CN102936277B

CN102936277B - Human blood vessel endothelial growth factor antigen epitope and epitope vaccine thereof

Info

Publication number: CN102936277B
Application number: CN201110452570.0A
Authority: CN
Inventors: 高建恩; 罗时伟; 钱军
Original assignee: Beijing Wu Kang Emerging Technology Co Ltd
Current assignee: Zuze regenerative medical technology (Beijing) Co., Ltd
Priority date: 2011-12-29
Filing date: 2011-12-29
Publication date: 2014-10-01
Anticipated expiration: 2031-12-29
Also published as: CN102936277A

Abstract

The invention relates to an antigen epitope and of a human blood vessel endothelial growth factor, and an epitope vaccine thereof, and belongs to the field of biotechnology. The invention discloses the antigen epitope of the human blood vessel endothelial growth factor, and also discloses the epitope vaccine of the human blood vessel endothelial growth factor built on the basis of a single domain antibody skeleton. The epitope vaccine can effectively induce animals to produce antibodies for the human blood vessel endothelial growth factor, presents relatively good antineoplastic growth effects in the bodies of the animals, and can be used for drugs of solid tumors such as tumors and the like.

Description

Human vascular endothelial growth factor epitope and epiposition vaccine thereof

Technical field

The invention belongs to biological technical field, be specifically related to a kind of epitope of human vascular endothelial growth factor, and utilize the human vascular endothelial growth factor epiposition vaccine of this epitope design, this epiposition vaccine can be used for the treatment of the solid tumors such as liver cancer.

Background technology

The tumor angiogenesis that vascular endothelial growth factor (vascular endothelial growth factor, VEGF) and acceptor thereof mediate plays an important role in the generation of tumour, development.It is one of important directions of current antitumor drug research and development that anti-tumor neovascularization generates.The most successful anti-tumor angiogenesis drug is the Avastin that Roche Holding Ag produces so far.Avastin is a kind of antibody drug, and its antigen molecule of identifying is exactly people VEGF.At present, Avastin is used for the treatment of advanced CRC, recurrent neurospongioma and tumor of kidney etc. by U.S. FDA approval.

Avastin produces by mammalian cell, its production cost is high, as a kind of antibody drug, its quantity is larger again, so very high with the cost of Avastin treatment tumour, common people are difficult to bear, therefore the cheap new type antineoplastic medicine of, finding, design taking people VEGF as target has realistic meaning.

Avastin is a kind of humanized antibody, and its people source degree is about 95%, and suppressing tumor growth with Avastin is a kind of passive immunotherapy method.If can design a kind of people VEGF vaccine, induction body self produces the neutrality antibody for people VEGF, can realize the active immunity treatment to tumour.But people VEGF is owing to being the molecule of human body self, human body has immunotolerance to it, can not effectively induce generated human antibody, therefore, can not directly people VEGF be used in human body as vaccine.

Synthetic peptide vaccine (synthetical peptide vaccine) is the little peptide that is similar to epitope (approximately 20～40 amino acid) with chemical synthesis synthetic.Synthetic peptide vaccine is generally made up of jointly multiple B cell epitopes and t cell epitope.At present, the effect of synthetic peptide vaccine is generally undesirable, and major cause comprises, is difficult to the three-dimensional structure of corresponding site in accurate analogue antigen molecule, and T, B cell epitope can not effectively be brought into play synergy, and synthetic peptide vaccine molecule is little, immunogenicity is poor etc.Therefore, the epitope of analyst VEGF, design, synthetic people VEGF polypeptide vaccine are difficult to the immune effect that reaches desirable.

If can design a kind of novel people VEGF recombination epitope vaccine, break the immunotolerance of body, allow body can effectively produce the neutrality antibody for people VEGF, just can realize the active immunity treatment to tumour.Give human immunity people VEGF epiposition vaccine induce in vivo generation have neutralization active antibody will be complete human antibody, there is biocompatibility completely with tumour patient self.And recombination epitope vaccine generally can be produced by prokaryotic expression systems such as intestinal bacteria, its manufacturing cost is lower, and its clinical application number of times is few, and consumption is low, so the active immunity treatment cost carrying out with the epiposition vaccine of restructuring is also lower.

1989, Ward etc. (Nature 1989,341:544-546) prepared the genetic engineering antibody being only made up of antibody heavy chain variable region (VH) structural domain first, and called after single domain antibody.1993, Hamers-Casterman etc. (Nature 1993,363:446-448) reported the antibody that has in a large number a kind of natural disappearance light chain in camel serum, i.e. heavy chain antibody.Heavy chain antibody plays an important role in the humoral immunization of camel.Compared with general antibody, heavy chain antibody is except lacking light chain, variable region of heavy chain (the variable domain of heavy chain of heavy antibody of heavy chain antibody, VHH) also there is the feature that is different from general antibody VH district, the VHH gene of camel, by maintained the normal function of heavy chain antibody at the selective evolution in several critical functions site, that is to say that the variable region of heavy chain (VHH) of heavy chain antibody can be folded to form separately stable and complete antigen binding site.The single domain antibody that variable region of heavy chain based on animal heavy chain antibodies such as camels builds has good structural stability, and the variable region of heavy chain of Huo Shu source, people source antibody is carried out after camelization transformation, also can build the single domain antibody that structure is more stable.

Though single domain antibody molecule is little, structure is relatively stable, particularly builds the single domain antibody from the heavy chain antibody such as camel, shark.At three antigen complementary determining region (complementarity-determining region of single domain antibody, CDR) in, antigen complementary determining region 3 (CDR3 district) with the combination of antigen in bring into play Main Function, the length variations scope of CDR3 is larger, can form abundant conformation to participate in conjugated antigen, changing a kind of angle sees, that is to say that CDR3 district can show the peptide section of different lengths, form various conformation, therefore, the CDR3 of single domain antibody should be that a kind of desirable epitope is shown position.

Based on above thinking, first the present invention has predicted the epitope of people VEGF, then, utilize the CDR3 district of single domain antibody skeleton to show this epitope, thereby build a kind of people VEGF epiposition vaccine, this vaccine can induce body to produce the antibody for people VEGF, can suppress in vivo the growth of tumour.

Summary of the invention

The object of the present invention is to provide a kind of novel people VEGF epiposition vaccine with potential medical science or pharmacy value, thereby realize the active immunity treatment to tumour, solve antibody heterology, the treatment cost high deficiency of the anti-human VEGF antibody drug using clinically at present to oncotherapy existence.

For achieving the above object, the invention process following technical scheme.

The invention provides the epitope of a kind of people VEGF, comprise the aminoacid sequence shown in SEQ ID NO:1.

The present invention also provides the epitope of a kind of people VEGF, comprises the aminoacid sequence shown in SEQ ID NO:2.

The present invention also provides the epitope of a kind of people VEGF, comprises the aminoacid sequence shown in SEQ ID NO:3.

The present invention also provides the epitope of a kind of people VEGF, and its aminoacid sequence is as shown in SEQ ID NO:3.

The present invention also provides a kind of albumen of the people of including VEGF epitope, and described albumen utilizes single domain antibody for molecular skeleton, and by the epitope sequence shows of arbitrary SEQ ID NO:1-3 described people VEGF in the CDR3 district of described single domain antibody.

Further, the present invention also provides a kind of albumen of the people of including VEGF epitope, and described albumen utilizes single domain antibody for molecular skeleton, and replaces the CDR3 district of described single domain antibody by the epitope sequence of the arbitrary described people VEGF of SEQ ID NO:1-3.

The amino acid of the 9th, 10 and 12 in the district of single domain antibody FR2 described in described albumen is replaced by respectively L-glutamic acid, arginine and glycine.

The present invention also provides a kind of albumen of the people of including VEGF epitope, and described single domain antibody builds from people's antibody, camel heavy chain antibody or murine antibody.

The present invention also provides a kind of albumen of the people of including VEGF epitope, and the aminoacid sequence of described albumen comprises the sequence shown in SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:10.

The present invention also provides the method for preparing above-mentioned albumen, and in the time that single domain antibody derives from the variable region of heavy chain of heavy chain antibody, the described basic step of preparing epiposition vaccine comprises:

(1) determine a kind of complete aminoacid sequence of variable region of heavy chain of heavy chain antibody, the aminoacid sequence using this sequence as single domain antibody, and analyze the sequence in its CDR3 district;

(2) replace the CDR3 region amino acid sequence of described single domain antibody by the epitope sequence of the arbitrary human vascular endothelial growth factor of SEQ ID NO:1-3, and the full length amino acid sequence after replacing is converted to its gene coded sequence, synthetic full-length gene order, and this full-length gene order is cloned in expression vector, obtain recombinant plasmid;

(3) by extremely corresponding the recombinant plasmid transformed obtaining host cell, screening obtains positive colony, and the recombinant protein that this positive colony is expressed is people VEGF epiposition vaccine albumen;

In the time that described single domain antibody derives from the variable region of heavy chain containing the conventional antibody of light chain antibody and heavy chain antibody, the described basic step of preparing epiposition vaccine comprises:

(1 ') determines the complete amino acid sequence containing the variable region of heavy chain of the conventional antibody of light chain and heavy chain, this sequence is the basic aminoacid sequence of single domain antibody, analyze the sequence in its framework region 2 (FR2) and CDR3 district, the amino acid of the 9th, 10 and 12 in Bing Jiang FR2 district replaces with respectively L-glutamic acid, arginine and glycine;

The present invention also provides a kind of people VEGF epiposition vaccine, and described epiposition vaccine comprises above-mentioned albumen.

The present invention also provides the purposes of above-mentioned albumen in the medicine for the preparation for the treatment of or prevention solid tumor.Especially as the purposes of vaccine medicine.

The present invention also provides the application in treatment of solid tumors field of above-mentioned people VEGF epiposition vaccine.

People VEGF epiposition vaccine of the present invention has shown good immunogenicity, has suppressed preferably the growth of liver cancer tumour, can develop into a kind of active immunity treatment medicine of the solid tumors such as a kind of Hepatoma therapy.Above-mentioned people VEGF epiposition vaccine provided by the invention has overcome the poor shortcoming of synthetic polypeptide vaccine immunogenicity, has shown good immunogenicity, has avoided heterology and the high deficiency for the treatment of cost of current anti-human VEGF antibody class anti-tumor medicine.It is complete human antibody that people VEGF epiposition vaccine of the present invention is induced the antibody producing in tumour patient body, there is 100% biocompatibility with patient itself, and people VEGF epiposition vaccine of the present invention can be realized high expression level in intestinal bacteria, production cost is lower, its clinical usage quantity is also low, so, clinical treatment cost will be lower, and this will overcome the existing high problem of clinical treatment cost that antibody drug production cost is very high, usage quantity causes greatly at eukaryotic expressions such as CHO.

Brief description of the drawings

For content of the present invention is more easily expressly understood, below according to a particular embodiment of the invention and by reference to the accompanying drawings, the present invention is further detailed explanation, wherein

A kind of mouse antibodies weight chain variable of Fig. 1 region amino acid sequence;

*: two line part WeiFR2 district, italicized item is CDR3 district;

Fig. 2 shows " PHQGQ " epitope with the single domain antibody skeleton that comes from mouse antibodies variable region of heavy chain;

*: single underscore part WeiFR2 district, italicized item is improved amino-acid residue;

Double underline part is the sequence that comprises " PHQGQ " epi-position after replacing;

Fig. 3 shows the mouse single domain antibody skeleton recombinant protein encoding gene of " PHQGQ " epitope;

*: single underscore part is restriction enzyme site;

The immune effect of the mouse single domain antibody skeleton recombinant protein of Fig. 4 displaying " PHQGQ " epi-position to mouse;

Fig. 5 shows " PHQGQHI " epitope with camel single domain antibody skeleton;

*: two line parts are the epitope sequence of displaying and the appended sequence of both sides;

Fig. 6 shows the encoding gene of the camel single domain antibody skeleton of " PHQGQHI " epitope;

*: underscore part is respectively BamHI and Xho I restriction enzyme site;

Fig. 7 induces and produces anti-human VEGF antibody with " PHQGQHI " epitope that camel single domain antibody skeleton is shown in Mice Body;

The aminoacid sequence of people's single domain antibody of Fig. 8 camelization transformation;

*: two line parts are framework region 2 (FR2);

Adding frame part is the improved amino acid of camelization;

Single underscore part is CDR3 district;

Fig. 9 people VEGF epiposition vaccine aminoacid sequence;

*: two line parts are the aminoacid sequence of people VEGF epitope;

Figure 10 people VEGF epiposition vaccine coding gene sequence;

*: double underline part is respectively BamHI and Xho I restriction enzyme site;

Figure 11 ELISA method detects the immune effect of people VEGF epiposition vaccine;

In Figure 12 people VEGF epiposition vaccine immune serum and activation analysis;

Figure 13 people VEGF epiposition vaccine immune group and the comparison of control group tumor weight.

Embodiment

Analysis, the checking of embodiment 1 people VEGF epitope

The Characterization of antigenic epitopes method of protein generally has synthetic peptide proof method, computer assisted program prediction procedure and the analytical method based on protein three-dimensional structure, but any method all exists limitation, wherein the Epitope prediction accuracy based on protein three-dimensional structure is the highest.At present, be generally to combine to use two or more analytical procedure to carry out the epitope of predicted protein matter, and finally determine whether an epitope is that real epitope also must be verified by experiment.

The present invention adopts computer assisted program prediction procedure and the analytical method based on protein three-dimensional structure to predict most probable people VEGF epitope, then, verifies by experiment whether this epi-position is real epitope.

First,, with the possible epitope of BepiPred (http://www.cbs.dtu.dk/services/BepiPred/) Epitope prediction program initial analysis people VEGF, predicting candidate epitope is as follows:

Table 1 people VEGF Antigen Epitope Prediction

Numbering	Zero position	Final position	Epitope peptide section	Peptide segment length
					1	1	11	APMAEGGGQNH	11
2	38	42	EYPDE	5
					3	66	75	LECVPTEESN	10
4	85	91	PHQGQHI	7
					5	103	122	ECRPKKDRARQENPCGPCSE	20
6	132	135	PQTC	4
					7	140	146	KNTDSRC	7

Crystalline structure (1FLT) in conjunction with people VEGF in PDB database (http://www.rcsb.org/pdb/home/home.do) and its receptor protein complex body is known, No. 4 candidate's epi-positions " PHQGQHI " (SEQ ID NO:2) in upper table are positioned at the bonding surface of people VEGF and acceptor, wherein, " PHQGQ " sequence forms ring-shaped area (Loop district), therefore the sequence (SEQ ID NO:1) that, comprises " PHQGQ " five amino acid is exactly likely an epitope of people VEGF.

In order to verify whether " PHQGQ " sequence is the real epitope of people VEGF, by " PHQGQ " sequence shows in a kind of structure on the single domain antibody skeleton of mouse antibodies, form a kind of new albumen, utilize this new protein immunization mouse, detect the antibody that whether has specific recognition people VEGF in serum.

Specific practice is as follows:

From PDB database (http://www.rcsb.org/pdb/home/home.do), find a kind of mouse antibodies structured data (3D69), therefrom determine that its weight chain variable region amino acid sequence is as shown in SEQ ID NO:4, (as Fig. 1, two line part WeiFR2 district, italicized item is CDR3 district), and basic aminoacid sequence using this sequence as mouse single domain antibody.

Single domain antibody basis, mouse source sequence in Fig. 1 is carried out to camelization transformation, wherein the 9th of FR2 the, the 10th and the 12nd amino acids replaces with respectively L-glutamic acid, arginine and glycine, simultaneously, " PHQGQ " sequence is substituted in CDR3 district, sequence after replacement completes, as shown in SEQ ID NO:5, (is shown in accompanying drawing 2, wherein, the amino acid of italicized item is the improved amino acid of FR2, and two line parts are the sequence that comprises " PHQGQ " epi-position after replacing).

SEQ ID NO:5 sequence after replacing is converted to its gene coded sequence (http://www.bioinformatics.org/sms2/rev_trans.html), BamHI and Xho I restriction enzyme site are added respectively in downstream thereon simultaneously, obtain full length sequence as shown in SEQ ID NO:6 (as Fig. 3, underscore part is respectively BamHI and Xho I restriction enzyme site), SEQ ID NO:6 sequence is transferred to Inovogen Tech Co., Ltd. to synthesize and is cloned in pUCE carrier called after pUCE-VE1.

BamH I for full length sequence (Takara) and Xho I (Takara) after synthetic are carried out to double digestion operation, and the enzyme system of cutting is:

Under 37 DEG C of conditions, enzyme is cut 6 hours.Then, reclaim goal gene fragment by electrophoresis.

Use equally BamH I (Takara) and Xho I (Takara) to carry out double digestion in pET24a carrier (purchased from Novagen company), the enzyme system of cutting is:

Under 37 DEG C of conditions, enzyme is cut 6 hours.Then, reclaim the large fragment of pET24a carrier by agarose electrophoresis.

The pET24a expression vector large fragment of the goal gene fragment of recovery and recovery is connected and is spent the night 16 DEG C of water-baths, and system is as follows:

10 μ l are connected to product to be added in the competence e. coli bl21 (DE3) of the fresh preparation of 100 μ l, on ice bath, place 30min, then, centrifuge tube is put into 42 DEG C of water-baths, after heat shock 90s, fast centrifuge tube is transferred in ice bath, make it cooling 2min, afterwards, add the LB substratum 500 μ l of antibiotic-free to every pipe, 45min is cultivated in 37 DEG C of concussions; Afterwards, bacterium liquid is applied on the LB agar plate that contains Kan resistance, is inverted plate overnight incubation in 37 DEG C of incubators, 3～5 single bacterium colonies of picking next day are cultivated to the LB substratum of Kan resistance, and 37 DEG C of shaking culture are to OD ₆₀₀reach at 0.5 o'clock, add 1mmol/L IPTG to carry out abduction delivering, abduction delivering, after 4 hours, is collected thalline, by electrophoretic analysis target protein expression, and retains the recombinant clone bacterium that expression amount is large.

By the recombinant bacterial strain after abduction delivering, be placed in ice bath supersound process, condition is electric current 350mA, pulse 5 seconds, 4 seconds, interval, ultrasonic 30 minutes altogether, then by the thalline after ultrasonic in 12000r/min, 4 DEG C are centrifugal 15 minutes, collecting precipitation and supernatant respectively, and carry out 12.5% SDS-polyacrylamide gel electrophoresis, and electrophoresis result shows, the main inclusion body form of target protein exists.

Use respectively 20mmol/L Tris-HCl damping fluid (pH8.0), 1mol/L NaCl, 1mmol/L EDTA, 0.1%Triton X-100,2mol/L urea to wash respectively 30 minutes inclusion body, finally, 12000r/min, centrifugal 15 minutes under 4 DEG C of conditions, collecting precipitation is inclusion body.

The inclusion body having washed is weighed, add the ratio of 20ml 8mol/L urea (being dissolved in 20mM Tris-HCl) by inclusion body cracking in every gram of inclusion body.Next day, by the at room temperature centrifugal 15min of 10000r/min of lysate, collect cracking supernatant.

Due to (His) 6 labels that the recombinant protein c end of expressing contains carrier sequence encoding, can, under denatured state, pass through Ni ²⁺affinity column carries out purifying, and concrete purification step is:

By Ni ²⁺after affinity column 20mM Tris-HCl, 8mol/L urea soln (level pad) balance, by sample on inclusion body cracking supernatant, loading is complete washes away uncombined foreign protein with level pad, then, carry out abundant wash-out with the 10mmol/L imidazoles being dissolved in level pad, then, then with the 100～200mmol/L imidazoles wash-out target protein being dissolved in level pad.The target protein of purifying is carried out to concentration determination with BCA determination of protein concentration test kit.

The renaturation of purified product: 20mM Tris-HCl, 8mol/L urea soln for the target protein of purifying are adjusted to protein concentration to 400-600 μ g/ml, then, with 4 times of dilutions of 20mmol/L Tris-HCl (pH8.0) damping fluid do, diluent is put to 4 DEG C of refrigerator overnight, within second day, fully dialyse with 20mmol/LTris-HCl (pH8.0) damping fluid, carry out concentration determination with BCA determination of protein concentration test kit more afterwards.

With the sample after renaturation, according to 5 of the female Balb/c mouse in 6～8 week age of ordinary method immunity.Basic skills is: immunity is for the first time that the target protein of 100 μ g adds the fully emulsified pneumoretroperitoneum injection of isopyknic Freund's complete adjuvant, after 15 days, adopt the target protein of 100 μ g to add the fully emulsified pneumoretroperitoneum injection of isopyknic freund 's incomplete adjuvant, after 7 days, still adopt the target protein of 100 μ g to add the fully emulsified pneumoretroperitoneum injection of isopyknic freund 's incomplete adjuvant, after 7 days, adopt the target protein of 100 μ g directly to carry out abdominal injection, after one week, from tail vein blood, detect serum antibody titer by ELISA method.

Detectable antigens behaviour VEGF, coated concentration is 2 μ g/ml.With the coated ELISA microwell plate of 2 μ g/ml people VEGF, 50 μ l/ holes, coated spending the night in 4 DEG C of refrigerators, next day, seal with 1% bovine serum albumin, 37 DEG C are sealed 1 hour, with Tris salt buffer (TBST solution) washing of 0.1%Tween 20 5 times, then add immune serum and the corresponding preimmune serum of 100 times of dilutions, 37 DEG C of incubations 1 hour, then, with TBST solution washing 5 times, add afterwards the sheep anti mouse two of horseradish peroxidase-labeled anti-, 37 DEG C of incubations 1 hour, use again TBST solution washing 5 times, afterwards, add horseradish peroxidase substrate tetramethyl benzidine two hydrochloric acid (TMB) lucifuge colour developing 10min, then, every hole adds stop buffer (2mol/L sulfuric acid) 50 μ l/ holes, termination reaction, survey CD ₄₅₀.

As shown in Figure 4, ELISA result shows, the mice serum after immunity can be identified people VEGF, and preimmune serum and people VEGF are reactionless.This shows, be showed in " PHQGQ " epi-position on the single domain antibody skeleton of mouse source effectively inducing mouse produce the antibody for people VEGF, the also epitope of the VEGF of people really in explanation " PHQGQ " Loop district.

In order further to verify whether No. 4 candidate's epi-positions " PHQGQHI " taking " PHQGQ " as core are the real epitopes of people VEGF, No. 4 candidate's epi-positions are illustrated in to the CDR3 district of the single domain antibody of a kind of camel, aminoacid sequence (accompanying drawing 5 as shown in SEQ ID NO:7 of the protein forming, wherein two line are partly the epitope sequence of displaying and the appended sequence of both sides), if this protein can induced animal produces the antibody of anti-human VEGF, this albumen can be considered to a kind of people VEGF vaccine.The aminoacid sequence of this protein is converted to its gene coded sequence (http://www.bioinformatics.org/sms2/rev_trans.html) by anti-translation program, BamHI and Xho I restriction enzyme site by synthetic this gene coded sequence of Inovogen Tech Co., Ltd. and two ends obtain full length sequence as shown in SEQ ID NO:8, (accompanying drawing 6, underscore part is respectively BamHI and Xho I restriction enzyme site) and be cloned in pUCE carrier called after pUCE-VE2.Because the single domain antibody of camel is the variable region of heavy chain that derives from heavy chain antibody completely, therefore without carrying out camelization transformation.

Use equally BamH I (Takara) and Xho I (Takara) to carry out double digestion in pET24a carrier (Novagen), the enzyme system of cutting is:

Under 37 DEG C of conditions, enzyme is cut 6 hours.Then, reclaim the large fragment of pET24a carrier by electrophoresis.

10 μ l are connected to product to be added in the competence e. coli bl21 (DE3) of the fresh preparation of 100 μ l, on ice bath, place 30min, then, centrifuge tube is put into 42 DEG C of water-baths, after heat shock 90s, fast centrifuge tube is transferred in ice bath, make it cooling 2min, afterwards, add the LB substratum 500 μ l of antibiotic-free to every pipe, 45min is cultivated in 37 DEG C of concussions; Afterwards, bacterium liquid is applied on the LB agar plate that contains Kan resistance, is inverted plate overnight incubation in 37 DEG C of incubators, 3～5 single bacterium colonies of picking next day to Kan resistance LB substratum in cultivate, 37 DEG C of shaking culture are to OD ₆₀₀reach at 0.5 o'clock, add 1mmol/L IPTG to carry out abduction delivering, abduction delivering, after 4 hours, is collected thalline, by its expression of electrophoretic analysis, and retains the recombinant clone bacterium that expression amount is large.

Because the target protein of expressing exists with inclusion body form, therefore, first break bacterium isolation of occlusion bodies.

The engineering bacteria that screening is obtained is placed in ice bath supersound process, and condition is electric current 350mA, pulse 5 seconds, 4 seconds, interval, ultrasonic 30 minutes altogether, then by the thalline after ultrasonic in 12000r/min, 4 DEG C centrifugal 15 minutes, collecting precipitation is inclusion body.Use respectively 20mmol/L Tris-HCl damping fluid (pH8.0), 1mol/L NaCl, 1mmol/L EDTA, 0.1%Triton X-100,2mol/L urea to wash respectively inclusion body, then 12000r/min under 4 DEG C of conditions, centrifugal 15 minutes, collecting precipitation is inclusion body.

Due to recombinant protein c end (His) that contain carrier sequence encoding expressing ₆label, can, under denatured state, pass through Ni ²⁺affinity column carries out purifying, and concrete purification step is:

Purified product can be by diluting and renaturation is carried out in dialysis, basic skills is that purified product is done to 4 times of dilutions, after dilution, protein concentration maintains 100 μ g/ml left and right, diluent is put to 4 DEG C of refrigerator overnight, within second day, fully dialyse with 20mmol/L Tris-HCl (pH8.0) damping fluid, carry out concentration determination with BCA determination of protein concentration test kit more afterwards.

With the sample after renaturation, according to 3 of the female Balb/c mouse in 6～8 week age of ordinary method immunity, after four immunity, detect immune serum by ELISA method.Detect with the antigen VEGF that behaves, coated concentration is 2 μ g/ml.ELISA result shows, the mice serum after immunity can be identified people VEGF, and preimmune serum can not be identified people VEGF (accompanying drawing 7).This shows, No. 4 candidate's epi-positions " PHQGQHI " that comprise " PHQGQ " core Loop district are the epitope of people VEGF really.

Can simulate better corresponding natural epi-position in people VEGF in order to ensure the epi-position of selecting, we have increased the sequence of " PHQGQ " core sequence both sides, whole epitope sequences is " QIMRIKPHQGQHIGEM ", builds people VEGF epiposition vaccine with this epitope sequences.

Embodiment 2: structure, the expression and purification of people VEGF epiposition vaccine expression vector

By people source single domain antibody BT32/A6 (Journal of Biology Chemistry 2001,276:24774-24780), carry out camelization transformation, the the 9th, the 10th and the 12nd amino acids by the FR2 of people source single domain antibody BT32/A6 replaces with respectively L-glutamic acid, arginine and glycine, its sequence is as shown in SEQ ID NO:9, (see accompanying drawing 8, wherein two line parts are framework region 2 (FR2); Adding frame part is the improved amino acid of camelization; Single underscore part is CDR3 district).

Employment VEGF epitope sequence " QIMRIKPHQGQHIGEM " is replaced " DRLKVEYYDSSGYYVSRFGA " aminoacid sequence in the CDR3 district in accompanying drawing 8, thereby form a kind of aminoacid sequence of new protein, its aminoacid sequence is as shown in SEQ ID NO:10, (as accompanying drawing 9, wherein two line parts are the aminoacid sequence of people VEGF epitope).This new protein sequence is the aminoacid sequence of people VEGF epiposition vaccine.

The aminoacid sequence of people VEGF epiposition vaccine is converted to its gene coded sequence (http://www.bioinformatics.org/sms2/rev_trans.html) by anti-translation program, the encoding gene of synthetic people VEGF epiposition vaccine, its sequence is as shown in SEQ ID NO:11, (as accompanying drawing 10, wherein underscore part is respectively BamHI and Xho I restriction enzyme site).

The gene coded sequence of above-mentioned people VEGF epiposition vaccine and the Bam HI of both sides thereof and Xho I restriction enzyme site sequence are synthesized by Inovogen Tech Co., Ltd., and are connected in pUCE carrier called after pUCE-VE3.

BamH I for full length sequence (Takara) and Xho I (Takara) after synthetic are carried out to double digestion, and the enzyme system of cutting is:

10 μ l are connected to product to be added in the competence e. coli bl21 (DE3) of the fresh preparation of 100 μ l, on ice bath, place 30min, then, centrifuge tube is put into 42 DEG C of water-baths, after heat shock 90s, fast centrifuge tube is transferred in ice bath, make it cooling 2min, afterwards, add the LB substratum 500 μ l of antibiotic-free to every pipe, 45min is cultivated in 37 DEG C of concussions; Afterwards, bacterium liquid is applied on the LB agar plate that contains Kan resistance, is inverted plate overnight incubation in 37 DEG C of incubators, 3～5 single bacterium colonies of picking next day to Kan resistance LB substratum in cultivate, 37 DEG C of shaking culture are to OD ₆₀₀reach at 0.5 o'clock, add 1mmol/L IPTG to carry out abduction delivering, abduction delivering, after 4 hours, is collected thalline, and electrophoretic analysis expression retains the large recombinant clone of expression amount.

Through electrophoretic analysis, the people VEGF epiposition vaccine of expression exists with the form of inclusion body.

By after phosphoric acid buffer, 0.5%Triton X-100,1mol/L NaCl and the washing of 2mol/L urea for inclusion body, with the cracking of 8mol/L urea, cracking supernatant is with using Ni ²⁺affinity column purifying.Concrete purification step is:

By Ni ²⁺after affinity column 20mM Tris-HCl, 8mol/L urea soln (level pad) balance, by sample on inclusion body cracking supernatant, loading is complete washes away uncombined foreign protein with level pad, then, carry out abundant wash-out with the 10mmol/L imidazoles being dissolved in level pad, then, then with the 100～200mmol/L imidazoles wash-out target protein being dissolved in level pad.Target protein is carried out to concentration determination with BCA determination of protein concentration test kit.

The people VEGF epiposition vaccine of the sex change in 8mol/L urea after purifying is carried out to renaturation, concrete renaturation condition is, under 4 DEG C of conditions, with renaturation buffer (20mmol/L Tris-HCl, pH8.0) carry out 4 times of dilutions, make protein final concentration maintain 100～150 μ g/ml, place after 24 hours for 4 DEG C, renaturation sample is fully dialysed with renaturation buffer, to remove residual urea.

By sample concentration, filtration sterilization after renaturation, obtain people VEGF epiposition vaccine, and with BCA determination of protein concentration kit measurement protein concn.After packing, 4 DEG C save backup.

Embodiment 3: people VEGF epiposition vaccine Analysis of Immunity Effect

With 5 of the female Balb/c mouse in 6～8 week age of people VEGF epiposition vaccine immunity obtaining in embodiment 2, observe immune effect of vaccine.

Immunizing dose is 50 μ g/, and immune position is abdominal cavity, and immune programme for children is the 0th, 14,21,35 days.Immunity for the first time adopts Freund's complete adjuvant, and second and third immunity adopts freund 's incomplete adjuvant, and last immunity does not add adjuvant.Latter 7 days of last immunity, gets blood, detects immune effect of vaccine.

Serum after the preimmune serum of 5 animals and immunity is all done to 10000 times of dilutions, then, adopt ELISA method to detect its identification to people VEGF, the coated concentration of people VEGF is 2 μ g/ml.Detected result shows, through 4 immunity, 5 mouse have all produced the antibody (accompanying drawing 11) of the anti-human VEGF of higher titre.

Embodiment 4: in people VEGF epiposition vaccine immune serum and activation analysis

By the antibody in the immune serum obtaining in protein G affinity chromatography column purification embodiment 3.First, 20mmol/L phosphoric acid buffer (pH7.0) for immune serum is carried out to 5 times of dilutions, filter, for subsequent use.

With fully balance Protein G-agarose affinity chromatography post (Protein G 1ml, GE) of 20mmol/L phosphoric acid buffer (pH7.0).Get sample 30ml upper prop to be purified, flow velocity is 0.5ml/min, upper sample is complete, by fully balance of 20mmol/L phosphoric acid buffer (pH7.0), then, antibody elution is got off with 0.1mol/L glycine-hydrochloride buffer (pH2.7), 1mol/LTris adjusts pH to 7.0 for the sample eluting.Antibody concentration after purifying with BCA determination of protein concentration kit measurement is 1000 μ g/ml.

The antibody of purifying is mixed with human epidermal growth factor's solution of isopyknic 0 μ g/ml and 100 μ g/ml, hatch 1 hour for 37 DEG C.2 groups of samples are added respectively in the Human umbilical vein endothelial cells of 12 hours of preculture (HUVEC) of cultivating in 96 well culture plates, continue to cultivate 24 hours, add MTT, measure the OD in each hole by microplate reader ₅₇₀value.

Result shows, adds merely the cell proliferation of people VEGF better, and poor with group cell growth state in antibody, the two has significant difference (accompanying drawing 12).This explanation, derive from antibody in antiserum(antisera) really have in and the activity of people VEGF, the epitope of the VEGF of people really of the epitope " QIMRIKPHQGQHIGEM " in people VEGF epiposition vaccine has also just been described, and has been a kind of neutrality epitope.

Embodiment 5: the antitumous effect analysis of people VEGF epiposition vaccine

According to the cDNA sequence of people VEGF, with reference to related documents, design, synthetic people VEGF upstream and downstream primer P-up and P-dh:

P-up：5′-GG GGATCCGCCGCCAGCC AACTTTCTGCTGTCTTG-3′(BamH I)

P-dn：5′-CC GAATTC CCGCCTCGGCTTGTCAC-3′(EcoR I)

Wherein, in upstream primer, before initiator codon ATG, add KOZAK sequence, to be conducive to the expression of people VEGF in eukaryotic cell.

From people's tire brain cDNA library, clone the people VEGF cDNA that contains signal peptide coding region of total length by PCR method.PCR condition is: on PCR instrument, react 30 circulations, the condition of each circulation is: 94 DEG C 30 seconds, 55 DEG C 45 seconds, 72 DEG C 1 point 30 seconds.After PCR finishes, PCR product is carried out to electrophoresis, obtaining molecular size is the band of 590bp left and right.PCR product is linked to (pT-sVEGF) in T carrier, show through order-checking, sequence is entirely true.

After the people VEGF cDNA enzyme that contains signal peptide coding region being cloned into is cut, be connected in carrier for expression of eukaryon pcDNA3.1 (+), build secretor type people VEGF carrier for expression of eukaryon pcDNA-hVEGF.The condition that concrete enzyme is cut, connected is as follows:

BamH I (Takara) for pT-sVEGF and EcoR I (Takara) are carried out to double digestion, and the enzyme system of cutting is:

Under 37 DEG C of conditions, enzyme is cut 6 hours.Then, by electrophoresis VEGF gene fragment.

Use equally BamH I (Takara) and EcoR I (Takara) to carry out double digestion in pcDNA3.1 (+) carrier, the enzyme system of cutting is:

Under 37 DEG C of conditions, enzyme is cut 6 hours.Then, reclaim the large fragment of pcDNA3.1 (+) carrier by electrophoresis.

The VEGF gene fragment reclaiming and pcDNA3.1 (+) the carrier large fragment of recovery are connected and are spent the night 16 DEG C of water-baths, and system is as follows:

10 μ l are connected to product to be added in the competence bacillus coli DH 5 alpha competent cell of the fresh preparation of 100 μ l, ice bath 30 minutes, then, centrifuge tube is put into 42 DEG C of water-baths, after heat shock 90s, fast centrifuge tube is transferred in ice bath, make it cooling 5 minutes, afterwards, add the LB substratum 700 μ l of antibiotic-free to every pipe, 37 DEG C of concussions are cultivated 50 minutes; Afterwards, bacterium liquid is applied on the LB agar plate that contains Amp resistance, is inverted plate overnight incubation in 37 DEG C of incubators, 3～5 clones of picking next day qualification of checking order.

Get the clone that order-checking is correct, extract plasmid, preserve, for subsequent use.

Use pcDNA-hVEGF carrier, require to specifications transfection H22 mouse tumor cell with Lipofectamine 2000 transfection reagents (Invitrogen), afterwards, with G418 resistance screening people VEGF stably express cell, the concentration of G418 is 100 μ g-1000 μ g/ml, filter out people VEGF stably express cell, called after hV-H22 cell by the G418 increasing gradually in substratum.

20 of the Balb/c mouse of getting 6～8 week age, are divided into two groups of A, B according to Stochastic Equilibrium principle, 10 every group.The people VEGF epiposition vaccine immunity as shown in SEQ ID NO:3 described in embodiment 24 times for A group mouse, immunizing dose is 100 μ g/ time, immunity position is abdominal cavity, immunity is fully emulsified by vaccine with equal-volume Freund's complete adjuvant for the first time, second and third immunity is fully emulsified by vaccine with equal-volume freund 's incomplete adjuvant, and last immunity does not add adjuvant.The immunity cycle is the 1st, 14,21,28 days.B isopyknic physiological saline immunity for group, program is with A group.Latter 3 days of last immunity, gives every right side of mice oxter inoculation 3 × 10 ⁶hV-H22 cell, prepare tumor model, inoculation after the 10th day, available hand touches tumour, the 20th day time, kill animals, gets tumour, measure tumor size.

Experimental result shows, injection people VEGF vaccine group animal tumor is significantly less than physiological saline control group (accompanying drawing 13), the average knurl of control group and vaccine group is heavily respectively 5.46g and 2.67g, vaccine reaches 51% to the inhibiting rate of tumour, illustrate that people VEGF epiposition vaccine can produce the neutrality antibody for people VEGF by induced animal really, thereby in and the activity of people VEGF that produces of tumour cell, and then the growth of inhibition tumour, this shows, people VEGF epiposition vaccine can be used for the treatment of noumenal tumour.

Obviously, above-described embodiment is to be only the example that clearly explanation is done, and the not restriction to embodiment.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here without also giving exhaustive to all embodiments.And the apparent variation of being extended out thus or variation are still among the protection domain in the invention.

Claims

1. one kind includes the albumen of human vascular endothelial growth factor epitope, it is characterized in that, it is molecular skeleton from the single domain antibody of people's antibody, camel heavy chain antibody or murine antibody that described albumen utilization builds, and use the epitope of the human vascular endothelial growth factor of aminoacid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 replace as described in the CDR3 district of single domain antibody.

2. albumen according to claim 1, the amino acid of the 9th, 10 and 12 in wherein said single domain antibody FR2 district is replaced by respectively L-glutamic acid, arginine and glycine.

3. albumen according to claim 2, is characterized in that: the aminoacid sequence of described albumen is as shown in SEQ ID NO:5 or SEQ ID NO:7 or SEQ ID NO:10.

4. a method of preparing the arbitrary described albumen of claim 1-3, is characterized in that, comprises the steps:

(1) determine the complete aminoacid sequence of the variable region of heavy chain of camel heavy chain antibody, the aminoacid sequence using this sequence as single domain antibody, and analyze the sequence in its CDR3 district;

Or

(1 ') determines the complete amino acid sequence containing light chain and people's antibody of heavy chain or the variable region of heavy chain of murine antibody, this sequence is the basic aminoacid sequence of single domain antibody, analyze the sequence in its framework region 2 (FR2) and CDR3 district, the amino acid of the 9th, 10 and 12 in Bing Jiang FR2 district replaces with respectively L-glutamic acid, arginine and glycine;

(2) use the epitope sequence of the human vascular endothelial growth factor of aminoacid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 replace as described in the CDR3 region amino acid sequence of single domain antibody, and the full length amino acid sequence after replacing is converted to its gene coded sequence, synthetic full-length gene order, and this full-length gene order is cloned in expression vector, obtain recombinant plasmid;

(3) by extremely corresponding the recombinant plasmid transformed obtaining host cell, screening obtains positive colony, and the recombinant protein that this positive colony is expressed is the arbitrary described albumen that includes human vascular endothelial growth factor epitope of claim 1-3.

5. a human vascular endothelial growth factor epiposition vaccine, is characterized in that, described epiposition vaccine comprises the arbitrary described albumen of claim 1-3.

6. the purposes of the arbitrary described albumen of claim 1-3 in the medicine for the preparation for the treatment of or prevention liver solid tumor.

7. purposes according to claim 6, wherein said medicine is vaccine.