CN100376690C - Screening method for human gastric cancer drug-resistant cell specific combination and drug-resistant reverse short peptide and sequence - Google Patents
Screening method for human gastric cancer drug-resistant cell specific combination and drug-resistant reverse short peptide and sequence Download PDFInfo
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Abstract
The present invention belongs to the field of biomedical technology, which specifically relates to a screening method for human gastric cancer drug-resistant cell specific combination and drug-resistant reverse short peptide, and a sequence. An in vitro susceptibility test of a random bacteriophage peptide library MTT is used for sieving to obtain cloned bacteriophage polypeptide which can reverse the drug resistance of the drug-resistant cells of gastric cancer, the survival rate and the IC50 value of the drug-resistant cells of gastric cancer can be reduced under the action of medicines for chemotherapeutics, and statistic difference cloning (p<0.05) is provided. Bacteriophage polypeptide is used for screening the ligands of all specific expression molecules on drug-resistant tumor cell membranes as well as screening the whole cells, the polypeptide which has bioactivity and can reverse the drug-resistant properties of the drug-resistant tumor cells can be directly obtained by screening, and simultaneously, a screening method for human gastric cancer drug-resistant polypharmacy cell specific combination and drug-resistant reverse short peptide, and a sequence (GMBPs) are provided.
Description
Technical field
The invention belongs to field of biomedicine technology, be specifically related to people's resistant Gastric Cancer specific combination and drug resistance inversion small peptide screening method and sequence.
Background technology
Cancer of the stomach is that China causes the malignant tumour that death toll is maximum, and chemotherapy is one of main means of current treatment cancer of the stomach, but its result of treatment does not still have major progress, and its major cause is exactly that stomach cancer cell produces multidrug resistance to chemotherapeutics.The multidrug resistance of tumour cell (multi-drug resistance, MDR) be meant that tumour cell produces resistance to a certain chemotherapeutics after, other chemical structure and the different chemotherapeutics of mechanism are also produced cross resistance.
Present studies show that, the generation of tumor multidrug-resistance is main relevant with following mechanism: 1. the ingestion of medicines that occurs in the cytolemma level reduces and effluxes and increases, and causes that the absolute concentration of medicine in the cell reduces, as the resistance that is caused by P-glycoprotein; 2. occur in the change of the medicine ubcellular distribution of tenuigenin and organoid level, make medicine cause that medicine effective concentration reduces near its action target spot; 3. drug target is expressed reduction in the change of quality and quantity as topoisomerase II, and having weakened with this enzyme is the cytotoxicity of the medicine of target spot; 4. cell detoxification system function is strengthened, and makes the rapid deactivation of medicine, as the resistance relevant with Thiadiazolidine isomerase (GST) etc.Mostly these mechanism are to concur, and wherein the too much expression of some membranin is the major cause that produces multidrug resistance on the tumour cell.The membranin of finding relevant with multidrug resistance has three kinds at present: P-glycoprotein (P-glycoprotein, P-gp), multidrug resistance albumen (multidrug resistance protein, MRP), mammary cancer resistance associated protein (Breastcancer resistance protein, BCRP), these three kinds of membranins are being exercised the function of " molecular pump " on tumor cell membrane, be about to chemotherapeutics " pump " in cell and go out, make tumour cell avoid the (NatureBiotechnology.2000 that kills and wounds of chemotherapeutics; 18 Suppl:IT18-20).
People adopt monoclonal antibody and single-chain antibody (scFv) to carry out the judgement of tumor drug resistance degree and prognosis at known resistance associated molecule on the tumor cell membrane at present, and the target of tumor drug resistance cell and reversing drug resistance treatment, (Proc.Natl.Acad.Sci.USA 1992 as anti-P-gp monoclonal antibody MRK-16; 89:5824-5829; JControl Release 2001; 77 (1-2): 77-86) can reversing tumor resistance, and anti-FAS (Urology 2001; 57 (5): 993-998) monoclonal antibody then can obviously improve chemotherapy drug concentrations in the tumour cell.But monoclonal antibody, single-chain antibody (scFv) and biotechnology antibody etc.But monoclonal antibody has the defective that is difficult to overcome, and it is more weak etc. to lack functional effect that the C district relies on and penetrativity more by force, relatively as immunogenicity.Though it is less that ScFv has molecular weight, a little less than the immunogenicity, seepage force is strong, and can be used for drug targeting, advantage such as neutralize a toxin, its no Antibody C region, other biological effect that can not mediate antibody.Monoclonal antibody that screens at present and single-chain antibody (scFv) treatment is just at known film surface resistance molecule, as P-gp, MRP, BCRP etc., and the molecule that is used for screening corresponding antibodies and monoclonal antibody is artificial the expression or the molecule of synthesizing and purifying, and the molecule under its conformation and functional epitope feature and the cell surface physiological status also has certain difference.And for expressing and the surface of cell membrane antigen of purification difficult is difficult to carry out the screening of associated ligands.
The effect of drug resistance inversion treatment at present is also undesirable, and its one of the main reasons is that also a lot of molecules relevant with resistance are still undiscovered, comprising a lot of films surface resistance molecule.We know at the drug-resistant tumor surface of cell membrane and exist the membrane molecule express spectra different with the susceptibility tumour cell, perhaps some membrane molecules change in the conformation of mdr cell, the change of these membrane molecules all is that tumour cell continues under the pressure of existence at chemotherapeutics, for the effect of antagonism chemotherapeutics pair cell by abduction delivering, the membrane molecule of these changes and the resistance of tumour cell take place exist necessary relation.Therefore seek can specificity in conjunction with and change the part of these resistance related film molecular biology functions, for the treatment of reversing drug resistance and the research of cancer of the stomach resistance associated molecule important reality and theory significance are arranged all.And the strategy that screens the reversing drug resistance part at present can only be the resistance molecule at the known membrane surface, can't directly find the part of unknown film surface resistance molecule.
In view of the deficiency that exists in present searching drug-resistant tumor cell-targeting and the reversal therapies molecule, we adopt the tumor drug resistance cell of the complete work of phage random peptide library screening, can screen with physiological status under surface of cell membrane molecule bonded polypeptide, can find the part of unknown resistance associated molecule.The peptide molecule molecular weight is littler, and penetrativity is strong, does not have antigenicity, but the characteristics of biologically active are more suitable for using in vivo.
Present the complete cell of peptide storehouse screening with phage random, the tumour cell complicated and changeable for cell surface molecule especially is fit to (Curr Opin Chem Biol.2000 Feb; 4 (1): 16-21).The screening of phage random peptide library viable cell has a lot of advantages, does not at first require the acceptor that pre-determines purpose; Except screening bonded acceptor, can also screen the acceptor of internalization.The phage that Johnston and its colleague has obtained to express 20 peptides in this way not only can with the fibroblast combination, and can internalization enter cell (Nat Genet 2001 Jan; 27 (1): 6-8).Application phage peptide libraries such as Hartley O successfully sieve with the CCR5 specificity bonded polypeptide that is expressed in CHO-CCR5 cell and HEK-CCR5 cell surface (J Virol.2003 Jun; 77 (12): 6637-44).Known technology utilizes and has screened in the phage random peptide library body and tumor vascular endothelial cell specificity bonded small peptide, the patent application of application number 200510042810.4, utilize the external screening gastric cancer vascular endotheliocyte in-vitro culture model of subduing of phage random peptide library, obtained and gastric cancer vascular endothelial cell specific bonded small peptide.This show with viable cell as the screening target can sieve can with surface of cell membrane molecular specific bonded polypeptide aglucon.But the influential polypeptide of cellular function is not screened in just simple screening of 200510042810.4 patent and cell-specific bonded polypeptide targetedly.And added screening in this research method for screening at cell function, be can directly screen than its advantage to the influential polypeptide of drug resistance of tumor cell function.
Summary of the invention
The object of the present invention is to provide a kind of people's resistant Gastric Cancer specific combination and drug resistance inversion small peptide screening method and sequence, it utilizes phage polypeptide to screen the part that full cell can screen all specific expressed molecules on the tumor drug resistance cytolemma simultaneously, and directly obtain bioactive polypeptide that can reversing tumor mdr cell resistant characterization by functional screening, a kind of people's Multidrug Resistance of Gastric Cancer cell-specific combination is provided simultaneously and has reversing drug resistance characteristic, bioactive short peptide and sequence thereof (GMBPs).
Technical scheme of the present invention is: people's resistant Gastric Cancer specific combination and drug resistance inversion small peptide screening method and sequence are provided, it is characterized in that: it is by the external drug sensitive test screening of phage random peptide library associating MTT, obtain can reversing stomach mdr cell resistant characterization the phage clone polypeptide, it can reduce survival rate and the IC50 value of resistant Gastric Cancer under the chemotherapeutics effect, and the clone (p＜0.05) of significant difference is arranged.
Described people's resistant Gastric Cancer specific combination and drug resistance inversion short peptide sequence are selected one of following sequence:
GMBP1： E T A P L S T M L S P Y；
GMBP2： T T F S P P S P L S S I；
GMBP3： F N N H Y P A A A T Y P；
GMBP4： E V F W P L N A P R L L；
GMBP5： S W Q I P Y P I S P R S；
GMBP6： K T A P T P Y A L V H D；
GMBP7： S N F M H N T R I W S H；
GMBP8： S W Q I T Y P I S P R S；
GMBP9： A W T W V L P S S I R A；
GMBP10：T D S Y H V A S A R Q P；
GMBP11：S T Y S H P L S L R P D；
GMBP12：Y T T W P F T S L Q L D；
GMBP13：A F M E T T S Q N A W L；
GMBP14：E T A P P T P Y S V M F；
GMBP15：T T F N P L Y L R L D T；
GMBP16：S S F P Q V I P L D Y L；
GMBP17：A P K Y S L S D L Y L N；
GMBP18：Q I E K I S Q H L D M H；
GMBP19：T W N Q P Y I P P L Y P；
GMBP20：Y A S P P N P S L R L T；
GMBP21：Y H G L T P V R Y V S V，
GMBP22：Y T F M P E L T T P R T；
GMBP23：N S F N Y A P L L M P R；
GMBP24：T S P Y H L L H A H L Q；
GMBP25：S S F V A L S I S P S M；
GMBP26：A N L S S H S S P G D S；
GMBP27：T T L Q F T G Q T N K T；
GMBP28：T P P P R D A S L S R W；
GMBP29：H N I G T W G P K S H L；
GMBP30：S G F F E P Q H S H P L；
GMBP31：Q I E E S F V R G H T T；
GMBP32：S P Q T D G L V S T P S；
GMBP33：Y T F D P Q I R P A G L；
GMBP34：Y E R S I L P F S H V F；
GMBP35：Y P S V T F P T Q T L L；
GMBP36：A S T N V F A R P M Y L；
GMBP37：S P W Y M T P S P N T A；
GMBP38：H I S V I N Y T T K I S；
GMBP39：M N V T V S G R L S G P；
GMBP40：T I P Y P F S L L N N P；
GMBP41：G P F L Y S Q V A W R S；
GMBP42：T A L P N H W S D A S P；
GMBP43：S V S V G M K P S P R P：
GMBP44：Y Q E E T P A S S F S R；
GMBP45：N S S Q L A P Y T T H R；
GMBP46：T L H P S V L S Y V L K：
GMBP47：H N G L P N F F Q T R L；
GMBP48：E A A P N F Y P P L T F；
GMBP49：D L F Q F A F P L N T I；
GMBP50：F T F S Y A E S V S Y F。
Proline(Pro) characteristic consensus sequence pattern that they are made up of 12 amino acid respectively, all have Threonine---(1-3) amino acid---, its aminoacid sequence such as one of following
GMBP1： E T A P L S T M L S P Y；
GMBP2： T T F S P P S P L S S I；
GMBP3： F N N H Y P A A A T Y P；
GMBP6： K T A P T P Y A L V H D；
GMBP8： S W Q I T Y P I S P R S；
GMBP9： A W T W V L P S S I R A；
GMBP11：S T Y S H P L S L R P D；
GMBP12：Y T T W P F T S L Q L D；
GMBP14：E T A P P T P Y S V M F；
GMBP15：T T F N P L Y L R L D T；
GMBP19：T W N Q P Y I P P L Y P；
GMBP22：Y T F M P E L T T P R T；
GMBP24：T S P Y H L L H A H L Q；
GMBP28：T P P P R D A S L S R W；
GMBP29：H N I G T W G P K S H L；
GMBP33：Y T F D P Q I R P A G L；
GMBP35：Y P S V T F P T Q T L L；
GMBP37： SP W Y M T P S P N T A；
GMBP40： TI P Y P F S L L N N P；
GMBP42：T A L P N H W S D A S P；
GMBP46： T L H P S V L S Y V L K。
Proline(Pro) characteristic consensus sequence pattern that they are made up of 12 amino acid respectively, all have Serine---(0-4) amino acid---, its aminoacid sequence such as one of following
GMBP5： S W Q I P Y P I S P R S；
GMBP10：T D S Y H V A S A R Q P；
GMBP16：S S F P Q V I P L D Y L；
GMBP20： Y A S P P N P S L R L T；
GMBP23： NS F N Y A P L L M P R；
GMBP25：S S F V A L S I S P S M；
GMBP26：A N L S S H S S P G D S；
GMBP30： S G F F E P Q H S H P L；
GMBP32： S P Q T D G L V S T P S；
GMBP34：Y E R S I L P F S H V F；
GMBP39：M N V T V S G R L S G P；
GMBP43：S V S V G M K P S P R P
GMBP45：N S S Q L A P Y T T H R。
They are made up of 12 amino acid respectively, all have phenylalanine---(0-2) amino acid---proline(Pro) and proline(Pro)---(0-2) amino acid---leucine characteristic consensus sequence pattern, its aminoacid sequence such as one of following
GMBP4： E V F W P L N A P R L L；
GMBP36： A S T N V F A R P M Y L；
GMBP48：E A A P N F Y P P L T F；
GMBP49：D L F Q F A F P L N T I。
Serine characteristic consensus sequence pattern that they are made up of 12 amino acid respectively, all have Threonine---(0-3) amino acid---, its aminoacid sequence such as one of following
GMBP7： S N F M H N T R I W S H；
GMBP13：A F M E T T S Q N A W L；
GMBP38：H I S V I N Y T T K I S；
GMBP50：F T F S Y A E S V S Y F。
They are made up of 12 amino acid respectively, all have leucine---(4) amino acid---glutamine---Threonine characteristic consensus sequence pattern, its aminoacid sequence such as one of following
GMBP27：T T L Q F T G Q T N K T；
GMBP47：H N G L P N F F Q T R L。
They are made up of 12 amino acid respectively,---Isoleucine---L-glutamic acid characteristic consensus sequence pattern that all has glutamine, its aminoacid sequence such as one of following
GMBP18：Q I E K I S Q H L D M H；
GMBP31：Q I E E S F V R G H T T。
Serine characteristic consensus sequence pattern that they are made up of 12 amino acid respectively, all have proline(Pro)---(1-4) amino acid---, its aminoacid sequence such as one of following
GMBP17： A P K Y S L S D L Y L N；
GMBP21： Y H G L T P V R Y V S V；
GMBP41： G P F L Y S Q V A W R S；
GMBP44：Y Q E E T P A S S F S R。
The external full cell of described (1) phage peptide library is subdued screening
Screening process is with normal gastric epithelial cell GES of people and the negative subtractive cell line of cancer of the stomach susceptibility cell SGC7901, hatch altogether with original peptide storehouse, get unconjugated phage again and resistant Gastric Cancer (SGC7901/ADR, SGC7901/VCR) hatch altogether, recovery is in conjunction with phage, the next round screening is dropped in the amplification back, and 4 take turns back picking phage mono-clonal carries out determined dna sequence;
(2) in-vitro screening step
GES cell 1 * 10 with commercially available EDTA cellular segregation liquid results logarithmic phase
7, and get original peptide storehouse 10 μ l (2 * 10
11Pfu) mix, hatch 1.5h for 4 ℃, 800rpm, centrifugal 5min collects supernatant, with same method the supernatant of SGC7901 and recovery is hatched altogether, and is centrifugal again, collects supernatant; With EDTA results SGC7901/ADR or SGC7901/VCR 1 * 10
7, hatch altogether with above-mentioned recovery supernatant, centrifugal, abandon supernatant, centrifugal again behind the DL 3min with resuspended SGC7901/ADR of BF or SGC7901/VCR, repeat to wash 7 times, 0.2%PBST washes 1 time, and PBS washes 2 times; 4 ℃ of wash-out 10min of glycine elution buffer with pH2.2, the Tris/HCl neutralization buffer that adds pH9.1, centrifugal, remove supernatant, again with 4 ℃ of wash-out 10min of glycine elution buffer of pH2.2, the Tris/HCl neutralization buffer that adds pH9.1 is collected supernatant, is membrane elution phage or film in conjunction with phage; To contain 4 ℃ of cracking wash-outs of TEA alkali elutriant 5min of PMSF, add the Tris/HCl neutralization buffer of pH7.4, centrifugal, collect supernatant, be cracking wash-out bacteriophage or internalization phage (AN or VN);
(3) amplification in vitro of phage
Shop system LB-Tet flat board, and add X-gal/IPTG, put upside down cultivation after, above, the well-grown blue plaque 400-500 of picking spacing 0.5cm at random, be divided into 5 parts and add the host bacterium respectively, 37 ℃, 250rpm shaking culture 4-6h; With 4 ℃ of cultures, the centrifugal 15min of 10000rpm, the PEG/NaCl solution of supernatant and 1/6 volume spends the night for 4 ℃; Centrifugal again, the resuspended precipitation of TBS, little centrifugal, the PEG/NaCl solution ice bath 1h of supernatant and 1/6 volume is centrifugal, abandon supernatant, with 200 μ l 0.02%NaN
3The resuspended throw out of solution is the phage of first round amplification;
(4) next round screening
Membrane elution phage and cracking wash-out bacteriophage after the amplification are respectively got 2 * 10
11Pfu drops into next round and screens respectively; Screening process is the same, carries out 4 altogether and takes turns screening, and increase proof strength gradually: the time of hatching with negative cells increases to 2h, is kept to 30mins with the incubation time of target cell; BF, PBST, PBS DL washing time increase to 5min, and PBST concentration is incremented to 0.2%, 0.3%, 0.5%;
(5) positive phage clones selects and the external drug sensitive test screening of MTT
Take turns screening through 4, the phage of reclaiming from resistant Gastric Cancer significantly increases, shop system LB flat board, be less than on 100 the flat board at random totally 200 of picking phage mono-clonals at clone's number, AN wherein, each 50 of AM, VN, each 50 of VM are numbered AN1-AN15, AN41-AN435, AM1-AM25, AM41-AM425, VN1-VN50, VN1-VN50; With AN2 is that example utilizes the external drug sensitivity assay of MTT to carry out the detection of phage to the influence of resistant Gastric Cancer resistant characterization; Gather in the crops the logarithmic growth SGC7901/ADR cell in mid-term, according to every hole 8 * 10
3Individual cell/200 μ L inoculate into 96 orifice plates, place overnight incubation in the cell culture incubator; Screening detects AN2-AN10 through MTT, AN41-AN43, AN45-AN418, AN420-AN425, AN428-AN430, AM1-AM13, AM15-AM23, AM41-AM45, AM47-AM413, AM415-AM425, VN1-VN14, VN17-VN21, VN23-VN30, VN34-VN42, VN45-VN50, VM1-VM7, VM9, VM11-VM14, VM16, VM17, VM19-VM30, these clones of VM35-VM47 can make resistant Gastric Cancer survival rate in the environment of 4 μ g/ml Zorubicins (ADR) descend, and compare with the resistant Gastric Cancer that does not add phage clone and add the irrelevant phage (the amplification liquid of original peptide storehouse Ph.D.-12) of Isodose that there were significant differences, and statistical significance (p＜0.05) is arranged;
(6) order-checking of positive phage clones
Adopt single stranded DNA to extract test kit and carry out the extraction of phage single-chain DNA, to be template with the single stranded DNA, adopt the sequencing primer that is provided in the phage random peptide library test kit then ,-96gIII, 5 '-CCC TCATAG TTA GCG TAACG-3 ' order-checking; According to the dna sequence dna that order-checking draws, derive and present the aminoacid sequence of peptide at random.
Characteristics of the present invention: people adopt monoclonal antibody and single-chain antibody (scFv) to carry out the judgement of tumor drug resistance degree and prognosis at known resistance associated molecule on the tumor cell membrane at present, and the target of tumor drug resistance cell and reversing drug resistance treatment, (Proc.Natl.Acad.Sci.USA 1992 as anti-P-gp monoclonal antibody MRK-16; 89:5824-5829; J Control Release 2001; 77 (1-2): 77-86) energy reversing tumor resistance, and anti-FAS (Urology2001; 57 (5): 993-998) monoclonal antibody then can obviously improve chemotherapy drug concentrations in the tumour cell.But monoclonal antibody, single-chain antibody (scFv) and biotechnology antibody etc.Monoclonal antibody has the defective that is difficult to overcome, and it is more weak etc. to lack functional effect that the C district relies on and penetrativity more by force, relatively as immunogenicity.Though it is less that ScFv has molecular weight, a little less than the immunogenicity, seepage force is strong, and can be used for drug targeting, advantage such as neutralize a toxin, its no Antibody C region, other biological effect that can not mediate antibody.
Monoclonal antibody that screens at present and single-chain antibody (scFv) treatment is just at known film surface resistance molecule, as P-gp, MRP, BCRP etc., and the molecule that is used for screening corresponding antibodies and monoclonal antibody is artificial the expression or the molecule of synthesizing and purifying, and the molecule under its conformation and functional epitope feature and the cell surface physiological status also has certain difference.And for expressing and the surface of cell membrane antigen of purification difficult is difficult to carry out the screening of associated ligands.The effect of drug resistance inversion treatment is also undesirable, and its one of the main reasons is that also a lot of molecules relevant with resistance are still undiscovered, comprising a lot of films surface resistance molecule.We know at the drug-resistant tumor surface of cell membrane and exist the membrane molecule express spectra different with the susceptibility tumour cell, perhaps some membrane molecules change in the conformation of mdr cell, the change of these membrane molecules all is that tumour cell continues under the pressure of existence at chemotherapeutics, for the effect of antagonism chemotherapeutics pair cell by abduction delivering, the membrane molecule of these changes and the resistance of tumour cell take place exist necessary relation.Therefore seek can specificity in conjunction with and change the part of these resistance related film molecular biology functions, for the treatment of reversing drug resistance and the research of cancer of the stomach resistance associated molecule important reality and theory significance are arranged all.And the strategy that screens the reversing drug resistance part at present can only be the resistance molecule at the known membrane surface, can't directly find the part of unknown film surface resistance molecule.
In view of the deficiency that exists in present searching drug-resistant tumor cell-targeting and the reversal therapies molecule, the present invention adopts the tumor drug resistance cell of the complete work of phage random peptide library screening, can screen with physiological status under surface of cell membrane molecule bonded polypeptide, find the part of unknown resistance associated molecule.The peptide molecule molecular weight is littler, and penetrativity is strong, does not have antigenicity, but the characteristics of biologically active are more suitable for using in vivo.Present the complete cell of peptide storehouse screening with phage random, the tumour cell complicated and changeable for cell surface molecule especially is fit to (Curr OpinChem Biol.2000 Feb; 4 (1): 16-21).The screening of phage random peptide library viable cell has a lot of advantages, does not at first require the acceptor that pre-determines purpose; Except screening bonded acceptor, can also screen the acceptor of internalization.The phage that Johnston and its colleague has obtained to express 20 peptides in this way not only can with the fibroblast combination, and can internalization enter cell (Nat Genet 2001 Jan; 27 (1): 6-8).Application phage peptide libraries such as Hartley O successfully sieve with the CCR5 specificity bonded polypeptide that is expressed in CHO-CCR5 cell and HEK-CCR5 cell surface (J Virol.2003 Jun; 77 (12): 6637-44).Utilize in the prior art and screened in the phage random peptide library body and tumor vascular endothelial cell specificity bonded small peptide, the patent application of application number 200510042810.4, utilize the external screening gastric cancer vascular endotheliocyte in-vitro culture model of subduing of phage random peptide library, obtained and gastric cancer vascular endothelial cell specific bonded small peptide.This show with viable cell as the screening target can sieve can with surface of cell membrane molecular specific bonded polypeptide aglucon.
But utilize phage polypeptide to screen full cell at present, the influential polypeptide of cellular function is not screened in just simple screening and cell-specific bonded polypeptide targetedly.The present invention then utilizes the biological sieve technology of washing in a pan of phage peptide library, sets about from the function of reversing drug resistance earlier, in conjunction with the external drug sensitive test screening scheme of MTT, screens the bioactive polypeptide that has of energy reversing tumor mdr cell resistant characterization targetedly.The present invention utilizes phage polypeptide to screen full cell, it can screen the part of all specific expressed molecules on the tumor drug resistance cytolemma simultaneously, directly obtaining bioactive polypeptide that can reversing tumor mdr cell resistant characterization by functional screening simultaneously, is a kind of method of seeking reversing tumor mdr cell resistant characterization polypeptide efficiently.
Description of drawings
The present invention is described in further detail below in conjunction with the accompanying drawing table.
Fig. 1 phage peptide library in-vitro screening schema;
Fig. 2 four-wheel screening pnagus medius enrichment diagram gradually on the Multidrug Resistance of Gastric Cancer cell;
The ELISA qualification result diagram of 7 phage clones of Figure 31 and SGC7901/ADR binding specificity;
The ELISA qualification result of Figure 47 phage clone and SGC7901/VCR binding specificity;
The external viable cell of Fig. 5 AN2 illustrates in conjunction with experimental result;
Fig. 6 GMBP1 combines competition and suppresses experiment with the AN2 cell;
The immunofluorescence of Fig. 7 AN2 phage and SGC7901/ADR binding specificity detects;
The synthetic peptide of Fig. 8 GMBP1 detects for the immunofluorescence of AN2 phage and the blocking-up of cancer of the stomach SGC7901/ADR cell binding specificity;
Table 4VCR acts on and is added with polypeptide GMBP42, to the cells survival rate (%) and the IC50 value of the SGC7901/VCR cell strain of rondom polypeptide and PBS;
Drug sensitivity test in the table 5 polypeptide GMBP42 nude mouse.
Embodiment
Resistant Gastric Cancer surface molecular marker is one of crucial target of Multidrug Resistance of Gastric Cancer reversal therapies, by the external full cell screening of phage random peptide library, the specificity combination and the reversing drug resistance small peptide GMBPs of lineup's resistant Gastric Cancer have successfully been obtained in conjunction with the external drug sensitive experiment Function Identification of MTT, and utilize the phage cell in conjunction with experiment, immunocyte/histochemical stain, cell suppresses experiment in conjunction with competition, methods such as immunofluorescence detection have been identified the binding specificity of GMBPs, further adopt the external drug sensitive experiment of polypeptide MTT, drug sensitivity assay has been determined the reversing drug resistance characteristic of GMBPs in the nude mouse, and concrete grammar is as follows:
1. technical scheme
1.1. the external full cell of phage peptide library is subdued screening
1.1.1. screening process
With normal gastric epithelial cell GES of people and the negative subtractive cell line of cancer of the stomach susceptibility cell SGC7901; hatch (company in New England Biolabs is purchased in original peptide storehouse) altogether with original peptide storehouse; get unconjugated phage again and resistant Gastric Cancer (SGC7901/ADR; SGC7901/VCR) hatch altogether; recovery is in conjunction with phage; the next round screening is dropped in the amplification back, and 4 take turns back picking phage mono-clonal carries out determined dna sequence (Shanghai Hua Nuo company) and homology analysis (see figure 1).
1.1.2. in-vitro screening step
GES cell 1 * 10 with EDTA (commercially available) cellular segregation liquid results logarithmic phase
7, and get original peptide storehouse 10 μ l (2 * 10
11Pfu) mix, hatch 1.5h for 4 ℃, 800rpm, centrifugal 5min collects supernatant, with same method the supernatant of SGC7901 and recovery is hatched altogether, and is centrifugal again, collects supernatant.With EDTA results SGC7901/ADR or SGC7901/VCR 1 * 10
7, hatch altogether with above-mentioned recovery supernatant, centrifugal, abandon supernatant, centrifugal again behind the DL 3min with resuspended SGC7901/ADR of BF or SGC7901/VCR, repeat to wash 7 times, 0.2%PBST washes 1 time, and PBS washes 2 times.4 ℃ of wash-out 10min of glycine elution buffer with pH2.2, the Tris/HCl neutralization buffer that adds pH9.1, centrifugal, remove supernatant, again with 4 ℃ of wash-out 10min of glycine elution buffer of pH2.2, the Tris/HCl neutralization buffer that adds pH9.1 is collected supernatant, is membrane elution phage or film in conjunction with phage (AM or VM).To contain 4 ℃ of cracking wash-outs of TEA alkali elutriant 5min of PMSF, add the Tris/HCl neutralization buffer of pH7.4, centrifugal, collect supernatant, be cracking wash-out bacteriophage or internalization phage (AN or VN).
1.1.3. the amplification in vitro of phage
Shop system LB-Tet flat board, and add X-gal/IPTG (purchase give birth in Shanghai worker), put upside down cultivation after, above, the well-grown blue plaque 400-500 of picking spacing 0.5cm at random, be divided into 5 parts and add the host bacterium respectively, 37 ℃, 250rpm shaking culture 4-6h.With 4 ℃ of cultures, the centrifugal 15min of 10000rpm, the PEG/NaCl solution of supernatant and 1/6 volume spends the night for 4 ℃.Centrifugal again, the resuspended precipitation of TBS, little centrifugal, the PEG/NaCl solution ice bath 1h of supernatant and 1/6 volume is centrifugal, abandon supernatant, with 200 μ l 0.02%NaN
3The resuspended throw out of solution is the phage of first round amplification.
1.1.4. next round screening
Membrane elution phage and cracking wash-out bacteriophage after the amplification are respectively got 2 * 10
11Pfu drops into next round and screens respectively.Screening process is the same, carries out 4 altogether and takes turns screening, and increase proof strength gradually: the time of hatching with negative cells increases to 2h, is kept to 30mins with the incubation time of target cell; BF, PBST, PBS DL washing time increase to 5min, and PBST concentration is incremented to 0.2%, 0.3%, 0.5%.
1.1.5. selecting and the external drug sensitive test screening of MTT of positive phage clones
Take turns screening through 4, the phage of reclaiming from resistant Gastric Cancer significantly increases, apparently higher than the control cells (see figure 2).Shop system LB flat board is less than on 100 the flat board at random totally 200 of picking phage mono-clonals at clone's number, AN wherein, and each 50 of AM, VN, each 50 of VM are numbered AN1-AN15, AN41-AN435, AM1-AM25, AM41-AM425, VN1-VN50, VN1-VN50.With AN2 is that example utilizes the external drug sensitivity assay of MTT to carry out the detection of phage to the influence of resistant Gastric Cancer resistant characterization.
Gather in the crops the logarithmic growth SGC7901/ADR cell in mid-term, according to every hole 8 * 10
3Individual cell/200 μ L inoculate into 96 orifice plates, place overnight incubation in the cell culture incubator.Be divided into 3 groups, each group is established 4 multiple holes, and every hole adds AN2 phage 1 * 10 respectively
10Pfu, with the irrelevant phage (the amplification liquid of original peptide storehouse Ph.D.-12) of same dosage and PBS in contrast, after 2 hours, every group of Zorubicin (ADR) that adds 4 μ g/ml concentration again continues to cultivate 3 days.Every hole adds fresh preparation MTT solution (5g/L) 20 μ L, and 37 ℃ are continued to cultivate 4 hours; The careful suction abandoned culture supernatant in the hole, and every hole adds the DMSO of 150 μ L, and vibration 10min fully dissolves crystallisate; Wavelength 490nm, on full-automatic microplate reader instrument, survey each hole absorbance value, get the mean number of per 4 repeating hole A values, calculate the survival rate of cell: cell survival rate=(experimental group A value-blank group A value)/(negative control group A value-blank group A value) * 100%; Nosa statistical software with statistics teaching and research room of The Fourth Military Medical University carries out test of significance.The result shows that AN2 can reverse the resistant characterization of SGC7901/ADR, and nothing to do with phage and PBS compare the survival rate that makes resistant Gastric Cancer and descend, and (p＜0.05) has statistical significance.
Screening detects AN2-AN10, AN41-AN43, AN45-AN418, AN420-AN425 through MTT, AN428-AN430, AM1-AM13, AM15-AM23, AM41-AM45, AM47-AM413, AM415-AM425, VN1-VN14, VN17-VN21, VN23-VN30, VN34-VN42, VN45-VN50, VM1-VM7, VM9, VM11-VM14, VM16, VM17, VM19-VM30, these clones of VM35-VM47 can make the survival rate of resistant Gastric Cancer descend, and statistical significance (p＜0.05) is arranged.
1.1.6. the order-checking of positive phage clones
Positive phage clones to the MTT experiment sieving checks order.The single stranded DNA that adopts Hua Shun company to produce extracts test kit and carries out the extraction of phage single-chain DNA, operate by process specifications: in containing the LB nutrient solution of single phage clone, add precipitated liquid, obtain the phage particle precipitation behind the high speed centrifugation, add lysate lytic phage coat protein, through resin purification, wash-out obtains phage single-chain DNA.With the single stranded DNA is template, and the sequencing primer that is provided in the phage random peptide library test kit is provided ,-96gIII, 5 '-CCC TCATAG TTA GCG TAA CG-3 ' delivers to the order-checking of Shanghai Hua Nuo company limited.Remove AM20, AM23, AM421, AM424, VN34, VN39, VN48, VM40, the failure of VM44 cloning and sequencing, because a part of cloned sequence repeats, and finally obtains 50 different phage clones (seeing Table 1), and it is presented small peptide called after GMBPs (Gastric cancer MDR cell Binding Peptides).According to the dna sequence dna that order-checking draws, derive and present the aminoacid sequence of peptide at random, with AN2 example.Dna sequencing is reported as:
CCTATGATTGACCGTCTGCGCCTCGTTCCGGCTAAGTAACATGGAGCAGGTCGC
GGATTTCGACACAATTTATCAGGCGATGATACAAATCTCCGTTGTACTTTGTTTC
GCGCTTGGTATAATCGCTGGGGGTCAAAGATGAGTGTTTTAGTGTATTCTTTTG
CCTCTTTCGTTTTAGGTTGGTGCCTTCGTAGTGGCATTACGTATTTTACCCGTTT
AATGGAAACTTCCTCATGAAAAAGTCTTTAGTCCTCAAAGCCTCTGTAGCCGTT
GCTACCCTCGTTCCGATGCTGTCTTTCGCTGCTGAGGGTGACGATCCCGCAAA
AGCGGCCTTTAACTCCCTGCAAGCCTCAGCGACCGAATATATCGGTTATGCGTG
GGCGATGGTTGTTGTCATTGTCGGCGCAACTATCGGTATCAAGCTGTTTAAGAA
ATTCACCTCGAAAGCAAGCTGATAAACCGATACAATTAAAGGCTCCTTTTGGA
GCCTTTTTTTTGGAGATTTTCAACGTGAAAAAATTATTATTCGCAATTCCTTA
G
TGGTACCTTTCTATTCTCACTCT
GAGACTGCTCCGCTTTCGACGATGTTGTC
GCCTTATGGTGGAGGTTCGGCCGAAACTGTTGAAAGTTGTTTAGCAAAATCCC
ATACAGAAAATTCATTTACTAACGTCTGGAAAGACGAC
Be the restriction enzyme site of restriction endonuclease KPN1 according to what italic had a underscore, find in its downstream on phage, to express and insert proteic dna sequence dna, the i.e. part of back underscore.And then according to dna sequence dna, going out its protein sequence according to the genetic code rule induction is ETAPLSTMLSPY.
Letter abbreviations and amino acid whose corresponding relation are as follows:
The A L-Ala; The R arginine; The N N; The D aspartic acid; The C halfcystine; The Q glutamine; E L-glutamic acid; The G glycine; The H Histidine; The I Isoleucine; The L leucine; K Methionin; The M methionine(Met); The F phenylalanine; The P proline(Pro); The S Serine; The T Threonine; The W tryptophane; Y tyrosine; The V Xie Ansuan.
Table 1 order-checking obtains 50 small peptides that insert fragment and coding thereof at random
| Sequence number | Phage clone | Clone's numbering | The small peptide numbering | Short peptide sequence (N → C) | |
| 1 | AN2-AN6/AN8/AN10/ AM1/AM3/AM6/AM10/ AM11/AM13/AM15/AM17/ AM18/AM19 | AN2 | *GMBP1 | ETAPLSTMLSPY | |
| 2 | AN7/AM4/AM9 | AN7 | *GMBP2 | TTFSPPSPLSSI | |
| 3 | AN9 | AN9 | GMBP3 | FNNHYPAAATYP | |
| 4 | AN41/AN42/AN43/ AN411/AN413/AN417/AN421/ AN423/AN425/AN429/AN430 | AN41 | *GMBP4 | EVFWPLNAPRLL | |
| 5 | AN45 | AN45 | GMBP5 | SWQIPYPISPRS | |
| 6 | AN46/AN47 | AN46 | *GMBP6 | KTAPTPYALVHD | |
| 7 | AN48 | AN48 | GMBP7 | SNFMHNTRIWSH | |
| 8 | AN49/AN412/AN416/AN424 AN428 | AN49 | *GMBP8 | SWQITYPISPRS | |
| 9 | AN410/AN415/AN420/AN422 | AN410 | *GMBP9 | AWTWVLPSSIRA | |
| 10 | AN414 | AN414 | GMBP10 | TDSYHVASARQP | |
| 11 | AN418 | | GMBP11 | STYSHPLSLRPD | |
| 12 | AM2 | AM2 | GMBP12 | YTTWPFTSLQLD | |
| 13 | AM5 | AM5 | GMBP13 | AFMETTSQNAWL | |
| 14 | AM7 | AM7 | GMBP14 | ETAPPTPYSVMF | |
| 15 | AM8 | AM8 | GMBP15 | TTFNPLYLRLDT | |
| 16 | AM12/AM16/AM21/AM22 | AM12 | *GMBP16 | SSFPQVIPLDYL | |
| 17 | AM41/AM42/AM43/AM415 /AM419/AM423/AM425 | AM41 | *GMBP17 | APKYSLSDLYLN |
| Sequence number | Phage clone | Clone's numbering | The small peptide numbering | Short peptide sequence (N → C) | |
| 18 | AM44 | AM44 | GMBP18 | QIEKISQHLDMH | |
| 19 | AM45 | | GMBP19 | TWNQPYIPPLYP | |
| 20 | AM47 | AM47 | GMBP20 | YASPPNPSLRLT | |
| 21 | AM48 | AM48 | GMBP21 | YHGLTPVRYVSV | |
| 22 | AM49/AM411/AM416/AM418 | AM49 | *GMBP22 | YTFMPELTTPRT | |
| 23 | AM410/AM420/AM422 | AM410 | *GMBP23 | NSFNYAPLLMPR | |
| 24 | AM412 | AM412 | GMBP24 | TSPYHLLHAHLQ | |
| 25 | AM413 | AM413 | GMBP25 | SSFVALSISPSM | |
| 26 | AM417 | | GMBP26 | ANLSSHSSPGDS | |
| 27 | VN1 | VN1 | GMBP27 | TTLQFTGQTNKT | |
| 28 | VN2/VN8/VN35/VN37/VN38/VN41 /VN47 | VN2 | *GMBP28 | TPPPRDASLSRW | |
| 29 | VN3 | | GMBP29 | HNIGTWGPKSHL | |
| 30 | VN4/VN46/VN50 | VN4 | *GMBP30 | SGFFEPQHSHPL | |
| 31 | VN5 | VN5 | GMBP31 | QIEESFVRGHTT | |
| 32 | VN6 | VN6 | GMBP32 | SPQTDGLVSTPS | |
| 33 | VN7 | VN7 | GMBP33 | YTFDPQIRPAGL | |
| 34 | VN9 | VN9 | GMBP34 | YERSILPFSHVF | |
| 35 | VN10 | VN10 | GMBP35 | YPSVTFPTQTLL | |
| 36 | VN11/VN14/VN20/VN21/ VN29/VN36/VN40/VN42 | VN11 | *GMBP36 | ASTNVFARPMYL | |
| 37 | VN12 | VN12 | GMBP37 | SPWYMTPSPNTA | |
| 38 | VN13 | VN13 | GMBP38 | HISVINYTTKIS | |
| 39 | VN17 | VN17 | GMBP39 | MNVTVSGRLSGP | |
| 40 | VN18 | VN18 | GMBP40 | TIPYPFSLLNNP | |
| 41 | VN19 | VN19 | GMBP41 | GPFLYSQVAWRS |
| Sequence number | Phage clone | Clone's numbering | The small peptide numbering | Short peptide sequence (N → C) |
| 42 | VN23/VN28/VN45/VN49 | VN23 | *GMBP42 | TALPNHWSDASP |
| 43 | VN24/VN25/VN27/ |
VN24 | *GMBP43 | SVSVGMKPSPRP |
| /VM21-VM30/VM41-VM43/VM47 | |||||
| 44 | VN26 | VN26 | GMBP44 | YQEETPASSFSR | |
| 45 | VN30 | VN30 | GMBP45 | NSSQLAPYTTHR | |
| 46 | VM2/VM3/VM5/ VM6/VM7/VM11/ VM13/VM16/VM17/VM19/ VM20/VM35-VM39/VM45/VM46 | VM2 | *GMBP46 | TLHPSVLSYVLK | |
| 47 | VM4 | VM4 | GMBP47 | HNGLPNFFQTRL | |
| 48 | VM9 | VM9 | GMBP48 | EAAPNFYPPLTF | |
| 49 | VM12 | | GMBP49 | DLFQFAFPLNTI | |
| 50 | VM14 | VM14 | GMBP50 | FTFSYAESVSYF |
*Represent the number of times that this fragment repeats in all order-checking clones, not marked being 1 time
1.2.GMBPs the homology analysis of aminoacid sequence
1.2.1.50 the homology analysis between individual GMBPs
Land ClustalW website (http://www.ebi.ac.uk/clustalw/), be analyzed between 12 small peptide aminoacid sequences, present higher homology between the partial sequence, find to have the consensus sequence pattern of 7 kinds of different characteristicss.Be shown in Table 2.
The consensus sequence pattern of 7 kinds of different characteristicss in 50 short peptide sequences of table 2
| The small peptide title | Aminoacid sequence | |||||||||||
| Threonine---(1-3) amino acid---proline(Pro) consensus sequence pattern | ||||||||||||
| GMBP1 | E | T | A | P | L | S | T | M | L | S | P | Y |
| GMBP2 | T | T | F | S | P | P | S | P | L | S | S | I |
| GMBP3 | F | N | N | H | Y | P | A | A | A | T | Y | P |
| GMBP6 | K | T | A | P | T | P | Y | A | L | V | H | D |
| GMBP8 | S | W | Q | I | T | Y | P | I | S | P | R | S |
| GMBP9 | A | W | T | W | V | L | P | S | S | I | R | A |
| GMBP11 | S | T | Y | S | H | P | L | S | L | R | P | D |
| GMBP12 | Y | T | T | W | P | F | T | S | L | Q | L | D |
| GMBP14 | E | T | A | P | P | T | P | Y | S | V | M | F |
| GMBP15 | T | T | F | N | P | L | Y | L | R | L | D | T |
1.2.2. carry out homology analysis by database retrieval
Land NCBI/BLAST website (http://www.ncbi.nlm.nih.gov/blast/), the search Protein Data Bank carries out homology analysis, the result does not find to contain and the on all four protein of these small peptide aminoacid sequences, and GMBPs and some protein molecule have homology (seeing Table 3) preferably.
The protein database search homology analysis of 50 GMBPs small peptides of table 3
| The small peptide title | The homology molecule | Homeopeptide hop count order | Homology (%) |
| *GMBP1 | Receptor type, albumen (matter) tyrosine phosphatase | 8/10 | 80 |
| *GMBP2 | Leukocytic immunity |
7/7 | 100 |
| GMBP3 | The sodium sulphur body that cotransports | 7/8 | 87 |
| *GMBP4 | Receptor tyrosine kinase | 7/8 | 87 |
| GMBP5 | Transient receptor |
7/9 | 77 |
| *GMBP6 | Ankyrin repeat | 6/6 | 100 |
| GMBP7 | Atp synthase | 7/10 | 70 |
| *GMBP8 | Transient receptor potential calcium channel 5 | 7/9 | 77 |
| *GMBP9 | Zine ion is specially transported body 3 | 6/6 | 100 |
| GMBP10 | CD8 | 7/9 | 77 |
| GMBP11 | Imagination albumen | 6/6 | 100 |
| GMBP12 | Ovarian cancer related neoplasms antigen | 7/8 | 87 |
| GMBP13 | α-NAC albumen | 6/9 | 66 |
| GMBP14 | The protein sugar based transferase | 7/10 | 70 |
| GMBP15 | Methyltransgerase | 6/6 | 100 |
| *GMBP16 | Iron ion is regulated albumen | 6/7 | 85 |
| *GMBP17 | CAT | 6/9 | 66 |
| GMBP18 | The GP-39 chondroprotein | 8/9 | 88 |
| GMBP19 | Mammary cancer and ovarian cancer susceptible protein | 7/9 | 77 |
| GMBP20 | Neurolin 3 | 8/11 | 72 |
| GMBP21 | SDK2 albumen | 6/7 | 85 |
| *GMBP22 | The non-integrin type i collagen of thrombocyte acceptor | 7/7 | 100 |
| *GMBP23 | The colorectal carcinoma associated protein | 6/7 | 85 |
| GMBP24 | Lipoxygenase | 6/8 | 75 |
| GMBP25 | The retinoblastoma correlation factor | 7/7 | 100 |
| GMBP26 | HIRA albumen | 7/7 | 100 |
| GMBP27 | Transmembrane channel sample albumen | 6/6 | 100 |
| *GMBP28 | 5-hydroxytryptamine receptor | 7/8 | 87 |
| GMBP29 | MAGE-E1 | 6/7 | 85 |
| GMBP30 | TXi Baoshouti | 6/7 | 85 |
| GMBP31 | Glycogen synthetase 1 | 7/9 | 77 |
| GMBP32 | Galactokinase | 7/9 | 77 |
| GMBP33 | MAP3K12 is in conjunction with arrestin | 7/11 | 63 |
| GMBP34 | Ubiquitin protein enzyme 9 | 7/11 | 63 |
| GMBP35 | Imagination albumen LOC400359 | 7/7 | 100 |
| *GMBP36 | Peptase a precursor | 6/8 | 75 |
| GMBP37 | Protein arginine takes off imido acid | 6/7 | 85 |
| GMBP38 | Cytolemma calcium ion ATP enzyme | 9/12 | 75 |
| GMBP39 | MGEA6 albumen | 7/7 | 100 |
| GMBP40 | Potassium-channel | 6/7 | 85 | |
| GMBP41 | RAMP2 albumen | 7/11 | 63 | |
| * | G CFS | 3 acceptors | 8/10 | 80 |
| *GMBP43 | death inducer-obliterator-3 | 6/7 | 85 | |
| GMBP44 | Metalloendoprotease | 7/8 | 87 | |
| GMBP45 | Seven transmembrane helix receptors | 6/7 | 85 | |
| *GMBP46 | Ring finger protein 38 | 7/9 | 77 | |
| GMBP47 | Promyelocytic leukemia albumen | 6/6 | 100 | |
| GMBP48 | Imagination albumen LOC65123 | 7/7 | 100 | |
| GMBP49 | The EGF-R ELISA substrate | 6/6 | 100 | |
| GMBP50 | Zinc finger protein 13 | 6/8 | 75 |
*Represent the number of times that this fragment repeats in all order-checking clones, not marked being 1 time
1.3. phage clone is identified in conjunction with active ELISA
According to order-checking and information analysis result, choose 24 clones and carry out the cell ELISA evaluation that phage clone carries out binding specificity, be 17 wherein with the clone that the SGC7901/ADR cell screening obtains, the clone who obtains with the SGC7901/VCR cell screening is 7.With SGC7901 is control cells, active with combining of SGC7901/VCR and SGC7901/ADR with the phage clone that the order-checking of ELISA method preliminary evaluation is successful, gets rid of false positive or indifference bonded phage.And establish the PBS contrast and phage (the amplification liquid of the original peptide storehouse Ph.D.-12) contrast that has nothing to do.Experimental procedure is as follows:
Collection is in SGC7901, SGC7901/VCR, the SGC7901/ADR of logarithmic phase, and (every porocyte number is 1 * 10 to be laid on 96 orifice plates
5), cultivate 4h and make cell attachment, stretching, extension, fixing 15min in 0.25% glutaraldehyde (commercially available), 4%H
2O
2Room temperature sealing 30min, 37 ℃ of sealings of 1%BSA (purchasing in Beijing ancient cooking vessel state company) 30min adds 1 * 10 respectively
10The pfu positive bacteriophage is hatched 2h for 37 ℃.Hatch 1h for 37 ℃ with the anti-M13IgG of HRP-(purchasing Pharmacia company) in the U.S., TMB (purchasing) 30min that develops the color in ten thousand safe medicine companies, termination reaction, the 450nm/630nm dual wavelength is measured the OD value down.Experiment repeats 3 times, calculates average OD value, and experimental group OD value is judged to be the positive with ratio 〉=2.1 of control group OD value.Data represent that with mean ± standard deviation SPSS software carries out statistical analysis.The result shows, AN2, and AN46, AN49, AN410, these 5 phage clones of AM410 have evident difference in conjunction with (see figure 3) on SGC7901 and SGC7901/ADR; VN4, VN23, these 3 phage clones of VM2 have evident difference in conjunction with (see figure 4) on SGC7901 and SGC7901/VCR.
1.4.8 individual difference is identified in conjunction with phage and the binding specificity that presents small peptide thereof
With the AN2 phage and to present small peptide GMBP1 be example, adopt the phage cell to identify that in conjunction with methods such as competition inhibition experiments it is in conjunction with activity and specificity in conjunction with experiment, immunocyte fluorescent dye, cell.The evaluation of other phage clone or small peptide is in kind carried out.
14.1. cell in vitro combination/internalization experiment
Be checking AN2 phage bonded specificity on SGC7901/ADR, with SGC7901, GES is a control cells, carries out the external combination experiment of phage.Method is as follows: SGC7901, SGC7901/ADR, the GES cell of results logarithmic phase each 1 * 10
7Individual, with 1 * 10
10PfuAN2 phage and irrelevant phage hatch altogether with three kinds of cells respectively, and phage, TEA alkaline lysis wash-out dissolve intracellular phage and titration in reclaiming on the glycine wash-out cytolemma of washing, pH2.2, compare the recovery phagocytosis scale of construction on three kinds of cells.Found that the AN2 phage titre of the internalization that reclaims from SGC7901/ADR is 34 times of irrelevant phage, is 32.4 times of control cells SGC7901,14.8 times of GES.And the AN2 phage nothing to do with phage no significant difference (see figure 5) of the internalization that reclaims from SGC7901 and GES cell.
1.4.2. vitro binding competitive inhibition experiment
The GMBP1 small peptide specific combination that whether presents by its surface for clear and definite AN2 phage is in SGC7901/ADR, can utilize the artificial synthesis peptide of GMBP 1 that the specificity combination of AN2 phage is intervened, suppress experiment by being at war with in conjunction with the internalization experiment with external viable cell.External viable cell suppresses experiment in conjunction with the internalization competition: combine the internalization experiment with cell in vitro similar, SGC7901/ADR1 * 10 that gather in the crops 14 groups of logarithmic phases
7, 6 experimental group cells are respectively synthetic peptide GMBP 1 (worker company is given birth in Shanghai) and the rondom polypeptide (12 peptide) of 50 μ M, 10 μ M, 5 μ M, 1 μ M, 0.1 μ M earlier with concentration, respectively with 1 * 10
10The AN2 phage of pfu hatches 2h for 4 ℃, and blocking buffer is a control group.Wash-out, recovery and titration are in conjunction with phage then.Calculate synthetic peptide GMBP1 to the competition inhibiting rate of AN2 phage in conjunction with internalization: quantity or OD value * 100% of inhibiting rate=(quantity or OD value-experimental group that control group reclaims phage reclaim quantity or the OD value of phage)/control group recovery phage, make inhibiting rate-synthesize peptide concentration curve.The result is along with GMBP1 concentration increases, and inhibiting rate rises gradually, and when 10-50 μ M, inhibiting rate maintains higher level, shows that the synthetic peptide of GMBP 1 has competitive inhibitory effect (see figure 6) in conjunction with internalization to the AN2 phage on SGC7901/ADR.
1.4.3.AN2 the immunofluorescence of phage and GMBP 1 synthetic peptide binding specificity thereof detects
Adopt immunofluorescence technique to identify AN2 phage and SGC7901/ADR bonded specificity, compare with SGC7901.Cell climbing sheet is after the normal serum sealing, earlier with AN2 phage 1 * 10
10Hatch 2h for pfu37 ℃, anti-ly hatch with the anti-mouse IgG two of mouse-anti M13 monomer (purchasing the Pharmacia company in the U.S.), FITC mark, DAPI dyeing then successively vibration washing 5min * 6 time.PBS replaces phage, makes negative control.Observe comparative result down in fluorescent microscope and show that the AN2 phage is positive on SGC7901/ADR, and the negative (see figure 7) of control cells.
Adopt immunofluorescence technique to identify GMBP 1 synthetic peptide and SGC7901/ADR bonded specificity.SGC7901/ADR and SGC7901 cell climbing sheet add AN2 phage 1 * 10 after the normal serum sealing
10The GMBP1 of pfu and 100 μ l, 10 μ M, another group adds AN2 phage 1 * 10 separately
10Pfu is hatched 2h for 37 ℃, and anti-ly hatch with the anti-mouse 1gG two of mouse-anti M13 monomer (purchasing the Pharmacia company in the U.S.), FITC mark, DAPI dyeing then successively vibration washing 5min * 6 time.Observe relatively down in fluorescent microscope.Found that, the SGC7901/ADR that adds GMBP1 is weak positive or negative, and SGC7901 adds GMBP1 or do not add the no significant difference of GMBP1, all be negative or the weak positive, this shows, the GMBP1 small peptide can specificity be incorporated into Multidrug Resistance of Gastric Cancer cell SGC7901/ADR really, and does not see with cancer of the stomach susceptibility cell and obviously to combine (see figure 8).
Comprehensive The above results, AN2, AN46, AN49, AN410, these 5 phage clones of AM410 have the evident difference combination on SGC7901 and SGC7901/ADR; VN4, VN23, these 3 phage clones of VM2 have the evident difference combination on SGC7901 and SGC7901/VCR.Wherein AN2, AN46, AN49, AN410, AM410, VN4, VN23, the corresponding small peptide GMBP1 of VM2 phage clone, GMBP6, GMBP8, GMBP9, GMBP23, GMBP30, GMBP42, GMBP46 all can suppress itself and the combining of drug-resistant tumor cell.
1.58 individual difference is in conjunction with phage and present small peptide and identify for the reversing drug resistance specificity
Variant obvious bonded 8 phage clone AN2, AN46, AN49, AN410, AM410, VN4, VN23, VM2 reversing drug resistance characteristic for above-mentioned experiment confirm are identified.Presenting small peptide GMBP42 with the VN23 phage is example, utilizes the external drug susceptibility experiment of MTT, and drug sensitivity assay is identified the effect that it reverses for Multidrug Resistance of Gastric Cancer cell resistance specificity in the nude mouse.The evaluation of other phage clone or small peptide is in kind carried out.
1.5.18 individual difference is in conjunction with phage and present the external drug susceptibility experiment of small peptide MTT
Gather in the crops the logarithmic growth SGC7901/VCR cell in mid-term, according to every hole 8 * 10
3Individual cell/200 μ L inoculate into 96 orifice plates, place overnight incubation in the cell culture incubator.Be divided into 3 groups, every hole adds the GMBP42 of 100 μ l10 μ M respectively in each group, with the rondom polypeptide of same dosage and 100ul PBS in contrast, every group of vincristine(VCR) (0.5ug/ml, 1ug/ml, 2ug/ml that adds different concns again, 4ug/ml), continue to cultivate 3 days.Every hole adds fresh preparation MTT solution (5g/L) 20 μ L, and 37 ℃ are continued to cultivate 4 hours; The careful suction abandoned culture supernatant in the hole, and every hole adds the DMSO of 150 μ L, and vibration 10min fully dissolves crystallisate; Wavelength 490nm, on full-automatic microplate reader instrument, survey each hole absorbance value, get the mean number of per 4 repeating hole A values, calculate the survival rate of cell: cell survival rate=(experimental group A value-blank group A value)/(negative control group A value-blank group A value) * 100%; Nosa statistical software with statistics teaching and research room of The Fourth Military Medical University calculates the IC50 value of cell to every kind of medicine, and carries out test of significance.The result shows that the GMBP42 polypeptide can reverse the resistant characterization of SGC7901/VCR, has compared notable difference with rondom polypeptide and PBS, and statistical significance is arranged.(table 4)
The external drug susceptibility experiment of MTT also shows GMBP8; GMBP46 also can the reversing stomach mdr cell resistant characterization, reduce its IC50 value, nothing to do with phage and rondom polypeptide have been compared notable difference, and statistical significance (p＜0.05) is arranged.And GMBP1; GMBP6; GMBP9; These 3 clones do not have obvious influence for the resistant characterization of SGC7901/VCR and SGC7901/ADR, and nothing to do with phage and rondom polypeptide result are approximate.
1.5.2 drug susceptibility experiment in the phage small peptide nude mouse
Collect resistant Gastric Cancer SGC7901/VCR cell, Digestive system digestion disperses cell, with serum free medium centrifuge washing twice, living cell counting number, adjusts cell count, with cell suspension in PBS.Extract cell suspension inoculation in the oxter of nude mice with syringe, contain viable count 2 * 10
612 4 the week age female BALB/c nude mice be divided into 4 groups at back subcutaneous vaccination SGC7901/VCR cell.(about the 0.3～0.4cm), 1st group of tail vein injection PBS of its size be as blank, the 2nd group of tail vein injection 500mg/kg5-FU (every day 1 time), continuously 5d when two all posterior tuberosity bodies can touch; The 3rd group of tail vein injection 500mg/kg 5-FU+200 μ M rondom polypeptide (every day 1 time), 5d continuously; The 4th group of tail vein injection 500mg/kg 5-FU+200 μ M GMBP42 (every day 1 time), 5d continuously; Put to death part mouse, vernier caliper measurement tumour size in the 6th day.
The result shows that GMBP42 can reverse the resistant characterization of SGC7901/VCR, and gross tumor volume is significantly less than and singly adds 5-FU, rondom polypeptide and PBS group, and statistical significance (p＜0.05) (table 5) is arranged.GMBP8; Drug susceptibility experiment in the GMBP46 nude mouse also confirms the resistant characterization that it can the reversing stomach mdr cell, dwindles gross tumor volume, with the rondom polypeptide group, singly compared notable difference with medicine group and PBS group, and statistical significance (p＜0.05) is arranged.
2. technique effect
Phage random peptide library associating MTT drug sensitivity assay of the present invention, the full cell of in-vitro screening, or obtain to combine with the Multidrug Resistance of Gastric Cancer cell-specific and the small peptide GMBPs of reversing drug resistance, identify in conjunction with methods such as experiment, vitro binding competitive inhibition experiment, immunofluorescence detections through cell ELISA, cell in vitro, confirm GMBPs small peptide and Multidrug Resistance of Gastric Cancer cell bonded specificity, and drug sensitivity assay confirms in the external drug sensitivity assay of MTT and the nude mouse, the resistant characterization that the GMBPs small peptide can the reversing stomach multidrug resistance cell.This shows, the phage polypeptide that utilizes that the present invention set up is united the external drug sensitive test in-vitro screening of MTT, the small peptide model that obtains combination of Multidrug Resistance of Gastric Cancer cell-specific and reversing drug resistance is a kind of successful model, can be used as the technology platform of people's Multidrug Resistance of Gastric Cancer surface of cell membrane resistance associated molecule research.Through the homology analysis of aminoacid sequence, do not finding to contain and the on all four protein molecular of aminoacid sequence of the present invention in the Protein Data Bank at present, so the present invention has uniqueness.This shows, the discovery of these small peptides is in the aspects such as diagnosis, targeted therapy, multi-medicine tolerant reversal treatment and Multidrug Resistance of Gastric Cancer associated molecule Study on Mechanism of cancer of the stomach, the potential using value and the theoretical significance that have.Now can be used for the following aspects:
.1. the screening of combination of tumor multi-medicine drug-resistant cell-specific and reversing drug resistance bioactive short peptide
With normal cell and the negative subtractive cell line of tumour susceptibility cell, hatch (company in New England Biolabs is purchased in original peptide storehouse) altogether with original peptide storehouse, getting unconjugated phage hatches altogether with the tumor drug resistance cell again, recovery is in conjunction with phage, the next round screening is dropped in the amplification back, 4 take turns back picking phage mono-clonal, directly carry out the MTT drug sensitivity test screening of single medicine concentration, the phage clone that can reduce tumor drug resistance cells survival rate that obtains carries out determined dna sequence and homology analysis again.And utilize the phage cell to suppress the binding specificity that methods such as experiment, immunofluorescence detection have been identified phage clone in conjunction with competition in conjunction with experiment, immunocyte/histochemical stain, cell, utilize the external drug sensitive experiment of MTT, the interior drug sensitivity assay of nude mouse to determine the reversing drug resistance characteristic of phage clone express polypeptide simultaneously.For seeking new targeted drug, the molecule of specificity reversing drug resistance and the new tumor multi-medicine drug-resistant related film molecule of tumor multi-medicine drug-resistant cell, provide new thinking and method.
.2. the development of stomach cancer diagnosis reagent box
With material couplings such as the specificity bound short peptide found among the present invention and fluorescent substance, isotropic substances, development is used for the test kit of diagnosing gastric cancer, if in bigger sample scope, be tested and appraised, a kind of approach that is expected to become the diagnosis of cancer of the stomach drug-resistant intensity and judges the cancer of the stomach prognosis.
.3. the targeted drug of resistant Gastric Cancer makes up
For specific binding capacity is arranged, but do not have the biologic activity small peptide, can be used for the targeted therapy of Multidrug Resistance of Gastric Cancer with its as target instrument, such as making up following stomach cancer target medicine at the Multidrug Resistance of Gastric Cancer cell.
The structure of the compound peptide of resistant Gastric Cancer target toxicity: these specificity small peptides among the present invention are connected with lps molecules such as diphtheria toxin, Pseudomonas exotoxin, clostridium perfringens enterotoxins, utilize the specificity small peptide that toxin is imported the Multidrug Resistance of Gastric Cancer cell, reach the purpose of killing the Multidrug Resistance of Gastric Cancer cell.
.4. the structure of Multidrug Resistance of Gastric Cancer reverse short peptide medicine
To reverse the small peptide with biologic activity to Multidrug Resistance of Gastric Cancer, directly research and development have the small peptide medicine of reversing drug resistance effect.
.5.GMBPs the research of small peptide binding target molecule
By to methods such as the screening of resistant Gastric Cancer cDNA expression library or affinity chromatographys, can be that bait angles and gets its binding target molecule or its encoding gene with the GMBPs small peptide, thereby illustrate small peptide and resistant Gastric Cancer specificity bonded molecular basis, and further study the function of this target molecule, if this target molecule plays a significant role, might become the new target molecule of curing gastric cancer in Multidrug Resistance of Gastric Cancer takes place.
3. description of drawings:
Fig. 1 is a phage peptide library in-vitro screening schema
Its expression is hatched with SGC7901/ADR or SGC7901/VCR more altogether with GES cell, the uncombined phage of SGC7901 cell, reclaims and amplification and its bonded phage, repeats the four-wheel screening.
Fig. 2 is four-wheel screening pnagus medius enrichment diagram R1 → R4 gradually on SGC7901/ADR or SGC7901/VCR, and the phage titre that reclaims from SGC7901/ADR or SGC7901/VCR has increased by 10
5-10
6Doubly.
Fig. 3 is in 17 phage clones of ELISA qualification result diagram of 17 phage clones and SGC7901/ADR binding specificity, and AN2, AN41, AN46, AN49, these 5 clones of AM410 can specificity be incorporated into SGC7901/ADR.
Fig. 4 is in 7 phage clones of ELISA qualification result of 7 phage clones and SGC7901/VCR binding specificity, and VN4, VN23, these 3 clones of VM2 can specificity be incorporated into SGC7901/VCR.
Fig. 5 is that external viable cell illustrates in conjunction with experimental result
The yield of AN2 phage on SGC7901/ADR is respectively control cells SGC7901 group, GES group 32.4,14.8 times.This prompting AN2 phage and SGC7901/ADR have the specific combination activity.
Fig. 6 is that cell suppresses experiment in conjunction with competition
Viable cell suppresses experiment in conjunction with competition and the competitive ELISA experiment all shows, along with increasing of the synthetic peptide concentration of GMBP1, inhibiting rate constantly rises, when GMBP1 concentration during at 10-50 μ M, inhibiting rate maintains higher level, and this shows that the synthetic peptide of GMBP1 has competitive inhibitory effect in conjunction with activity to the AN2 phage on SGC7901/ADR.
Fig. 7 is that immunofluorescence detection (* 400) SGC7901/ADR of AN2 phage and SGC7901/ADR binding specificity is positive, and after birth, endochylema send green fluorescence, and SGC7901 is negative.This shows that AN2 phagocytosis physical efficiency specific combination is on SGC7901/ADR.Karyon is dyed blueness by DAPI.
Fig. 8 is the immunofluorescence detection (* 400) of the synthetic peptide of GMBP1 for AN2 phage and the blocking-up of cancer of the stomach SGC7901/ADR cell binding specificity
Add the AN2 phage for the SGC7901/ADR cell and add the synthetic peptide of GMBP1 simultaneously, people's cancer of the stomach SGC7901/ADR is negative, and is not positive and add the synthetic peptide of GMBP1.
And that the SGC7901 cell adds the synthetic peptide of GMBP1 is all negative.This shows the synthetic Toplink specific combination of GMBP1 in cancer of the stomach SGC7901/ADR cell, and can block combining of AN2 phage and SGC7901/ADR.Karyon is dyed blueness by DAPI.
Table 4 be VCR act on be added with polypeptide GMBP42, to the cells survival rate (%) and the IC50 value of the SGC7901/VCR cell strain of rondom polypeptide and PBS.
| Vincristine(VCR) (ug/ml) | Survival rate ± standard deviation (%) | ||
| PBS | Rondom polypeptide | GMBP42 | |
| 0.5 1 2 4 | 0.98±0.091 0.59±0.11 0.28±0.026 0.24±0.007 | 0.83±0.037 0.55±0.136 0.31±0.051 0.25±0.047 | 0.70±0.08 0.39±0.012 0.21±0.042 0.14±0.021 |
| IC50 value (mean ± standard deviation, μ g/mL) | 1.51±0.23 | 1.35±0.22 | 0.96±0.17 * |
*P＜0.05 table 4
The SGC7901/VCR cell that is added with the GMBP42 polypeptide when equal drug level, survival rate and IC50 value be starkly lower than add the contrast phage, rondom polypeptide reaches and only adds the SGC7901/VCR cell of vincristine(VCR), and significant difference (p＜0.05) is arranged.Illustrate that VN23 phage polypeptide GMBP42 has reverse effect for the resistant characterization of SGC7901/VCR cell.
Table 5 is drug sensitivity tests in the nude mouse
Polypeptide GMBP42 is to the influence of tumor-bearing mice tumour.Administration after 6 days 5-FU+GMBP42 group mouse tumor diameter be significantly less than the PBS group, singly add 5-FU group and 5-FU+ rondom polypeptide group, and significant difference (p＜0.05) is arranged.
| Tumour size ± standard deviation (cm) | ||
| PBS 5-FU 5-FU+ rondom polypeptide 5-FU+GMBP42 | Administration preceding 0.23 ± 0.042 0.28 ± 0.025 0.24 ± 0.032 0.26 ± 0.027 | After the administration 6 days 0.56 ± 0.079 0.36 ± 0.054 0.33 ± 0.066 0.28 ± 0.042 * |
*P＜0.05 table 5
Claims (1)
1. people's resistant Gastric Cancer specific combination and drug resistance inversion small peptide is characterized in that being selected from following sequence:
GMBP8： S W Q I T Y P I S P R S；
GMBP42： T A L P N H W S D A S P；
GMBP46： T L H P S V L S Y V L K。
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