and can antagonism reorganization BLyS peptide section (rhBLyS _112-285) stimulate lymphopoietic activity.

The present invention provides a kind of screening method of bone-marrow-derived lymphocyte stimulating factor inhibiting peptide simultaneously, may further comprise the steps:

1. with the bone-marrow-derived lymphocyte stimulating factor, promptly BLyS is hatched with phage peptide library, then wash-out specific combination phage and amplification;

2. repeat aforesaid operations and carry out 2-5 affine screening, obtain a phage set higher with BLyS avidity;

3. the dna sequence dna of determining the specific combination phage promptly gets this inhibiting peptide.

Preferably, with human soluble BLyS extracellular region 112 to 285 amino acid fragment, the i.e. rhBLyS that recombinate _112-285, screening people phage conformation type 7 peptide storehouses.

Preferably, the screening method of a kind of bone-marrow-derived lymphocyte stimulating factor inhibiting peptide of the present invention may further comprise the steps:

1. with human soluble BLyS extracellular region 112 to 285 amino acid fragment, the i.e. rhBLyS that recombinate _112-285, be added in culture dish or other containers and cultivate, after washing, add people phage conformation type 7 peptide storehouses and hatch;

2. in TBS/Tween20 washing, HCl-Gly and bovine serum albumin damping fluid dissociate wash-out and Tris-HCl neutralization buffer and after, from culture dish or other containers, collect the specific combination phage, be used for the screening of next round after the amplification, use and carry out 2-5 altogether with quadrat method and take turns affine screening;

3. the mono-clonal phage that screening is obtained is increased, and derives the peptide binding sequence of BLyS by the dna sequencing result, and after it is suppressed lymphopoietic situation and detect, can determine this inhibiting peptide.

Wherein, when washing, use the TBS/Tween20 of 1-5mL/L to wash 6-10 time, the concentration of Tween20 is 5mL/L in second takes turns and subsequent screen.

Wherein, be the HCl-Gly elution buffer of 2.2 the 0.2mol/L 5-10min that at room temperature dissociates during wash-out with the pH value; With 140-160 μ L pH value is that the 1mol/L Tris-HCl neutralization buffer of 8.9-9.3 neutralizes.

Preferred, preparation method of the present invention may further comprise the steps:

1. with the rhBLyS of 100-200mg/L _112-285Be added in culture dish or other containers and cultivate, after the TBS/Tween20 of 1-5mL/L washing 7-8 time, add 8-12 μ L people phage conformation type 7 peptide storehouses, in room temperature jog absorption 30-60min;

2. wash-out specific combination phage, after the TBS/Tween20 of 1-5mL/L washing 6-10 time, add 1mL pH value and be 2.2 0.2mol/L HCl-Gly elution buffer and the bovine serum albumin of 1g/L, the room temperature 5-10min that dissociates, add the 1mol/L Tris-HCl neutralization buffer of 140-160 μ L pH value again for 8.9-9.3, neutralization is also collected, and repeats 3 and takes turns affine screening;

3. the mono-clonal phage that screening is obtained is increased, and derives the peptide binding sequence of BLyS by the dna sequencing result, and after it is suppressed lymphocytic propagation and detect, can determine this inhibiting peptide.

Most preferred, preparation method of the present invention may further comprise the steps:

1. with the rhBLyS of 1.5ml 100-200mg/L _112-285Be added in overall plastic culture dish or other polystyrene container, 4 ℃ are spent the night, and abandon protein liquid, add 5g/L bovine serum albumin room temperature sealing 1-2h.

After the TBS/Tween20 of 1-5mL/L washing 6-10 time, add 8-12 μ L people phage conformation type 7 peptide storehouses, wherein contain 2 * 10 ¹¹The phage virus particle is in room temperature jog absorption 30-60min.

2. wash-out specific combination phage, after the TBS/Tween20 of 1-5mL/L washing 6-10 time, add 1mL pH value and be 2.2 0.2mol/L HCl-Gly elution buffer and the BSA of 1g/L, the room temperature 5-10min that dissociates, add the 1mol/L Tris-HCl neutralization buffer of 140-160 μ L pH value again for 8.9-9.3, neutralization is also collected, and repeats 3 and takes turns affine screening.

3. from resulting phage set, isolate one phage mono-clonal, and to its with BlyS combine that activity is carried out ELISA experiment, competition suppresses experimental identification, observe positive phage clones and suppress BLyS and lymphocyte bonded situation, promote the positive bacteriophage of lymphocyte proliferation activity to carry out dna sequencing to suppressing BlyS, and analyze the contrast sequence.

The retarding effect of little peptide of the present invention when combined action is better than separately effect separately, this may be because BLyS combination and activated b, T cell relate to its a plurality of functional sites, the a plurality of different functional sites of combined utilization seal little peptide in conjunction with little peptide or functional site, can strengthen the function that little peptide suppresses BLyS.

The acquisition of this antagonism peptide is that the research and development of little peptide type BLyS inhibitor are laid a good foundation, prove by experimentation on animals, activity test in vitro and clinical trial, the inhibiting peptide that this little peptide can be used as BLyS is used to prepare the medicine of treatment BLyS relative disease, thereby can be used for therapy system lupus erythematosus (SLE), rheumatoid arthritis (RA) and sjogren syndrome (Sj gren ' s syndrom, the relevant disease of BLyS such as SS) can be used as the candidate new molecule of control BlyS relative disease.

The little peptide of BLyS inhibition of the present invention possesses following advantage:

1. little peptide of the present invention can be blocked the biologic activity of BLyS, thereby can make two kinds of lymphocytic activation be subjected to press down, be applied to clinically can competitive suppress combining of acceptor on BLyS and the lymphocyte (or closure), reach and suppress BLyS activated b, the lymphocytic purpose of T, thereby created condition at the little peptide type inhibitor of BLyS for further researching and developing.

Because SLE, RA and SS etc. are to relate to B, the overactive autoimmune disorder of T lymphocyte simultaneously, and BLyS has participated in the lymphocytic excessive activation of B, T simultaneously, thereby little peptide of the present invention is prevented and treated the new formulation of various autoimmune diseases such as BLyS hyperfunction relevant SLE, RA and SS candidate target is provided for development, can be above-mentioned various autoimmune treatment of diseases and recurring prevention provides new (and even specific) method and medicine.

2. three classes of little peptide of the present invention and existing blocking-up BLyS biological action may preparation (soluble receptors of BLyS, anti--BLyS antibody and only the BLyS mutant of activated lymphocyte not in conjunction with the BLyS acceptor) compares: easy in conjunction with the synthetic and the purifying of little peptide by the BLyS that screening phage conformation type peptide library obtains; accomplish scale production easily, when synthetic, can add some characteristic that alpha-non-natural amino acid improved and improved little peptide simultaneously.All can only use 20 kinds of natural amino acids because be used to produce the protokaryon and the eukaryotic cell of first three class preparation, can't accomplish so this is first three class preparation.

3. little peptide molecule of the present invention is little, and penetrating power is strong, arrives whole body each tissue and organ easily; Little peptide is few accumulating of kidney simultaneously, is difficult for causing toxic side effect, uses comparatively safe.The immunogenicity of resulting free little peptide is extremely low, generally can not cause the immunne response of anti-little peptide.

Description of drawings

Fig. 1 is YG-clone 60-DNA sequencer map

Fig. 2 is PM-clone 67-DNA sequencer map

The positive phage clone of Fig. 3 combines active ELISA and identifies with BLyS

The competition of the positive phage clone of Fig. 4 suppresses ELISA and identifies

The positive phage of Fig. 5 is to the restraining effect of BLyS propagation lymphocyte function

Fig. 6 is the restraining effect of the little peptide of synthetic to BLyS propagation lymphocyte function

Embodiment

Embodiment 1

One. with BLyS is target molecule screening phage conformation type peptide library

1, the affine screening of phage peptide library

1. with the rhBLyS of 1.5ml 100mg/L _112-285Be added in the overall plastic culture dish, 4 ℃ are spent the night.Abandon protein liquid, add 5g/LBSA (bovine serum albumin) room temperature sealing 1h.

2. after the TBS/Tween20 of 1mL/L washing 8 times, add 10 μ L people phage conformation types, 7 peptide storehouses and (contain 2 * 10 ¹¹Virion), people phage conformation type 7 peptide storehouses comprise host bacterium ER2738, available from NewEngland Biolabs company, in room temperature jog absorption 55min.

Wherein the prescription of TBS damping fluid is Tris-HCl 50mmol/L, NaCl150mM/L.

3. after the TBS/Tween20 of 1mL/L washing 7 times, add the 1mLpH value and be 2.2 0.2mol/LHCl-Gly (glycine) elution buffer and the BSA of 1g/L, the room temperature 7min that dissociates adds 150 μ LpH values and is 9.1 1mol/L Tris-HCl neutralization buffer again, and neutralization is also collected.

Tris wherein is tris (hydroxymethyl) aminomethane Tutofusin tris.

4. collect phage and measure titre, be used for the screening of next round after the amplification.

2, the amplification of mono-clonal phage

1. with the 3rd take turns the screening the phage of being obtained by different extent of dilution bed boards (LB/IPTG/X-Gal agar plate), join the ER2738 bacterium (the special host bacterium of the phage library that this screening is selected) of logarithmic phase less than picking locus coeruleus on 100 the flat board from clone's number, cultivate 4-5h with the 200-300r/min jolting.

2. the centrifuging and taking supernatant adds the PEG8000/NaCl precipitation and spends the night, the centrifugal supernatant redeposition of abandoning, and (Phosphate-buffered saline, a kind of phosphate buffered saline buffer contain NaCl 8g among every 1000ml PBS, KCl0.2g, Na to be dissolved in PBS ₂HPO ₄7H ₂O0.5g, KH ₂PO ₄0.2g, pH7.4) in, measure the titre of each mono-clonal phage.

3. in order to obtain and rhBLyS _112-285Has high-affinity bonded phage, with rhBLyS _112-285Be target molecule, encircle in the 7 peptide storehouses in phage and carry out affine screening, carry out 3 altogether and take turns affine screening.Add greatly 3mL/L in second concentration of taking turns in the screening Tween20 by 1mL/L, washing times increases to 10 times.After screening, the rate of recovery (referring to take turns in the affine screening at each phage clone that elutes and the ratio that drops into the phage clone number that screens) is from 8.5 * 10 ^-4Be increased to 2.1 * 10 ^-2, illustrate that phage obtains effective enrichment (table 1).

Table 1BLyS high-affinity is in conjunction with the enrichment of phage

Round	Drop into phage	The output phage	Rate of recovery %
				1	2×10 11	1.7×10 6	8.5×10 -4
2	1.8×10 11	7.6×10 7	4.2×10 -3
				3	1.2×10 12	2.5×10 10	2.1×10 -2

Two. identify the binding peptide of BLyS

The phage of reclaiming in the affine screening all is with screening aglucon mortise and can tolerates the clone of rinse cycle.Therefore has certain specificity and avidity.In the affine screening, even, can not get rid of the existence of non-specific binding phage clone through multi-turns screen, thereby the further single clone of picking, determine that it and the specific adsorption of screening target molecule are the experiment necessary procedure.ELISA (enzyme linked immunosorbent assay enzyme-linked immunosorbent assay) can semiquantitative determination binding constant between screening target molecule and phage clone, be method comparatively commonly used.Adopt bovine serum albumin in contrast, avoided because the phage consumption is excessive and the washing insufficient strength the false positive that may cause.

1. the ELISA of positive colony identifies

With rhBLyS _112-285(20mg/L) join in the 96 hole enzyme plates, every hole 200 μ L spend the night in 4 ℃ in wet box.Abandon protein liquid next day, every hole adds 200 μ L20g/L skim-milk room temperatures sealing 1h.

The mono-clonal phage supernatant of amplification (is contained 1 * 10 approximately ¹²Virion) respectively with isopyknic 20g/L skim-milk mixing, behind the incubated at room 30min, add in each hole of enzyme plate (every hole 100 μ L), in room temperature jog absorption 1h.

After PBST washes 5 times, the anti-M13 antibody (antibody of anti-M13 phage that adds HRP (horseradish peroxidase) mark, HRP enzyme mark mouse anti M13 monoclonal antibody, available from Pharmacia company) (100 μ L/ hole) incubated at room 1h, after washing 6 times with PBST, add O-Phenylene Diamine (OPD) (100 μ L/ hole) colour developing, read A with microplate reader (the Austrian Tecan Sunrise of company microplate reader) _492nmValue (numerical value when absorbancy is 492nm).Each mono-clonal phage all with PBS bag by control wells, establish simultaneously affine screening for the first time not with rhBLyS _112-285The bonded phage contrasts as negative phage, and all the other operations are identical.

Take turns the flat board of screening phage from the 3rd, 80 phage clones of picking, amplification back bed board is measured its titre.Identify each phage mono-clonal and rhBLyS with ELISA _112-285In conjunction with active, by control wells, (negative control phage NP) be the unconjugated negative phage of screening for the first time to the negative control phage to each mono-clonal phage with not protein-contg coating buffer bag.The result shows, 15 clones can with rhBLyS _112-285Produce the high-affinity combination, and do not have association reaction (A with not protein-contg coating buffer _492nmFig. 3 is seen in value P/N 〉=2).

2. the competitive ELISA of phage clone is identified

Bag is by rhBLyS _112-285And sealing (method is the same).

With rhBLyS _112-28550mg/L (contains 1 * 10 approximately with each positive bacteriophage of having identified ¹²Virion) equal-volume mixes, and hatches 1h for 37 ℃, adds (every hole 100 μ L) in 96 each hole of hole enzyme plate, in room temperature jog absorption 1h.

After PBST washes 5 times, add the anti-M13 antibody (100 μ L/ hole) of HRP mark, incubated at room 1-2h.

Add OPD (100 μ L/ hole) colour developing, read A with microplate reader _492nmValue.Control wells adds not and rhBLyS _112-285The phage particle of the equal amts of hatching, all the other operations are all identical.

With rhBLyS _112-285Competition suppresses phage and wraps by rhBLyS _112-285Combination, judge phage and rhBLyS _112-285Bonded specificity, control wells add not and rhBLyS _112-285Bonded phage in advance.The result shows that 15 ELISA identify among the male clone, with rhBLyS _112-285In advance in conjunction with can block 12 clones with the bag by rhBLyS _112-285Combination (with A _492nmThe positive phage in value P/N 〉=2 sees Fig. 4).

3. the order-checking of positive bacteriophage

The screening gained positive phage clones that will increase precipitates with PEG8000/NaCl, extracts phage single-chain DNA, after agarose gel electrophoresis is identified, delivers Shanghai biotechnology company limited and checks order.After 12 positive phage clones that screening is obtained carry out sequential analysis, find 3 groups of sequences, wherein one group has 7 identical clones, and another group has 3 identical clones, and last group has 2 identical clones.3 acceptors of retrieval BLyS are not all found and above-mentioned 3 groups of homologous sequences in the GenBank database.

The phage clone that screening is obtained extracts single stranded DNA, and M13 phage single-chain DNA extraction agent box is available from Shanghai China Shun biotechnology company limited.What carry out the dna sequencing use is dideoxy sequencing method, is finished by Shanghai bio-engineering corporation.

Three. phage clone suppresses lymphopoietic detection

Separate human peripheral lymphocytes with lymphocyte separation medium, lymphocyte is made into 10 with RPMI1640 nutrient solution (be used for the lymphocytic a kind of liquid of vitro culture, purchase company) in GIBCO ⁵The suspension of/L adds in the 96 hole circle floor cells culture plates, in 37 ℃, 50mL/L CO by every hole 200 μ L cell suspensions ₂Cultivate under the condition.

With 5 μ g/ μ L rhBLyS _112-285(be respectively 0,10 with three different numbers of phage clone ¹⁰, 10 ¹¹, 10 ¹²And 10 ¹³Individual virion) phage hatches 1-2h for 37 ℃ after mixing, and joins in the Tissue Culture Plate, establishes two multiple holes, continues to cultivate 14h for every group.

Add MTT (tetrazolium bromide by 20 μ L/ holes then, MTT can act on viable cell, the black and blue color product that generates is directly proportional with the extent of metabolism of cell, be used for analysis of cells hyperplasia level) solution (5g/L), the centrifugal nutrient solution of abandoning behind the 8h, DMSO (the inferior maple of dimethyl) the vibration 15-20min that every hole adds 150 μ L measures photoabsorption (A) value with microplate reader in wavelength 570nm.Control group is detect to confirm not and rhBLyS through ELISA _112-285The negative phage of bonded is established simultaneously and singly adds phage or groups of cells, with RP MI1640 nutrient solution as blank.

The rhBLyS that adds _112-285When not combining in advance, the lymphopoietic activity of stimulation is arranged all with phage clone.The result shows that the irrelevant phage of contrast is to rhBLyS _112-285The activity that stimulates cellular proliferation does not have obvious influence.In 3 clones, clone 1 couple of rhBLyS _112-285The activity that stimulates cellular proliferation does not have obvious influence; Clone 2, clone 3 are along with adding increasing of phage particle number, to rhBLyS _112-285The active restraining effect that stimulates cellular proliferation strengthens (see figure 5).Add separately the phage particle group then with cell independence growth group no significant difference (result slightly), illustrate that phage particle can suppress rhBLyS _112-285Stimulate lymphopoietic activity.

Wherein can suppress BlyS and promote that lymphopoietic clone's 2 sequences are P-M-K-M-R-T-M, cloning 3 sequences is Y-E-P-K-I-R-G, with reference to figure 1.YG-clone's 60-DNA sequencer map and Fig. 2 .PM-clone 67-DNA sequencer map.

Four. the little peptide of synthetic and it is suppressed lymphocytic propagation detect

1, the little peptide of synthetic

The aminoacid sequence that two inhibited phages present the little peptide of type is transferred to Shanghai Bo Ya biotech company; adopt the Fmoc solid-phase synthesis, synthetic corresponding free little peptide on peptide synthesizer is behind resin cutting and deprotection; synthetic little peptide is carried out purifying, measure its concentration then.

2, the little peptide of synthetic is to suppressing lymphopoietic detection

Separate human peripheral lymphocytes with lymphocyte separation medium, lymphocyte is made into 10 with the RPMI1640 nutrient solution ⁵The suspension of/L adds in the 96 hole circle floor cells culture plates, in 37 ℃, 50mL/L CO by every hole 200 μ L cell suspensions ₂Cultivate under the condition.

With 5 μ g/ μ L rhBLyS _112-285After little peptide different concns mixes, hatch 1h for 37 ℃, join in the Tissue Culture Plate, establish three multiple holes, continue to cultivate 14h for every group.

Add MTT solution (5g/L) by 20 μ L/ holes then, the centrifugal nutrient solution of abandoning behind the 8h, the DMSO vibration 15min that every hole adds 150 μ L measures photoabsorption (A) value with microplate reader in wavelength 570nm.With the solution that does not dissolve little peptide is contrast, establish simultaneously singly to add little polypeptide cell group, with the RPMI1640 nutrient solution as blank.

Two little peptides of synthetic are the above purity of HPLC95%, called after P-A and P-B.The rhBLyS that adds _112-285When not combining in advance, the lymphopoietic activity of stimulation is arranged all with little peptide.The result shows that P-A is along with adding increasing of concentration, to rhBLyS _112-285The active restraining effect that stimulates cellular proliferation strengthens; P-B is to rhBLyS _112-285The activity inhibition that stimulates cellular proliferation relatively a little less than; When P-A and P-B add fashionable to rhBLyS simultaneously _112-285The activity inhibition that stimulates cellular proliferation is higher than P-A and P-B adds (see figure 6) separately.Add separately little peptide group then with cell independence growth group no significant difference, illustrate that little peptide is playing a role under the interactional prerequisite earlier with the BlyS fragment, if do not act in advance lymphocyte is not seen obvious influence (result slightly), little peptide and rhBLyS are described with BlyS _112-285In advance in conjunction with having suppressed rhBLyS _112-285Stimulate lymphopoietic activity.

Embodiment 2

One. with BLyS is target molecule screening phage conformation type peptide library

1, the affine screening of phage peptide library

1. with the rhBLyS of 120mg/L _112-285Be added in the polystyrene container, 4 ℃ are spent the night.Abandon protein liquid, add 5g/LBSA (bovine serum albumin) room temperature sealing 1h.

2. after the TBS/Tween20 of 1mL/L washing 9 times, add 10 μ L people phage conformation types, 7 peptide storehouses and (contain 2 * 10 ¹¹Virion), people phage conformation type 7 peptide storehouses comprise host bacterium ER2738, available from NewEngland Biolabs company, in room temperature jog absorption 60min.

Wherein the prescription of TBS damping fluid is Tris-HCl 50mmol/L, NaCl150mM/L.

3. after the TBS/Tween20 of 1mL/L washing 6 times, add the 1mLpH value and be 2.2 0.2mol/LHCl-Gly (glycine) elution buffer and the BSA of 1g/L, the room temperature 8min that dissociates adds 150 μ LpH values and is 9.1 1mol/L Tris-HCl neutralization buffer again, and neutralization is also collected.

Tris wherein is tris (hydroxymethyl) aminomethane Tutofusin tris.

4. collect phage and measure titre, be used for the screening of next round after the amplification.

Repeat above step and carry out 5 altogether and take turns affine screening, the concentration of taking turns in back 4 in the screening Tween20 adds greatly 3mL/L by 1mL/L.Washing times increases to 10 times.

Remaining operation is with embodiment 1.

Embodiment 3

One. with BLyS is target molecule screening phage conformation type peptide library

1, the affine screening of phage peptide library

1. with the rhBLyS of 150mg/L _112-285Be added in the polystyrene container, 4 ℃ are spent the night.Abandon protein liquid, add 5g/LBSA (bovine serum albumin) room temperature sealing 1h.

2. after the TBS/Tween of 1mL/L 20 washings 10 times, add 10 μ L people phage conformation types, 7 peptide storehouses and (contain 2 * 10 ¹¹Virion), people phage conformation type 7 peptide storehouses comprise host bacterium ER2738, available from NewEngland Biolabs company, in room temperature jog absorption 50min.

Wherein the prescription of TBS damping fluid is Tris-HCl 50mmol/L, NaCl150mM/L.

3. after the TBS/Tween of 1mL/L 20 washings 9 times, add the 1mLpH value and be 2.2 0.2mol/LHCl-Gly (glycine) elution buffer and the BSA of 1g/L, the room temperature 6min that dissociates adds 150 μ LpH values and is 9.1 1mol/L Tris-HCl neutralization buffer again, and neutralization is also collected.

Tris wherein is tris (hydroxymethyl) aminomethane Tutofusin tris.

4. collect phage and measure titre, be used for the screening of next round after the amplification.

Repeat above step and carry out 3 altogether and take turns affine screening, the concentration with Tween20 in the two-wheeled screening of back adds greatly 4mL/L by 1mL/L.Washing times increases to 10 times.

Remaining operation is with embodiment 1.

Embodiment 4

One. with BLyS is target molecule screening phage conformation type peptide library

1, the affine screening of phage peptide library

1. with the rhBLyS of 200mg/L _112-285Be added in the polystyrene container, 4 ℃ are spent the night.Abandon protein liquid, add 5g/LBSA (bovine serum albumin) room temperature sealing 2h.

2. after the TBS/Tween20 of 1mL/L washing 7 times, add 10 μ L people phage conformation types, 7 peptide storehouses and (contain 2 * 10 ¹¹Virion), people phage conformation type 7 peptide storehouses comprise host bacterium ER2738, available from NewEngland Biolabs company, in room temperature jog absorption 30min.

Wherein the prescription of TBS damping fluid is Tris-HCl 50mmol/L, NaCl150mM/L.

3. after the TBS/Tween20 of 1mL/L washing 10 times, add 1mL pH value and be 2.2 0.2mol/LHCl-Gly (glycine) elution buffer and the BSA of 1g/L, the room temperature 5min that dissociates adds 150 μ L pH values and is 9.1 1mol/L Tris-HCl neutralization buffer again, and neutralization is also collected.

Tris wherein is tris (hydroxymethyl) aminomethane Tutofusin tris.

4. collect phage and measure titre, be used for the screening of next round after the amplification.

Repeat above step and carry out 4 altogether and take turns affine screening, the concentration of taking turns in back 3 in the screening Tween20 adds greatly 5mL/L by 1mL/L.Washing times increases to 10 times.

Remaining operation is with embodiment 1.

Embodiment 5

One. with BLyS is target molecule screening phage conformation type peptide library

The affine screening of l, phage peptide library

1. with the rhBLyS of 180mg/L _112-285Be added in the polystyrene container, 4 ℃ are spent the night.Abandon protein liquid, add 5g/LBSA (bovine serum albumin) room temperature sealing 2h.

2. after the TBS/Tween of 1mL/L 20 washings 6 times, add 10 μ L people phage conformation types, 7 peptide storehouses and (contain 2 * 10 ¹¹Virion), people phage conformation type 7 peptide storehouses comprise host bacterium ER2738, available from NewEngland Biolabs company, in room temperature jog absorption 40min.

Wherein the prescription of TBS damping fluid is Tris-HCl 50mmol/L, NaCl 150mM/L.

3. after the TBS/Tween20 of 1mL/L washing 8 times, add the 1mLpH value and be 2.2 0.2mol/LHCl-Gly (glycine) elution buffer and the BSA of 1g/L, the room temperature 10min that dissociates adds 150 μ LpH values and is 9.1 1mol/L Tris-HCl neutralization buffer again, and neutralization is also collected.

Tris wherein is tris (hydroxymethyl) aminomethane Tutofusin tris.

4. collect phage and measure titre, be used for the screening of next round after the amplification.

Repeat above step and carry out 5 altogether and take turns affine screening, the concentration of taking turns in back 4 in the screening Tween 20 adds greatly 5mL/L by 1mL/L.Washing times increases to 10 times.

Remaining operation is with embodiment 1.

Claims (10)

1. a little peptide molecule is characterized in that, has following aminoacid sequence: P-M-K-M-R-T-M or Y-E-P-K-I-R-G.

2. the preparation method of the described a kind of little peptide molecule of claim 1 is characterized in that, may further comprise the steps:

1. with the 9-fluorenylmethyloxycarbonyl group amino acid whose alpha-amino group is protected, be connected on the insoluble macromolecule resin upholder carrier by a support arm, use the piperidines base with the alpha-amino group deprotection subsequently, remove the protectiveness of Fmoc, washing amino acid-support arm-resin;

2. the amino acid of activatory alpha-amino group protection in advance is connected to amino acid-support arm-resin by coupled reaction, repeats deprotection, washing, coupling connection then, up to obtaining the purpose peptide;

With peptide-support arm-resin cracking, behind resin cutting and deprotection, synthetic little peptide is carried out purifying at last.

3. the preparation method of a kind of little peptide molecule according to claim 2 is characterized in that, may further comprise the steps:

1. with the 9-fluorenylmethyloxycarbonyl group alpha-amino group of proline(Pro) is protected, by a support arm it is attached on the insoluble macromolecule resin upholder carrier, use the piperidines base with the alpha-amino group deprotection subsequently, remove the protectiveness of Fmoc, wash amino acid-support arm-resin with dichloromethane solution;

2. the methionine(Met) of activatory alpha-amino group protection in advance is connected to amino acid-support arm-resin by coupled reaction, carries out deprotection, washing then;

3. repeat 2., Methionin, methionine(Met), arginine, Threonine and the methionine(Met) of the protection of activatory alpha-amino group in advance carried out the coupling connection successively, up to the little peptide that obtains tool P-M-K-M-R-T-M sequence;

With peptide-support arm-resin cracking, behind resin cutting and deprotection, synthetic little peptide is carried out purifying at last.

4. the preparation method of a kind of little peptide molecule according to claim 2 is characterized in that, may further comprise the steps:

1. with the 9-fluorenylmethyloxycarbonyl group alpha-amino group of tyrosine is protected, by a support arm it is attached on the insoluble macromolecule resin upholder carrier, use the piperidines base with the alpha-amino group deprotection subsequently, remove the protectiveness of Fmoc, wash amino acid-support arm-resin with dichloromethane solution;

2. the L-glutamic acid of activatory alpha-amino group protection in advance is connected to amino acid-support arm-resin by coupled reaction, carries out deprotection, washing then;

3. repeat 2., proline(Pro), Methionin, Isoleucine, arginine and the glycine of the protection of activatory alpha-amino group in advance carried out the coupling connection successively, up to the little peptide that obtains tool Y-E-P-K-I-R-G sequence;

With peptide-support arm-resin cracking, behind resin cutting and deprotection, synthetic little peptide is carried out purifying at last.

5. the screening method of a bone-marrow-derived lymphocyte stimulating factor inhibiting peptide is characterized in that, may further comprise the steps:

1. with the bone-marrow-derived lymphocyte stimulating factor, promptly BLyS is hatched with phage peptide library, then wash-out specific combination phage and amplification;

2. repeat aforesaid operations and carry out 2-5 affine screening, obtain a phage set higher with BLyS avidity;

3. the dna sequence dna of determining the specific combination phage promptly gets this inhibiting peptide.

6. the screening method of a kind of bone-marrow-derived lymphocyte stimulating factor inhibiting peptide according to claim 5 is characterized in that, with human soluble BLyS extracellular region 112 to 285 amino acid fragment, the i.e. rhBLyS of reorganization _112-285, screening people phage conformation type 7 peptide storehouses.

7. the screening method of a kind of bone-marrow-derived lymphocyte stimulating factor inhibiting peptide according to claim 5 is characterized in that, may further comprise the steps:

1. with human soluble BLyS extracellular region 112 to 285 amino acid fragment, the i.e. rhBLyS that recombinate _112-285, be added in culture dish or other containers and cultivate, after washing, add people phage conformation type 7 peptide storehouses and hatch;

2. through TBS/Tween 20 washings, HCl-Gly and bovine serum albumin buffer solution elution dissociate and the Tris-HCl neutralization buffer in and after, from culture dish or other containers, collect the specific combination phage, be used for the screening of next round after the amplification, use and carry out 2-5 altogether with quadrat method and take turns affine screening;

3. the mono-clonal phage that screening is obtained is increased, and derives the peptide binding sequence of BLyS by the dna sequencing result, and after it is suppressed lymphocytic propagation and detect, can determine this inhibiting peptide.

8. the screening method of a kind of bone-marrow-derived lymphocyte stimulating factor inhibiting peptide according to claim 5 is characterized in that, when washing, uses the TBS/Tween20 of 1-5mL/L to wash 6-10 time, and the concentration of Tween 20 is 5mL/L in second takes turns and subsequent screen.

9. the screening method of a kind of bone-marrow-derived lymphocyte stimulating factor inhibiting peptide according to claim 5 is characterized in that, is the HCl-Gly elution buffer of 2.2 the 0.2mol/L 5-10min that at room temperature dissociates with the pH value during wash-out; With 140-160 μ L pH value is that the 1mol/L Tris-HCl neutralization buffer of 8.9-9.3 neutralizes.

10. according to the screening method of any one described a kind of bone-marrow-derived lymphocyte stimulating factor inhibiting peptide of claim 5-9, it is characterized in that, may further comprise the steps:

1. with the rhBLyS of 100-200mg/L _112-285Be added in culture dish or other containers and cultivate, after the TBS/Tween of 1-5mL/L 20 washings 7-8 time, add 8-12 μ L people phage conformation type 7 peptide storehouses, in room temperature jog absorption 30-60min;

2. wash-out specific combination phage, after the TBS/Tween of 1-5mL/L 20 washings 6-10 time, add 1mL pH value and be 2.2 0.2mol/L HCl-Gly elution buffer and the bovine serum albumin of 1g/L, the room temperature 5-10min that dissociates, add the 1mol/L Tris-HCl neutralization buffer of 140-160 μ L pH value again for 8.9-9.3, neutralization is also collected, and repeats 3 and takes turns affine screening;

3. the mono-clonal phage that screening is obtained is increased, and derives the peptide binding sequence of BLyS by the dna sequencing result, and after it is suppressed lymphocytic propagation and detect, can determine this inhibiting peptide.

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