CN109467602A

CN109467602A - Human protein binding to CXCR4 and its application

Info

Publication number: CN109467602A
Application number: CN201811308248.9A
Authority: CN
Inventors: 魏星; 梁广铨; 刘静; 林泽雄
Original assignee: Jinan University
Current assignee: Jinan University
Priority date: 2018-11-05
Filing date: 2018-11-05
Publication date: 2019-03-15

Abstract

The source of people albumen and its application that the invention discloses a kind of in conjunction with CXCR4.The present invention screens the binding protein library of people by yeast-two hybrid technique, it finally screens to obtain the source of people albumen in conjunction with CXCR4, is one of entitled NB97 binding protein, NB226 binding protein and NB412 binding protein or the binding protein that at least two combinations are formed.The source of people albumen application in preparation of anti-tumor drugs that the invention also discloses above-mentioned in conjunction with CXCR4.Yeast two-hybrid method can sensitively detect the interaction between albumen, the binding protein high specificity of acquisition.Binding protein provided by the present invention is full source of people section, and immunogenicity is low, can be preferably applied to anticancer drug exploitation and clinical application.

Description

Source of people albumen and its application in conjunction with CXCR4

Technical field

The invention belongs to binding protein field, in particular to a kind of source of people albumen and its application in conjunction with CXCR4.

Background technique

Malignant tumour is always medically to be difficult to the problem of capturing, even more " number one killer " of human health, therefore for The treatment of tumour always is the hot and difficult issue of research.Traditional treatment anti-tumor regimen mainly passes through radiotherapy, chemotherapy and operation Treatment etc. combines.Although these treatment means can inhibit or eliminate tumour cell, also have to the normal cell of body Damage and toxic side effect.In recent years, the targeted therapy of tumour becomes research hotspot, and this treatment method is by the albumen of specificity It is integrated on tumour cell, can play the role of killing tumour cell, and the degree of injury of organism normal cell is dropped to It is minimum.

7 transmembrane glycoproteins that Chemokine receptor4 (CXCR4) is made of 352 amino acid residues, belong to G-protein Coupled receptor, size about 40kDa.It has now been found that CXCR4 has expression in 23 kinds of different type tumours, is tumour cell Express chemokine receptors the most universal.The biological axis of CXCR4 and its ligand SDF-1 composition can be logical by activation multi-signal Road and migration, invasion, apoptosis, angiogenesis and the proliferation etc. of mediate tumor cell.Based on chemotactic factor (CF) SDF-1/CXCR4 and swell The substantial connection of tumor just blocks the drug development of SDF-1/CXCR4 axis to cause the highest attention of people at present.It is treating In sarcoma, glioma and brain tumor, the antagonist AMD3100 of CXCR4 has come into the clinical I and II phase.Meanwhile it is existing The antibody of anti-CXCR4 is used for oncotherapy, MDX-1338 monoclonal antibody blocks SDF-1 combination CXCR4, effectively inhibition tumour Transfer has been used to acute leukemia, non-Hodgkin lymphoma, the treatment of the clinical I phase of Huppert's disease at present.Therefore it utilizes Target spot of the CXCR4 as treatment of cancer has very high medical value.

Summary of the invention

The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide a kind of people in conjunction with CXCR4 Source protein.

The application for the source of people albumen that another object of the present invention is to provide above-mentioned in conjunction with CXCR4.

The purpose of the invention is achieved by the following technical solution: a kind of source of people albumen in conjunction with CXCR4, is entitled The combination egg that one of NB97 binding protein, NB226 binding protein and NB412 binding protein or at least two combinations are formed It is white；

The NB97 binding protein includes amino acid sequence heavy chain variable region and amino acid as shown in SEQ ID NO:1 Sequence light chain variable region as shown in SEQ ID NO:2；

The NB226 binding protein includes amino acid sequence heavy chain variable region and amino as shown in SEQ ID NO:3 Acid sequence light chain variable region as shown in SEQ ID NO:4；

The NB412 binding protein includes amino acid sequence heavy chain variable region and amino as shown in SEQ ID NO:5 Acid sequence light chain variable region as shown in SEQ ID NO:6.

The nucleotide sequence of the coding protein-bonded heavy chain variable region of NB97 is as shown in SEQ ID NO:7.

The nucleotide sequence of the coding protein-bonded light chain variable region of NB97 is as shown in SEQ ID NO:8.

The nucleotide sequence of the coding protein-bonded heavy chain variable region of NB226 is as shown in SEQ ID NO:9.

The nucleotide sequence of the coding protein-bonded light chain variable region of NB226 is as shown in SEQ ID NO:10.

The nucleotide sequence of the coding protein-bonded heavy chain variable region of NB412 is as shown in SEQ ID NO:11.

The nucleotide sequence of the coding protein-bonded light chain variable region of NB412 is as shown in SEQ ID NO:12.

Since protein-bonded bioactivity is by the gene of the hypervariable region specificity in antibody light chain and heavy chain variable region What sequence determined, it therefore, can be by gene engineering method to encoding heavy chain variable region as provided above and coding light chain variable region Nucleotide sequence recombinated, obtain different types of binding protein.

The source of people albumen in conjunction with CXCR4, is made of heavy chain variable region, connection chain and light chain variable region.

The protein-bonded amino acid sequence of the NB97 is as shown in SEQ ID NO:13.

The protein-bonded amino acid sequence of the NB226 is as shown in SEQ ID NO:14.

The protein-bonded amino acid sequence of the NB412 is as shown in SEQ ID NO:15.

The coding protein-bonded nucleotide sequence of NB97 is respectively as shown in SEQ ID NO:18.

The coding protein-bonded nucleotide sequence of NB226 is respectively as shown in SEQ ID NO:19.

The coding protein-bonded nucleotide sequence of NB412 is respectively as shown in SEQ ID NO:20.

Coding NB97 binding protein, NB226 binding protein and the protein-bonded gene of NB412 be respectively by 759, 774 and 744 base compositions are separately encoded 253,258 and 248 amino acid.

The gene of the coding protein-bonded heavy chain variable region of NB97 encodes 126 amino by 378 base compositions 3 complementary determining region areas (CDR): CDR1 GFTFSSYA are contained in acid, variable region；CDR2 is ISYDGSNK；CDR3 is ARDREYSGYDFRSEY。

The gene of the coding protein-bonded light chain variable region of NB97 encodes 109 amino by 327 base compositions 3 complementary determining region areas (CDR): CDR1 NIGTKS are contained in acid, variable region；CDR2 is FDT；CDR3 is QVWDSTSDH.

The gene of the coding protein-bonded heavy chain variable region of NB226 encodes 127 ammonia by 381 base compositions Base acid, 3 complementary determining region areas (CDR): CDR1 GGSFSGYY are contained in variable region；CDR2 is INHSGST；CDR3 is AR.

The gene of the light chain variable region of the coding NB226 encodes 113 amino acid by 339 base compositions, can be changed Contain 3 complementary determining region areas (CDR): CDR1 QSLLHSNGYNY in area；CDR2 is LGS；CDR3 is MQALQPP.

The heavy chain variable region of the coding NB412 encodes 122 amino acid, variable region is contained by 366 base compositions 3 complementary determining region areas (CDR): CDR1 GGSFSGYY；CDR2 is INHSGSTNY；CDR3 is AR.

The gene of the light chain variable region of the coding NB412 encodes 108 amino acid by 324 base compositions, and 3 Complementary determining region area (CDR): CDR1 QSISSW；CDR2 is KAS；CDR3 is QQYDDYS.

The preparation method of the source of people albumen in conjunction with CXCR4, include the following steps: will to encode it is described with CXCR4 combine source of people albumen gene cloning into expression vector, then be transformed into host cell carry out inclusion body expression it is pure Change, the source of people albumen in conjunction with CXCR4 can be obtained；Or it is synthesized by protein synthetic method.

The source of people albumen application in preparation of anti-tumor drugs in conjunction with CXCR4.

The present invention has the following advantages and effects with respect to the prior art:

1. the present invention screens the knot with CXCR4 interaction by yeast-two hybrid technique from the binding protein library of people Hop protein.Yeast two-hybrid method facilitates easy to operate, high conversion efficiency, the fusion protein containing the building of destination protein gene order Carrier expression quantity is high, so as to sensitively detect the interaction between albumen, the binding protein high specificity of acquisition.

2. binding protein provided by the present invention is full source of people section, immunogenicity is low, can be preferably applied to anticancer drug Exploitation and clinical application.

Detailed description of the invention

Fig. 1 is using the protein-bonded expression of SDS-PAGE electrophoresis detection and purification result figure；Wherein, figure A is NB97 knot The electrophoresis result figure of hop protein, figure B is the protein-bonded electrophoresis result figure of NB226, and figure C is the protein-bonded electrophoresis knot of NB412 Fruit figure, arrow represent the position where destination protein；Swimming lane M: albumen marker, swimming lane 1: the mycoprotein not induced, swimming lane 2: the mycoprotein of induction, swimming lane 3: the precipitating after the thallus ultrasonication of induction, swimming lane 4: after the thallus ultrasonication of induction Supernatant, swimming lane 5: the precipitating denaturing liquid after the bacterial cell disruption of induction, swimming lane 6: crossing column liquid, swimming lane 7: washing miscellaneous liquid, swimming lane 8~ 12: eluent.

Fig. 2 is the combination situation map using ELISA method identification binding protein and antigens c XCR4.

Fig. 3 is the influence result figure using MTT method detection binding protein to tumor cell proliferation ability；Wherein, figure A is For binding protein to the influence result figure of cell DU145 proliferative capacity, figure B is influence of the binding protein to cell PC-3 proliferative capacity Result figure, figure C is influence result figure of the binding protein to cell MDA-MB-231 proliferative capacity；* 0 μ g/mL and * * of p < 0.05vs 0 μ g/mL (n=3) of p < 0.01vs.

Fig. 4 is the result photo figure using cell scratch method detection binding protein to tumor cell migration；Wherein, scheme A- C is the migration results photo figure of tumour cell DU145, PC-3 and MDA-MB-231 respectively.

Fig. 5 is the mobility result figure obtained according to the scratch length computation of Fig. 4；Wherein, figure A-C is tumour cell respectively DU145, PC-3 and MDA-MB-231；* 0 μ g/mL, * * p < 0.01vs of p < 0.05vs, 0 μ g/mL (n=3).

Fig. 6 is the result photo figure using Transwell invasion method detection binding protein to tumor cell invasion；Its In, the result that figure A-C distinguishes each concentration binding protein treated tumour cell DU145, PC-3 and MDA-MB-231 invasion is shone Piece figure.

Fig. 7 is the binding protein light absorption value trend chart obtained according to Fig. 6；Wherein, figure A-C is tumour cell respectively DU145, PC-3 and MDA-MB-231；* 0 μ g/mL, * * p < 0.01vs of p < 0.05vs, 0 μ g/mL (n=3).

Fig. 8 is the result photo figure using Transwell moving method detection binding protein to tumor cell migration；Its In, figure A-C is the result photo figure that binding protein migrates tumour cell DU145, PC-3 and MDA-MB-231 respectively.

Fig. 9 is the binding protein light absorption value trend chart obtained according to Fig. 8；Wherein, figure A-C is tumour cell respectively DU145, PC-3 and MDA-MB-231；* 0 μ g/mL, * * p < 0.01vs of p < 0.05vs, 0 μ g/mL (n=3).

Figure 10 is using stream type cell analyzer and the bis- dye detection binding proteins of Annexin V/PI to apoptosis of tumor cells Influence result figure；Wherein, figure A-C is shadow of the binding protein to tumour cell DU145, PC-3 and MDA-MB-231 apoptosis respectively Ring result figure.

Figure 11 is that the binding protein obtained according to Figure 10 promotes the histogram of apoptosis of tumor cells ratio；Wherein, A-C points of figure It is not tumour cell DU145, PC-3 and MDA-MB-231；* p < 0.05vs (no binding protein control group) and * * p < 0.01vs (nothing Binding protein control group) (n=3).

Figure 12 is that NB97 binding protein and NB226 binding protein inhibit swollen in DU145 prostate gland cancer cell mouse model Tumor growth results figure；Wherein, figure A is the volume change curve graph of tumour after administration, and figure B is the swollen of each group at the end of dosage period The big small photo figure of tumor, figure C are the tumor weights of each group after dosage period；* p < 0.05vs (negative binding protein control group) (n=5).

Specific embodiment

The present invention is described in further detail with attached drawing combined with specific embodiments below, but embodiments of the present invention It is without being limited thereto.

If unspecified, embodiment is referring to conventional laboratory conditions, or the specification referring to kit manufacturer It carries out.Used engineering bacteria, cell strain are commercialized bacterial strain or cell strain in embodiment.Competent escherichia coli cell Preparation prepared according to " molecular cloning ".

The building of 1 bait plasmid pGBKT7-CXCR4 of embodiment

Second ring cDNA sequence of mankind CXCR4 extracellular fragment is inquired from NCBI, and yeast is carried out by Suzhou Jin Weizhi company It synthesizes after expression optimization and is inserted into pGBKT7 plasmid by EcoRI and BamHI restriction enzyme site, form pGBKT7-CXCR4, And provide the bacillus coli DH 5 alpha containing antigen plasmid pGBKT7-CXCR4.The sequence of second ring of CXCR4 extracellular fragment of synthesis It is as follows:

AACGTCAGCGAGGCAGATGACAGATATATCTGTGACCGCTTCTACCCCAATGACTTGTGGGTGGTTGT GTTCCAGTTTCAGTAA。

2 bait plasmid pGBKT7-CXCR4 transformed yeast competence AH109 of embodiment

Specifically according to 2 specification of clontech company Yeastmaker TM Yeast Transformation System Operation.

(1) preparation of yeast cells competence.

1) barms AH109 (being purchased from clontech company) is placed in 30 DEG C of incubators and is trained in YPDA lining out It supports 2 days.

2) glucose of final concentration of 2% (w/v) is added into 5mL YPDA culture medium in picking monoclonal strain inoculated, 30 DEG C, cultivate 12h in the shaking table of 220rpm.

3) 5 μ L bacterium solutions is taken to be inoculated into 50mL YPDA culture medium, 30 DEG C, cultivate 20h or so in the shaking table of 220rpm, until OD₆₀₀Value reaches between 0.15-0.3.

4) by obtained bacterium solution, 700g is centrifuged 5min at room temperature, outwells supernatant, retains precipitating.

5) precipitating is resuspended with 100mL YPDA culture medium, is then placed on 30 DEG C of shaking tables and shakes 5h or so, makes OD₆₀₀Value reaches Between 0.4-0.6.

6) bacterium solution is dispensed into 50mL centrifuge tube, room temperature 700g is centrifuged 5min, outwells supernatant, retains precipitating.It uses respectively Precipitating is resuspended in the deionized water of 30mL sterilizing, and room temperature 800g is centrifuged 6min, abandons supernatant, retains precipitating.

7) every pipe uses 1.1 × TE/LiAc of 1mL that precipitating is resuspended respectively, is transferred in two 1.5mL EP pipes, at room temperature 16000g is centrifuged 15s, abandons supernatant.

8) precipitating mixed in EP pipe is resuspended with 500 1.1 × TE/LiAc of μ L respectively, obtains competent yeast.

(2) bait plasmid pGBKT7-CXCR4 is transformed into competent yeast.

1) Carrier DNA (being purchased from Guangzhou roc occasion biology Co., Ltd) is placed on 100 DEG C of heater and heats 5min, Then 3min in ice face, then 100 DEG C of inactivation 5min are placed on, are finally placed on spare on ice.

2) 100ng and 5 μ L Carrier DNA is taken to add together the bait plasmid pGBKT7-CXCR4 that embodiment 1 obtains Enter in EP pipe, mix, 50 μ L of competent yeast is added, adds the 500 μ LPEG/LiAC (preparations of 10mL PEG/LiAc: lmL L0 × TE LiAc adds 8mL 50%PEG-3350 after lmL10 × TE buffer is added), it mixes well.

It 3) will the EP pipe equipped with step 2) mixed liquor the water-bath 30min in 30 DEG C of water-baths, every 5min reverse 1 after mixing It is secondary.30 μ L DMSO are added, mix.

4) 42 DEG C of water-bath heat shocks 15min, every 5min are 1 time reverse.16000g is centrifuged 20s, abandons supernatant, retains precipitating, adds The YPD Plus culture medium of 1mL is resuspended, and then EP pipe is put into 30 DEG C of shaking tables and shakes 45min.

5) 16000g abandons supernatant after being centrifuged 15s, and precipitating is resuspended with the NaCl solution of 1mL 0.9% (w/v).

6) bacterium solution is diluted into 1000 times, 100 times, 10 times, is coated on SD/-Trp plate respectively, is put into 30 DEG C of Bacteria Cultures It is cultivated 5 days in case.

The albumen of 3 yeast two-hybrid screening of embodiment and CXCR4 interaction

(1) binding protein library screening

1) the AH109 bacterial strain containing bait plasmid pGBKT7-CXCR4 that picking embodiment 2 is prepared is in SD/-Trp plate Upper scribing line is put into 37 DEG C of bacteriological incubators and cultivates 5 days.As described in Example 2, bait plasmid pGBKT7-CXCR4 will be contained AH109 bacterial strain be prepared into competent yeast cells.

2) by 100 μ L Carrier of 30 μ g binding protein Library plasmids (being purchased from hundred Ao Tai biotech firm of Guangzhou) and inactivation DNA is added together in 15mL centrifuge tube, and 3mL is then added and contains the competent yeast of bait plasmid and the PEG/LiAC of 12mL, 30 DEG C of water-bath 45min after mixing well.

3) 0.8mL DMSO is added, mixes, 42 DEG C of water-bath 20min.

4) 2000g is centrifuged 5min, abandons supernatant, and precipitating is resuspended with 15mL YPD plus culture medium, is then placed on 30 DEG C of shaking tables Shake 1.5h.

5) 2000g is centrifuged 5min, abandons supernatant, and precipitating is resuspended with 8mL 0.9%NaCl (w/v), will dilute 100 and 1000 times Dilution on SD/-Trp/-Leu plate coated plate, for measuring conversion ratio.

6) left re-suspension liquid 2000g is centrifuged 5min, and 1mL 0.9%NaCl (w/v) is added and is resuspended, respectively 0.5mL is taken to exist Coated plate on SD/-Trp/-Leu/-His/-Ade plate containing 10mM 3-AT.All plates are put into 37 DEG C of bacteriological incubators Middle inversion is cultivated one week.

7) picking positive colony is further cultured for 7 days in same flat lining out, and excretion is weaker or adiaphorous vacation It is positive.

(2) the scribing line screening of candidate positive colony.

The well-grown clone strain of picking is in SD/- on SD/-Trp/-Leu/-His/-Ade+10mM3-AT plate It crosses on Trp/-Leu, SD/-Trp/-Leu/-His/-Ade+10mM 3-AT plate, 30 DEG C of incubators are placed 5-7 days, Preliminary screening goes out positive colony.In scratching process, negative control is set, some act on relatively weak or adiaphorous albumen meeting It is not grown during crossing screening, therefore some clones can be excluded.

The candidate positive colony plasmid of embodiment 4 carries out revolution verifying

(1) yeast plasmid of candidate positive colony is extracted using yeast plasmid extracts kit, the specific steps are as follows:

1) positive monoclonal that picking embodiment 3 obtains is added 5mL SD/-Trp/-Leu/-His/-Ade tetra- and lacks culture Base, 30 DEG C, 220rpm shake bacterium for 24 hours, bacterium solution is transferred in EP pipe, 16000g be centrifuged 15s.

2) supernatant is abandoned, 480 μ L Buffer SE, 10 μ L β mercaptoethanols and 20 μ L lywallzymes are added into precipitating (Lyticase), 40min is stood after resuspension.

3) 4000g is centrifuged 5min, abandons supernatant, is resuspended after the Buffer YPI/Rnase A of 250 μ L is added into precipitating.

4) 50mg bead is added, vortex 5min draws 1.5mL supernatant into centrifuge tube.

5) add 250 μ LBufferYP II, stand 2min after mixing of turning upside down.

6) add the BufferYP III of 350 μ L, mixed up and down until there is rope appearance, 16000g is centrifuged 10min.

7) supernatant is drawn on clean microcylinder, below nesting 2mL collecting pipe, 10000g be centrifuged 1min.

8) column liquid was outwelled, 0.5mL Buffer HB is added, 10000g is centrifuged 1min.

9) column liquid was outwelled, 700 μ L DNA Wash Buffer (DNA cleaning buffer solution) are added, 10000g is centrifuged 1min. Repetitive operation is primary.

10) column liquid was outwelled, the sub- 16000g of void column is centrifuged 2min.

11) collecting pipe is changed into empty EP to manage, the ddH of 30 μ L is added₂O, stands 2min, and 10000g is centrifuged 1min, EP pipe In the liquid that is collected into be plasmid.

12) plasmid concentration that detection extracting obtains, and be placed in -20 DEG C of refrigerators and store.

(2) candidate positive colony be turned back to respectively containing control vector, bait plasmid, irrelevant antigen yeast strain AH109 is verified

The method of revolution verifying is transformed into identical in AH109 with bait plasmid.It is set in the experimentation for excluding false positive Set negative control.Negative control: the AH109 containing control vector pGBKT7 contains irrelevant antigen body pGBKT7-EpCAM's AH109.PGBKT7-EpCAM is synthesized after carrying out Yeast expression optimization by Suzhou Jin Weizhi company, and passes through EcoRI and BamHI enzyme Enzyme site is inserted into pGBKT7.Wherein, the visible NCBI of the sequence of EpCAM, the number of logging in: NP_002345.2 sequence 27-59 Base.The entire form for excluding to titrate during false positive using grid: the bacterium solution for taking 20 μ L to convert is added dropwise in SD/- In the grid finished on Trp/-Leu and SD/-Trp/-Leu/-His/-Ade+10mM 3-AT plate, each sub-box drips 2-3 drop. Be added dropwise on SD/-Trp/-Leu plate is that whether successful conversion is into AH109 in order to verify bait plasmid and binding protein plasmid. SD/-Trp/-Leu/-His/-Ade+10mM 3-AT plate is to exclude false positive.By turning round confirmatory experiment, it is obtained 3 Positive colony.

(3) DNA sequence analysis of positive colony

Obtain 3 clones are sent to company's sequencing, and combined with the website Ig BLAST NCBI find with same text It screens protein-bonded document and 3 positive colonies is analyzed in library.The sequence measured is counted in the lg blast of NCBI According to comparing, it is found that this 3 binding proteins have complete CDR region and the area FR, and CDR region between them and the area FR not phases Together.3 albumen that can be interacted with CXCR4 obtained by yeast two-hybrid screening and sequencing, are respectively designated as NB97 knot Hop protein, NB226 and NB412.It is as follows by the way that 3 obtained protein-bonded nucleotide sequence informations difference are sequenced:

NB97:GAAGTGCAGCTGGTGGAGTCTGGAGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCC TGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGT GGGTGGCAGTTATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAG AGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGCG AGAGATCGGGAATATAGTGGCTACGATTTCCGGTCGGAGTACTACTTTGACTACTGGGGCCAGGGAACCCTGGTCA CCGTCTCCTCAGCGGCCGCAATAACTTCGTATAATGTGTACTATACGAAGTTATTGGCGCGCCAGTCCTATGAGCT GATGCAGCCACCCTCAGTGTCAGTGGCCCCAGGACAGACGGCCAGCATTACCTGTGGGGGAAACAACATTGGAACT AAAAGTGTACACTGGTACCAGCAGAAGCCAGGCCAGGCCCCTGTATTGGGCATCTATTTTGATACTGACCGGCCCT CAGGGATCCCTGAGCGATTCTCTGGCTCCAACTCTGGGAACACGGCCACCCTGACCATCAGCAGGGTCGAAGCCGG GGATGAGGCCAACTATTCTTGTCAGGTGTGGGATAGTACTAGTGATCATGTGGTGTTTGGCGGAGGCACCCAGCTG ACCGTCCTCGCA；

NB226:CAGGTGCAGCTACAGCATTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCAC CTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAG TGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAG ACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAG AAGGGTAGGGGACTGGGGTCCCGTGTCTGGCCCGCAACGGACTTGGTACTTCGATCTCTGGGGCCGTGGCACCCTG GTCACTGTCTCCTCAGCGGCCGCAATAACTTCGTATAATGTGTACTATACGAAGTTATTGGCGCGCCAGGATATTG TGATGACACAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAG CCTCCTGCATAGTAATGGATACAACTATTTGCATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATC TATTTGGGTTCTCATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGA AAATCAGCAGAGTGGAGGCTGAGGATGTTGGGATTTATTACTGCATGCAAGCTCTACAACCTCCGCTCACTTTCGG CGGAGGGACCAAGCTGGAGATCAAAGCA；

NB412:CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCAC CTGCACTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAG TGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAG ACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTGTATTACTGTGCGAG AGTAGGGATTTTTGGAGTGGCTAGGGGGGGCTACTTTGACTACTGGAGCCAGGGAACCCTGGTCACTGTCTCCTCA GCGGCCGCAATAACTTCGTATAATGTGTACTATACGAAGTTATTGGCGCGCCAGGTCATCCGGATGACCCAGTCTC CTTCCACCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCCAGTCAGAGTATTAGTAGCTGGTT GGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAAGGCGTCTAGTTTAGAAAGTGGGGTC CCATCTAGGTTCAGCGGCAGTGGATCTGGGACAGAATTCACTCTCACCATCAGCAGCCTGCAGCCTGATGATTTTG CAACTTATTACTGCCAACAGTATGATGATTATTCTCACACGTTCGGCCAAGGGACCAAGCTGGAGATCAAAGCA。

The building of 5 expression vector of embodiment and protein-bonded expression and purification

(1) building of binding protein expression carrier

1) in order to which 3 protein-bonded target gene are cloned on pET-28a-sumo carrier, expanded by the method for PCR Increase target gene.Prepare PCR reaction system: 5 × PrimerStar buffer, 10 μ L, dNTP mixture (every kind of 2.5mM) 4 μ L, 1.5 μ L of upstream primer, 1.5 μ L of downstream primer, 0.5 μ L of template 10ng, PrimerStarDNA polymerase, aseptic deionized water are mended Enough to 50 μ L.PCR reaction: 96 DEG C of 10min；95 DEG C of 10sec, 60 DEG C of 10sec, 72 DEG C of 30sec, 30 circulations；72℃5min.

Primer is as follows:

NB97-F:5 '-TATAAGCTTGCGAAGTGCAGCTGGTGGAG-3 '；

NB97-R:5 '-TATCTCGAGGAGGACGGTCAGCTGGG-3 '；

NB226-F:5 '-TATAAGCTTGCCAGGTGCAGCTACAGCATT-3 '；

NB226-R:5 '-TATCTCGAGTTTGATCTCCAGCTTGGTCCC-3 '；

NB412-F:5 '-TATCCATGGATCAGGTGCAGCTACAGCAGT-3 '；

NB412-R:5 '-TATCTCGAGCTTTGATCTCCAGCTTGGTCC-3 '.

2) by the target gene of PCR amplification and pET-28a-sumo plasmid (being purchased from EMD Biosciences (Novagen)) Carry out double digestion, restriction enzyme site NcoI, XhoI.PCR product is purified by PCR product purification kit.

3) pET-28a-sumo carrier and PCR product are subjected to double enzyme digestion reaction, digestion system is as follows:

2 μ g of pET-28a-sumo vector plasmid, 5 μ L of enzyme cutting buffering liquid, 2 NcoI μ L, 2 XhoI μ L, sterile water complement to 50μL；1.4 μ g of PCR product, 3 μ L of enzyme cutting buffering liquid, 2 NcoI μ L, 2 μ L of Xho I, sterile water complement to 60 μ L.

Above-mentioned reaction system is respectively placed in 37 DEG C and keeps the temperature 15 minutes.Up to pET-28a-sumo double enzyme digestion product and purpose Segment double enzyme digestion product.Digestion products are purified by digestion products purification kit.

4) it is then attached reaction, system is as follows: the double enzymes of 0.1 μ g of pET-28a-sumo double enzyme digestion product, target fragment Cut 0.07 μ g of product, enzyme connects 2.5 μ L of buffer, 1 μ L of T4 DNA ligase, sterile water complements to 25 μ L.4 DEG C of connections overnight, obtain To connection product.

5) connection product is transformed into bacillus coli DH 5 alpha competent cell, be coated on containing 100 μ g/mL ampicillins On LB plate, 37 DEG C of overnight incubations carry out bacterium solution PCR identification after picking single colonie culture.It (is purchased from using plasmid extraction kit Omega the DH5 α containing target gene plasmid) is extracted.

(2) expression of binding protein inclusion body

1) pET 28a-sumo-NB (NB is NB97 or NB226 or NB412) plasmid containing target gene that extracting obtains 100 μ L e. coli bl21 competent bacterias (being purchased from Shanghai Wei Di Bioisystech Co., Ltd), finger gently springing is added in 1 μ g Centrifuge tube mixes.

2) 40min in ice face is placed, 42 DEG C of water-bath thermal shock 2min are then placed in 2min in ice, add 900 μ L LB liquid Body culture medium, 37 DEG C, cultivate 1h in 200rpm shaking table.

3) 10000g is centrifuged 1min, absorbs 900 μ L supernatants, and remaining 100 μ L bacterium solution is coated on LB/Kana⁺(contain 50 μ g/ ML kanamycins) on plate, puts upside down in 37 DEG C of bacteriological incubators and cultivate 12h.

4) one well-grown monoclonal of picking, is seeded to 5mL LB/Kana⁺In, in 37 DEG C, the shaking table of 200rpm Cultivate 12h.

5) bacterium solution 4mL is taken to be seeded to 400mL LB/Kana⁺In, 37 DEG C, 2.5h to OD is cultivated in 200rpm shaking table₆₀₀For 0.6-1.0.The bacterium solution of 1mL is taken to examine dye identification (the mycoprotein sample not induced) for subsequent destination protein.

6) IPTG (the final concentration of 0.5mM of IPTG) is added in bacterium solution, 30 DEG C, 220rpm inducing expression 6h.Take 1mL bacterium Liquid examines dye identification (the mycoprotein sample of induction) for subsequent destination protein, and remaining bacterium solution is centrifuged in 4 DEG C, 5000g 5min outwells supernatant, retains precipitating.

7) 20~40mL that pre-cooling is added breaks bacterium buffer and (prepares: by 14.6g sodium chloride and 2.42g Tris- alkali soluble solution In 900mL ultrapure water, pH value is adjusted to 7.45 using dilute hydrochloric acid, is settled to 1L, 4 DEG C of preservations use 100 × PMSF of preceding addition 10mL.) be resuspended be deposited in 50mL beaker.

8) ultrasonication bacterium, ultrasonication machine power are 650W, and setting ultrasonic disintegrator power is 40%, and work 4s, Stop 8s, working time 40min.

9) after being crushed, 4 DEG C, 13000g centrifugation 30min take 20 μ L supernatants to examine dye identification with destination protein later (Supernatant samples after the bacterial cell disruption of induction), remaining supernatant abandon, and retain precipitating, this precipitating is inclusion body.

10) it cleans inclusion body: 30mL inclusion body cleaning solution A is added and (prepares: 1.21g Tris, 1.75g NaCl, 0.8mL 190mL distilled water is added, after completely dissolution constant volume to 200mL in the above reagent by EDTA and 4mL Triton X-100,4 DEG C of storages Deposit) cleaning is stayed overnight, until not observing precipitating, take 20 μ L destination protein later examines dye identification (after the bacterial cell disruption of induction Deposit sample).

11) 4 DEG C, 13000g centrifugation 30min, abandon supernatant；Addition 30mL inclusion body cleaning solution B (it prepares: 1.21g Tris, The above reagent is added 190mL distilled water, sufficiently dissolved by 1.75g NaCl, 100 2mL × PMSF, 4mL Triton X-100 Constant volume is stored to 200mL, 4 DEG C afterwards) cleaning 1.5h, 4 DEG C, 13000g centrifugation 30min, abandoning supernatant.Inclusion body cleaning at this time finishes, It can be spare at any time in -80 DEG C of refrigerator long-term preservations.

12) pour into 20mL denaturation buffer (prepare: 0.968gTris, 5.84g NaCl, 100 × PMSF of 4mL, 380mL distilled water, the stirring and dissolving in 30 DEG C of water-bath is added in the above reagent by 192.192g urea, 0.276mL2-ME Afterwards, pH to 7.5 is adjusted with HCl, with 0.2 μm of membrane filtration, 4 DEG C of storages) dissolution inclusion body, it is transferred in 50mL beaker, is put into Rotor, is stirred at room temperature 2.5h, and 4 DEG C, 20000g centrifugation 40min retain supernatant, as solubilization of inclusion bodies denaturing soln.Take 20 μ L Dye identification (the precipitating denaturing liquid sample after the bacterial cell disruption of induction) is examined with destination protein later, 4 DEG C can save 3 days left sides It is right.

(3) protein-bonded purifying.

1) it balances pillar: taking a Ni-NTA HisBind Resin Purification Resin, 10mL denaturation buffer mistake is added Column.

2) loading: crossing column for solubilization of inclusion bodies denaturing soln, and destination protein is adsorbed on pillar, takes 20 μ L to cross column liquid and is used for The dye of examining of destination protein is identified and (crosses column liquid) below.

3) other 10mL denaturation buffer is added and crosses column, the protein sample left on pillar side wall is cleaned.

4) be added 20mL wash miscellaneous buffer (prepare: weigh 0.68g imidazoles, sufficiently dissolved with 5mL denaturation buffer, take with Upper 200 μ L of solution is added in 19.8mL denaturation buffer and mixes to get miscellaneous buffer is washed) column is crossed, each 8mL takes 20 μ L to wash Miscellaneous buffer destination protein later examines dye identification (washing miscellaneous liquid).

5) be added 10mL elution buffer (prepare: weigh 0.68g imidazoles, sufficiently dissolved with 5mL denaturation buffer, take with Upper solution 1mL is added in 9mL denaturation buffer and mixes to get eluent) destination protein is eluted, it is received with 1.5mL EP pipe Collection, every pipe collected column liquid 1mL, collected 5 pipes, and 20 μ L destination protein later is in addition taken in each EP pipe examines dye identification (eluent).

6) with the protein concentration of ultramicrospectrophotometer Nanodrop measurement sample.

(4) protein-bonded renaturation.

1) purified albumen is added in 5kDa bag filter, tightens bag filter both ends with rubber band, prevent that liquid seeps Leakage, should be there are a certain amount of air, in order to avoid bag filter spalling in dialysis in bag filter.

2) the whole protein renaturation liquid that is immersed in is prepared (: 20mM Tris-HCl (pH 9.0), 150mM NaCl, 3mM GSH (reduced glutathione), 1mM GSSG (oxidized form of glutathione), 8M-0M urea), Renaturation solution volume should be 100 times of protein sample volume, dialyse under the conditions of 4 DEG C.Urea concentration in the protein renaturation liquid from 8M-0M successively successively decreases, the dialysed overnight in each urea concentration gradients renaturation solution.

3) after dialysing, sample is taken out, detect protein concentration and is put into -80 DEG C of refrigerators preservations.

(5) PAGE gel electrophoresis and coomassie brilliant blue staining (referred to as examining dye).

1) compound concentration is the separation gel of 12% (w/v), and prepared separation gel is added to encapsulating die apart from top There are also the positions of 2-3cm or so, and dehydrated alcohol crimping is then added, and place 20min, wait separation gelling solid.

2) the concentration glue that 5% (w/v) is added after dehydrated alcohol is outwelled to top, plugs comb, static 30min waits concentration Gelling is solid, pulls out comb, prepares loading.

3) prepared offset plate is placed in electrophoresis tank, electrophoresis liquid is added.

4) by the protein sample being collected into above-mentioned binding protein expression purification step be added 5 × SDS-PAGE albumen on Sample buffer, make buffer final concentration of 1 ×, 100 DEG C of heating 10min, sample preparation finishes.

5) the 5 μ L of sample prepared is loaded on the offset plate of electrophoresis tank, albumen marker is added, 80V runs through concentration glue Changing 120V runs through separation gel later.

6) glue is taken out, coomassie brilliant blue staining vacuole 10min is added, changes dyeing liquor with clear water, is placed in micro-wave oven and uses It is cooked by medium heat 5min.

7) destainer is added, is placed on decolorization swinging table and decolourizes overnight, destainer is changed in not timing, until seeing clearly Purpose band, photographic analysis.As a result as shown in Figure 1, figure A is the protein-bonded electrophoresis result figure of NB97, combination egg after purification White molecular weight is about 43.2kDa；Scheming B is the protein-bonded electrophoresis result figure of NB226, and binding protein molecular weight after purification is big About 44kDa；Scheming C is the protein-bonded electrophoresis result figure of NB412, and binding protein molecular weight after purification is about 43kDa.

Embodiment 6 detects the combination of binding protein and antigens c XCR4 using ELISA

(1) using 100 μ L CXCR4 antigens-PBS buffer solution, (concentration of antigen is safe biological purchased from upper hypo for 2 μ g/mL Company) coating NUNC96 hole elisa Plates, 37 DEG C of standing 2h.Use PBS buffer solution coating as blank control.

(2) three times with PBS rinsing, the excess liq of elisa plate is abandoned on blotting paper by overturning.Again with 200 μ L 3% 37 DEG C of (w/v) BSA lock solution closing 2h, is washed three times with PBS, and excessive buffer is lost on blotting paper by overturning It abandons.

(3) binding protein-PBS mixed liquor that 100 μ L concentration are 20 μ g/mL is added to every hole, ELISA Plate is incubated at room temperature 60min is educated, hole is washed three times with the PBS containing 0.05% (w/v) Tween-20.

(4) 1:5000 is diluted in the PBS of 3% (w/v) BSA anti-His-HRP conjugate by volume, and 100 μ L is taken to add to In each hole, elisa plate 60min under incubation at room temperature, 100 μ L tmb substrate developing solutions are added in each hole and in dark Room incubates 10min, and OD value is read at wavelength 450nm.As a result as shown in Fig. 2, binding protein NB97, NB226, NB412 points Not in conjunction with antigens c XCR4, and do not combined substantially with PBS negative control group.

Embodiment 7 obtains the protein-bonded amino acid of negative control and DNA sequence dna

By display technique of bacteriophage, human epidermal growth factor receptor 2 (Her2) and blood is respectively adopted in this laboratory early period Endothelial tube growth factor (VEGF) synthesis polypeptide is the nano antibody NB277 and NB352 that antigen obtains, as negative right According to.Nano antibody NB277 and NB352 are respectively provided with the amino acid sequence as shown in SEQ ID NO:16 and SEQ ID NO:17；It compiles The nucleotide sequence of code NB277 and NB352 is respectively as shown in SEQ ID NO:21 and SEQ ID NO:22.In Escherichia coli, Retain supernatant by solubility expression of protein, purifying (with embodiment 5, in addition to the expression of (2) binding protein inclusion body, 9) step, Discard precipitating, 10) -12) it does not do, remaining step is the same), obtain nano antibody NB277 and NB352.

Embodiment 8 analyzes influence of the binding protein to tumor cell proliferation after purification using MTT experiment

(1) DU145 (Human Prostate Cancer Cells), PC-3 (Human Prostate Cancer Cells) and the MDA- of logarithmic growth phase are collected MB-231 (human breast cancer cell) cell (purchased from Shanghai Bo Gu biology), adjustment cell density is 5000/mL, is inoculated into 96 holes On tissue culture plate, every hole adds 200 μ L cell suspensions.

(2) in 37 DEG C, 5%CO₂Cell incubator in overnight incubation keep cell adherent, the 2nd day with contain 1% fetal calf serum DMEM culture medium starvation cultivate 6h.

(3) with 5 binding proteins, (NB97, NB226, NB412, NB277 and NB352, last 2 combine for negative control Albumen) under different concentration (0,25,50,100 μ g/mL) respectively with the 1 (SDF- of ligand matrices cell derived factor of CXCR4 1,100ng/mL, it is purchased from the Divine Land Beijing Yi Qiao Science and Technology Ltd.) co-incubation cell DU145, PC-3 and MDA-MB-231, 37 DEG C, 5%CO₂Cell incubator in cultivate 72h.

(4) 30 μ L MTT solution are added in every hole, are put into cell incubator and continue to cultivate 4h.

(5) 96 orifice plates are taken out, cell culture fluid is sucked out.200 μ L DMSO are added in every hole, are placed on Quick shaking on shaking table 10min makes crystal measure the OD value at wavelength 570nm in microplate reader after completely dissolution.As a result as shown in figure 3, binding protein NB97, NB226 and NB412 can inhibit the proliferation of DU145, PC-3 and MDA-MB-231 cancer cell, and with antibody concentration Increase, it is in apparent dose-dependence that inhibiting effect, which is more obvious, and 2 kinds of negative control antibody NB277 and NB352 to this 3 The no inhibiting effect of proliferation of kind cancer cell.

Embodiment 9 analyzes influence of the CXCR4 binding protein to tumor cell migration after purification using cell scratch experiment

(1) it is inoculated with DU145, PC-3 and MDA-MB-231 cell 2 × 10 of logarithmic growth phase⁵A cells/well is to 12 orifice plates.

(2) after the DMEM culture medium culture 12h containing 10% (v/v) FBS being added, the DMEM culture containing 1% (v/v) FBS is changed Base starvation cultivates 6h.

(3) it is crossed using 200 μ L pipette tips, PBS is washed 2 times, the cell crossed out is washed, takes pictures and measures under the microscope The width (L1) of scratch.

(4) with 5 binding proteins, (NB97, NB226, NB412, NB277 and NB352, last 2 are negative control respectively Binding protein) it is common with the ligand SDF-1 (100ng/mL) of CXCR4 respectively under different concentration (0,25,50,100 μ g/mL) Cell DU145, PC-3 and MDA-MB-231 are cultivated, in 37 DEG C, 5%CO₂It is cultivated in incubator and takes pictures afterwards for 24 hours and measure scratch Width (L2).

(5) computation migration rate, calculation formula: (L1 ﹣ L2)/L1 × 100%.As a result as shown in Figures 4 and 5, with 3 CXCR4 binding protein concentration increases, and the mobility of DU145, PC-3 and MDA-MB-231 cell reduces, it was demonstrated that after purification 3 A CXCR4 binding protein (NB97, NB226, NB412) is able to suppress the migration of DU145, PC-3 and MDA-MB-231 cell.

Experimental example 10 analyzes influence of the binding protein to tumor cell invasion after purification using Transwell Matrigel

(1) inner surface of the cell Transwell (8 μm of apertures, 24 orifice plates) is coated with 50 hole μ g/ of Matrigel matrigel, It is put into plastic 12h in cell incubator.

(2) the DMEM culture medium of DU145, PC-3 and MDA-MB-231 cell serum-free of logarithmic growth phase is hungry Cell will be resuspended with the DMEM culture medium containing 1% (v/v) FBS after cell dissociation, centrifugation in 6h.

(3) it is jointly processed by respectively with various concentration binding protein (0,25,50 and 100 μ g/mL) and SDF-1 (100ng/mL) 200 μ L (5 × 10 of cell suspension⁴A cell contains the serum of 1% (v/v)) upper chamber of the cell Transwell, 24 orifice plates are added The cell culture medium for containing 20% (v/v) FBS, 37 DEG C, 5%CO are added in lower room₂Incubator in cultivate for 24 hours.

(4) cell is taken out, wipes the cell that upper chamber is not invaded with cotton swab.4% (w/v) poly methanol is added in lower room to fix Cell 10min abandons fixer, and 500 μ L, 0.1% crystal violet dye liquor is added in lower room, dyes 30min, distillation washing 3 times will be small Room is just placed on glass slide, is taken pictures under microscope, and then 33% (w/v) acetic acid solution, 100 μ L is added in every hole, is sufficiently surveyed after oscillation Determine the light absorption angle value OD at 570nm.As a result as shown in Figures 6 and 7, binding protein NB97, NB226 and NB412 can inhibit The invasion of DU145, PC-3 and MDA-MB-231 cancer cell, and with the increase of antibody concentration, inhibiting effect is more obvious, in bright Aobvious dose-dependence, and 2 kinds of negative control antibody NB277 and NB352 do not inhibit to make to the invasion of this 3 kinds of cancer cells With.

Binding protein influence to tumor cell migration of the experimental example 11 using Transwell migration experimental analysis after purification

Step (1) middle berth Matrigel base is only omitted with Transwell Matrigel in embodiment 10 in method and step One step of matter glue.As a result as shown in FIG. 8 and 9, binding protein NB97, NB226 and NB412 can inhibit DU145, PC-3 and MDA- The migration of MB-231 cancer cell, and with the increase of antibody concentration, inhibiting effect is more obvious, and is closed in apparent dose-dependant System, and 2 kinds of negative control antibody NB277 and NB352 are to the no inhibiting effect of the invasion of this 3 kinds of cancer cells.

Binding protein of the experimental example 12 using stream type cell analyzer and the bis- dyeing method analyses of Annexin V/PI after purification Influence to apoptosis of tumor cells

(1) cancer cell of logarithmic growth phase cultivates 6h with the DMEM culture medium starvation of no FBS.

(2) respectively with concentration be 50 μ g/mL 5 binding proteins (NB97, NB226, NB412, NB277 and NB352, most Afterwards 2 be negative control binding protein) and the ligand SDF-1 (100ng/mL) of CXCR4 be jointly processed by tumour cell DU145, PC- 3 and MDA-MB-2313, in 37 DEG C, 5%CO₂Incubator in cultivate 48h.

(3) cancer cell is digested, and is cleaned cell 1 time with PBS.

(4) ddH is used₂O is 4 × combination buffer (Sangon Biotech (Shanghai) Co., Ltd., Annexin V/PI Apoptosis kit) it is diluted to 1 ×.

(5) cell is resuspended with 195 μ 1 × combination buffers of L, obtaining cell concentration is 5 × 10⁵The suspension of a cell/mL.

(6) 5 μ L fluorescein isothiocynates (Annexin V-FITC) are added, room temperature, which is protected from light, is incubated for 20min.

(7) after 1000rpm is centrifuged 5min, cleaning cell is resuspended with 200 μ L1 × combination buffer.

(8) 1000rpm is centrifuged 5min, abandons supernatant.

(9) cell is resuspended with 190 μ 1 × combination buffers of L.

(10) 10 μ L propidium iodide (PI) dyeing liquors are added.

(11) with the apoptosis situation of machine testing cancer cell on stream type cell analyzer.As a result as shown in FIG. 10 and 11, with nothing Binding protein control group is compared, and 3 binding proteins NB97, NB226 and NB412 can promote this DU145, PC-3 and MDA-MB- The apoptosis of 231 cancer cells, and 2 kinds of negative control antibody NB277 and NB352 do not have facilitation to the apoptosis of this 3 kinds of cancer cells.

Effect of the experimental example 13CXCR4 binding protein to Prostate Carcinoma of Mice model

(1) according to the external contractile studies of CXCR4 binding protein, selection building mouse DU145 cell prostate cancer mould Type chooses binding protein NB97 and NB226 and carries out zoopery.

(2) secondary culture is carried out to DU145 cell, when cell grows into logarithmic phase, pancreatin digests and collect cell, PBS After washing cell, appropriate PBS is added by cell density and is adjusted to 5 × 10⁷/mL。

(3) 100 μ L cell suspensions are drawn, suspension is injected in mouse with injected s.c., and (4 week old male Balb/C are naked Mouse is purchased from Guangdong Medical Lab Animal Center) oxter carries out making tumor.Mouse is raised in SPF grades of Animal Lab.s.

(4) a mouse tumor volume, gross tumor volume calculation formula: ab are measured every three days²×0.5。

(5) when 20 mouse tumor volumes rise to average 100mm³Left and right, is randomly divided into 4 groups, every group 5.Its In 1 group be positive controls (cis-platinum, DDP), 2 groups for experiment binding protein group (NB97, NB226), 1 group for negative control combine Protein groups (NB277).Using insulin syringe, it is administered by tail vein.Wherein test binding protein group and negative control knot Every mouse dosage of hop protein group is 10mg/kg, and every dosage of positive controls is 2mg/kg.Dosage period is 24 days, It is administered once and records mouse tumor volume within every 3 days.

(6) mouse is put to death after full experiment, strips each group mouse tumor, is measured tumor weight and is taken pictures.It is soft using SPSS Part carries out statistical analysis to data, as a result represents Mean ± S.D. (n=5).As a result as shown in figure 12, test and combine after administration Pushing the speed for protein groups gross tumor volume is obviously slowed down relative to negative control binding protein group, and final volume also may be significantly smaller, NB97 the and NB226 binding protein of proof after purification is able to suppress the tumour of DU145 mouse cell model of human prostate carcinoma.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, other any changes, modifications, substitutions, combinations, simplifications done without departing from the spirit and principles of the present invention, It should be equivalent substitute mode, be included within the scope of the present invention.

Sequence table

<110>Ji'nan University

<120>the source of people albumen in conjunction with CXCR4 and its application

<130> 1

<160> 29

<170> SIPOSequenceListing 1.0

<210> 1

<211> 126

<212> PRT

<213>people (Homo sapiens)

<220>

<223>amino acid sequence of the heavy chain variable region of NB97

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Glu Tyr Ser Gly Tyr Asp Phe Arg Ser Glu Tyr Tyr

100 105 110

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 2

<211> 109

<212> PRT

<213>people (Homo sapiens)

<220>

<223>amino acid sequence of the light chain variable region of NB97

<400> 2

Ser Tyr Glu Leu Met Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

1 5 10 15

Thr Ala Ser Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser Val

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Gly Ile Tyr

35 40 45

Phe Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

65 70 75 80

Asp Glu Ala Asn Tyr Ser Cys Gln Val Trp Asp Ser Thr Ser Asp His

85 90 95

Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Ala

100 105

<210> 3

<211> 127

<212> PRT

<213>people (Homo sapiens)

<220>

<223>amino acid sequence of the heavy chain variable region of NB226

<400> 3

Gln Val Gln Leu Gln His Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Arg Val Gly Asp Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 4

<211> 113

<212> PRT

<213>people (Homo sapiens)

<220>

<223>amino acid sequence of the light chain variable region of NB226

<400> 4

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Ala

85 90 95

Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

Ala

<210> 5

<211> 122

<212> PRT

<213>people (Homo sapiens)

<220>

<223>amino acid sequence of the heavy chain variable region of NB412

<400> 5

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Val Gly Ile Phe Gly Val Ala Arg Gly Gly Tyr Phe Asp Tyr Trp

100 105 110

Ser Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 6

<211> 108

<212> PRT

<213>people (Homo sapiens)

<220>

<223>amino acid sequence of the light chain variable region of NB412

<400> 6

Val Ile Arg Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asp Tyr Ser His

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ala

100 105

<210> 7

<211> 378

<212> DNA

<213>people (Homo sapiens)

<220>

<223>nucleotide sequence of NB97 heavy chain variable region is encoded

<400> 7

gaagtgcagc tggtggagtc tggaggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagcaa taaatactac 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagatcgg 300

gaatatagtg gctacgattt ccggtcggag tactactttg actactgggg ccagggaacc 360

ctggtcaccg tctcctca 378

<210> 8

<211> 327

<212> DNA

<213>people (Homo sapiens)

<220>

<223>nucleotide sequence of NB97 light chain variable region is encoded

<400> 8

tcctatgagc tgatgcagcc accctcagtg tcagtggccc caggacagac ggccagcatt 60

acctgtgggg gaaacaacat tggaactaaa agtgtacact ggtaccagca gaagccaggc 120

caggcccctg tattgggcat ctattttgat actgaccggc cctcagggat ccctgagcga 180

ttctctggct ccaactctgg gaacacggcc accctgacca tcagcagggt cgaagccggg 240

gatgaggcca actattcttg tcaggtgtgg gatagtacta gtgatcatgt ggtgtttggc 300

ggaggcaccc agctgaccgt cctcgca 327

<210> 9

<211> 381

<212> DNA

<213>people (Homo sapiens)

<220>

<223>nucleotide sequence of NB226 heavy chain variable region is encoded

<400> 9

caggtgcagc tacagcattg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagggtaggg 300

gactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg gggccgtggc 360

accctggtca ctgtctcctc a 381

<210> 10

<211> 339

<212> DNA

<213>people (Homo sapiens)

<220>

<223>nucleotide sequence of NB226 light chain variable region is encoded

<400> 10

gatattgtga tgacacagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60

atctcctgca ggtctagtca gagcctcctg catagtaatg gatacaacta tttgcattgg 120

tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc tcatcgggcc 180

tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc 240

agcagagtgg aggctgagga tgttgggatt tattactgca tgcaagctct acaacctccg 300

ctcactttcg gcggagggac caagctggag atcaaagca 339

<210> 11

<211> 366

<212> DNA

<213>people (Homo sapiens)

<220>

<223>nucleotide sequence of NB412 heavy chain variable region is encoded

<400> 11

caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcactg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agtagggatt 300

tttggagtgg ctaggggggg ctactttgac tactggagcc agggaaccct ggtcactgtc 360

tcctca 366

<210> 12

<211> 324

<212> DNA

<213>people (Homo sapiens)

<220>

<223>nucleotide sequence of NB412 light chain variable region is encoded

<400> 12

gtcatccgga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60

atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatct 180

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 240

gatgattttg caacttatta ctgccaacag tatgatgatt attctcacac gttcggccaa 300

gggaccaagc tggagatcaa agca 324

<210> 13

<211> 253

<212> PRT

<213>people (Homo sapiens)

<220>

<223>NB97 antibody

<400> 13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Arg Glu Tyr Ser Gly Tyr Asp Phe Arg Ser Glu Tyr Tyr

100 105 110

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala

115 120 125

Ala Ile Thr Ser Tyr Asn Val Tyr Tyr Thr Lys Leu Leu Ala Arg Gln

130 135 140

Ser Tyr Glu Leu Met Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln

145 150 155 160

Thr Ala Ser Ile Thr Cys Gly Gly Asn Asn Ile Gly Thr Lys Ser Val

165 170 175

His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Gly Ile Tyr

180 185 190

Phe Asp Thr Asp Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

195 200 205

Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Val Glu Ala Gly

210 215 220

Asp Glu Ala Asn Tyr Ser Cys Gln Val Trp Asp Ser Thr Ser Asp His

225 230 235 240

Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Ala

245 250

<210> 14

<211> 258

<212> PRT

<213>people (Homo sapiens)

<220>

<223>NB226 antibody

<400> 14

Gln Val Gln Leu Gln His Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Arg Val Gly Asp Trp Gly Pro Val Ser Gly Pro Gln Arg Thr Trp

100 105 110

Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser Ala

115 120 125

Ala Ala Ile Thr Ser Tyr Asn Val Tyr Tyr Thr Lys Leu Leu Ala Arg

130 135 140

Gln Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro

145 150 155 160

Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His

165 170 175

Ser Asn Gly Tyr Asn Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln

180 185 190

Ser Pro Gln Leu Leu Ile Tyr Leu Gly Ser His Arg Ala Ser Gly Val

195 200 205

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

210 215 220

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

225 230 235 240

Ala Leu Gln Pro Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

245 250 255

Lys Ala

<210> 15

<211> 248

<212> PRT

<213>people (Homo sapiens)

<220>

<223>NB412 antibody

<400> 15

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Tyr Gly Gly Ser Phe Ser Gly Tyr

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Val Gly Ile Phe Gly Val Ala Arg Gly Gly Tyr Phe Asp Tyr Trp

100 105 110

Ser Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala Ile Thr Ser

115 120 125

Tyr Asn Val Tyr Tyr Thr Lys Leu Leu Ala Arg Gln Val Ile Arg Met

130 135 140

Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp Arg Val Thr

145 150 155 160

Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu Ala Trp Tyr

165 170 175

Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser

180 185 190

Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly

195 200 205

Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp Phe Ala

210 215 220

Thr Tyr Tyr Cys Gln Gln Tyr Asp Asp Tyr Ser His Thr Phe Gly Gln

225 230 235 240

Gly Thr Lys Leu Glu Ile Lys Ala

245

<210> 16

<211> 129

<212> PRT

<213>people (Homo sapiens)

<400> 16

Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Val Ser

20 25 30

Ser Glu Asn Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Gly Ile Leu Ala Gly Asp Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Phe Thr Ser Gly Gln Gly Ser Leu Arg Ser Asp Pro

100 105 110

Ile Arg Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala

115 120 125

Ala

<210> 17

<211> 128

<212> PRT

<213>people (Homo sapiens)

<400> 17

Met Ala Gln Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro

1 5 10 15

Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Ser Val Ser

20 25 30

Asn Glu Ala Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu

35 40 45

Trp Val Ser Ser Ile Thr Asp Gln Ser Gly Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Gly Gln Arg Arg Arg Gln Met His Ser Tyr Lys Val

100 105 110

Ser Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala Ala

115 120 125

<210> 18

<211> 759

<212> DNA

<213>people (Homo sapiens)

<220>

<223>nucleotide sequence of NB97 antibody is encoded

<400> 18

gaagtgcagc tggtggagtc tggaggaggc gtggtccagc ctgggaggtc cctgagactc 60

tcctgtgcag cctctggatt caccttcagt agctatgcta tgcactgggt ccgccaggct 120

ccaggcaagg ggctggagtg ggtggcagtt atatcatatg atggaagcaa taaatactac 180

gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgag agctgaggac acggctgtgt attactgtgc gagagatcgg 300

gaatatagtg gctacgattt ccggtcggag tactactttg actactgggg ccagggaacc 360

ctggtcaccg tctcctcagc ggccgcaata acttcgtata atgtgtacta tacgaagtta 420

ttggcgcgcc agtcctatga gctgatgcag ccaccctcag tgtcagtggc cccaggacag 480

acggccagca ttacctgtgg gggaaacaac attggaacta aaagtgtaca ctggtaccag 540

cagaagccag gccaggcccc tgtattgggc atctattttg atactgaccg gccctcaggg 600

atccctgagc gattctctgg ctccaactct gggaacacgg ccaccctgac catcagcagg 660

gtcgaagccg gggatgaggc caactattct tgtcaggtgt gggatagtac tagtgatcat 720

gtggtgtttg gcggaggcac ccagctgacc gtcctcgca 759

<210> 19

<211> 774

<212> DNA

<213>people (Homo sapiens)

<220>

<223>nucleotide sequence of NB226 antibody is encoded

<400> 19

caggtgcagc tacagcattg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag aagggtaggg 300

gactggggtc ccgtgtctgg cccgcaacgg acttggtact tcgatctctg gggccgtggc 360

accctggtca ctgtctcctc agcggccgca ataacttcgt ataatgtgta ctatacgaag 420

ttattggcgc gccaggatat tgtgatgaca cagtctccac tctccctgcc cgtcacccct 480

ggagagccgg cctccatctc ctgcaggtct agtcagagcc tcctgcatag taatggatac 540

aactatttgc attggtacct gcagaagcca gggcagtctc cacagctcct gatctatttg 600

ggttctcatc gggcctccgg ggtccctgac aggttcagtg gcagtggatc aggcacagat 660

tttacactga aaatcagcag agtggaggct gaggatgttg ggatttatta ctgcatgcaa 720

gctctacaac ctccgctcac tttcggcgga gggaccaagc tggagatcaa agca 774

<210> 20

<211> 744

<212> DNA

<213>people (Homo sapiens)

<220>

<223>nucleotide sequence of NB412 antibody is encoded

<400> 20

caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcactg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca gtagacacgt ccaagaacca gttctccctg 240

aagctgagct ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agtagggatt 300

tttggagtgg ctaggggggg ctactttgac tactggagcc agggaaccct ggtcactgtc 360

tcctcagcgg ccgcaataac ttcgtataat gtgtactata cgaagttatt ggcgcgccag 420

gtcatccgga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 480

atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca 540

gggaaagccc ctaagctcct gatctataag gcgtctagtt tagaaagtgg ggtcccatct 600

aggttcagcg gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct 660

gatgattttg caacttatta ctgccaacag tatgatgatt attctcacac gttcggccaa 720

gggaccaagc tggagatcaa agca 744

<210> 21

<211> 387

<212> DNA

<213>people (Homo sapiens)

<220>

<223>nucleotide sequence of NB277 antibody is encoded

<400> 21

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggatatagc gttagctctg agaatatggg ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaggcattt tggcgggaga cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaga 300

tttacgtcgg gtcaggggtc gttgcggtcc gaccccatcc ggtcttgggg tcagggaacc 360

ctggtcaccg tctcgagcgc ggccgca 387

<210> 22

<211> 384

<212> DNA

<213>people (Homo sapiens)

<220>

<223>nucleotide sequence of NB352 antibody is encoded

<400> 22

atggcccagg tgcagctgtt ggagtctggg ggaggcttgg tacagcctgg ggggtccctg 60

cgtctctcct gtgcagcctc cggagttagc gttagcaatg aggctatggg ctgggtccgc 120

caggctccag ggaagggtct agagtgggta tcaagcatta ctgaccaaag cggtagcaca 180

tactacgcag actccgtgaa gggccggttc accatctccc gtgacaattc caagaacacg 240

ctgtatctgc aaatgaacag cctgcgtgcc gaggacaccg cggtatatta ttgcgcgaga 300

gggcagcgtc gtaggcagat gcattcgtac aaggtcagct cttggggtca gggaaccctg 360

gtcaccgtct cgagcgcggc cgca 384

<210> 23

<211> 84

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>the CXCR4 sequence after optimizing

<400> 23

aacgtcagcg aggcagatga cagatatatc tgtgaccgct tctaccccaa tgacttgtgg 60

gtggttgtgt tccagtttca gtaa 84

<210> 24

<211> 29

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>primer NB97-F

<400> 24

tataagcttg cgaagtgcag ctggtggag 29

<210> 25

<211> 26

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>primer NB97-R

<400> 25

tatctcgagg aggacggtca gctggg 26

<210> 26

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>primer NB226-F

<400> 26

tataagcttg ccaggtgcag ctacagcatt 30

<210> 27

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>primer NB226-R

<400> 27

tatctcgagt ttgatctcca gcttggtccc 30

<210> 28

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>primer N412-F

<400> 28

tatccatgga tcaggtgcag ctacagcagt 30

<210> 29

<211> 30

<212> DNA

<213>artificial sequence (Artificial Sequence)

<220>

<223>primer N412-R

<400> 29

tatctcgagc tttgatctcc agcttggtcc 30

Claims

1. a human protein combined with CXCR4, it is characterized in that: described human protein combined with CXCR4 is one or at least two in combination of NB97 binding protein, NB226 binding protein and NB412 binding protein. the binding protein;

The NB97 binding protein comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 1 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO: 2;

The NB226 binding protein comprises a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:3 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:4;

The NB412 binding protein includes a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO:5 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO:6.

2 . The human protein binding to CXCR4 according to claim 1 , wherein the amino acid sequence of the NB97 binding protein is shown in SEQ ID NO: 13. 3 .

3 . The human protein binding to CXCR4 according to claim 1 , wherein the amino acid sequence of the NB226 binding protein is shown in SEQ ID NO: 14. 4 .

4 . The human protein binding to CXCR4 according to claim 1 , wherein the amino acid sequence of the NB412 binding protein is shown in SEQ ID NO: 15. 5 .

5. The nucleotide sequence encoding the human protein binding to CXCR4 according to claim 1, characterized in that: the nucleotide sequence encoding the NB97 binding protein, the nucleotide sequence encoding the NB226 binding protein A nucleotide sequence formed by one or at least two of the acid sequence and the nucleotide sequence encoding the NB412 binding protein.

6. The nucleotide sequence encoding a human protein that binds to CXCR4 according to claim 5, wherein the nucleotide sequence encoding the NB97 binding protein comprises the encoding shown in SEQ ID NO:7 The nucleotide sequence of the heavy chain variable region and the nucleotide sequence encoding the light chain variable region as shown in SEQ ID NO: 8;

The nucleotide sequence encoding the NB226 binding protein includes the nucleotide sequence encoding the variable region of the heavy chain as shown in SEQ ID NO: 9 and the variable region encoding the light chain as shown in SEQ ID NO: 10. nucleotide sequence;

The nucleotide sequence encoding the NB412 binding protein includes the nucleotide sequence encoding the variable region of the heavy chain as shown in SEQ ID NO: 11 and the variable region encoding the light chain as shown in SEQ ID NO: 12. Nucleotide sequence.

7 . The nucleotide sequence encoding a human protein that binds to CXCR4 according to claim 6 , wherein the nucleotide sequence encoding the NB97 binding protein is shown in SEQ ID NO: 18. 8 .

8 . The nucleotide sequence encoding a human protein that binds to CXCR4 according to claim 6 , wherein the nucleotide sequence encoding the NB226 binding protein is shown in SEQ ID NO: 19. 9 .

9 . The nucleotide sequence encoding a human protein that binds to CXCR4 according to claim 6 , wherein the nucleotide sequence encoding the NB412 binding protein is shown in SEQ ID NO: 20. 10 .

10. The application of the human protein binding to CXCR4 according to claim 1 in the preparation of antitumor drugs.