CN102936277A

CN102936277A - Human blood vessel endothelial growth factor antigen epitope and epitope vaccine thereof

Info

Publication number: CN102936277A
Application number: CN2011104525700A
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: 2013-02-20
Anticipated expiration: 2031-12-29
Also published as: CN102936277B

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 the human vascular endothelial growth factor epiposition vaccine that utilizes 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 present 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 drugs approved by FDA.

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 seeking, designing take 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, induce body self generation for the neutrality antibody of people VEGF, then can realize the active immunity treatment to tumour.But people VEGF is owing to be the molecule of human body self, and 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 (about 20～40 amino acid) with the chemical synthesis synthetic.Synthetic peptide vaccine generally is made of jointly a plurality of 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 the accurate analogue antigen molecule, and T, B cell epitope can not effectively be brought into play synergy, and the 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 neutrality antibody for people VEGF, just can realize the active immunity treatment to tumour.Inducing in vivo the antibody with neutralization activity of generation for human immunity people VEGF epiposition vaccine will be complete human antibody, have completely biocompatibility with tumour patient self.And recombination epitope vaccine generally can be produced by prokaryotic expression systems such as intestinal bacteria, and its manufacturing cost is lower, and its clinical application number of times is few, and consumption is low, so the active immunity treatment cost that carries out with the epiposition vaccine of recombinating is also lower.

1989, (Nature 1989,341:544-546) prepared first the genetic engineering antibody that only is comprised of antibody heavy chain variable region (VH) structural domain, and the called after single domain antibody for Ward etc.1993, (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 for Hamers-Casterman etc.Heavy chain antibody plays an important role in the humoral immunization of camel.Compare with general antibody, heavy chain antibody is except lacking light chain, the variable region of heavy chain of heavy chain antibody (variable domain of heavy chain of heavy antibody, VHH) also has the feature that is different from general antibody VH district, the VHH gene of camel that is to say that by kept the normal function of heavy chain antibody at the selective evolution in several critical functions site 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 makes up based on the variable region of heavy chain of the animal heavy chain antibodies such as camel has preferably structural stability, and after the variable region of heavy chain of people source or mouse source antibody carried out the camelization transformation, also can make up the more stable single domain antibody of structure.

Though the single domain antibody molecule is little, structure is relatively stable, particularly makes up the single domain antibody from heavy chain antibodies such as camel, sharks.Three antigen complementary determining region (complementarity-determining region at 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 the 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 showed the position.

Based on above thinking, the present invention has at first predicted the epitope of people VEGF, then, utilize the CDR3 district of single domain antibody skeleton to show this epitope, thereby made up a kind of people VEGF epiposition vaccine, this vaccine can induce the body generation for the antibody of 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 the high deficiency of antibody heterology, treatment cost that the present anti-human VEGF antibody drug that uses clinically exists oncotherapy.

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 the SEQ ID NO:1.

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

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

The present invention also provides the epitope of a kind of people VEGF, and its aminoacid sequence is 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 to be molecular skeleton, and with the epitope sequence shows of the arbitrary described people VEGF of SEQ ID NO:1-3 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 to be molecular skeleton, and replaces the CDR3 district of described single domain antibody with the epitope sequence of the arbitrary described people VEGF of SEQ ID NO:1-3.

The the 9th, 10 and 12 the amino acid in the FR2 district of single domain antibody described in the 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 makes up 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 the SEQ ID NO:10.

The present invention also provides the method for preparing above-mentioned albumen, and when single domain antibody derived from the variable region of heavy chain of heavy chain antibody, the basic step of described preparation epiposition vaccine comprised:

(1) determines a kind of complete aminoacid sequence of variable region of heavy chain of heavy chain antibody, with the aminoacid sequence of 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 with 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 will replacing is converted to its gene coded sequence, synthetic full-length gene order, and this full-length gene order is cloned in the expression vector, obtain recombinant plasmid;

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

When described single domain antibody derived from the variable region of heavy chain of the conventional antibody that contains light chain antibody and heavy chain antibody, the basic step of described preparation epiposition vaccine comprised:

(1 ') determines to contain the complete amino acid sequence of 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, and the 9th, 10 and 12 the amino acid in FR2 district is replaced 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 provide above-mentioned albumen for the preparation of the treatment or the prevention solid tumor medicine in purposes.Especially as the purposes of vaccine medicine.

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

People VEGF epiposition vaccine of the present invention has shown preferably 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 preferably immunogenicity, has avoided heterology and the high deficiency for the treatment of cost of present anti-human VEGF antibody class anti-tumor medicine.People VEGF epiposition vaccine of the present invention is induced the antibody of generation in the tumour patient body be complete human antibody, has 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, the clinical treatment cost will be lower, and this will overcome existing in the high problem of the clinical treatment cost that the antibody drug production cost is very high, usage quantity causes greatly of the eukaryotic expressions such as CHO.

Description of drawings

Content of the present invention is easier to be expressly understood in order to make, the 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 parts are the FR2 district, and italicized item is the CDR3 district;

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

*: single underscore partly is the FR2 district, and italicized item is improved amino-acid residue;

Double underline partly 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 partly is restriction enzyme site;

Fig. 4 shows that the mouse single domain antibody skeleton recombinant protein of " PHQGQ " epi-position is to the immune effect of 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 partly is respectively BamHI and Xho I restriction enzyme site;

" PHQGQHI " epitope that Fig. 7 shows with camel single domain antibody skeleton is induced in Mice Body and is produced anti-human VEGF antibody;

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

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

Add frame and partly be the improved amino acid of camelization;

Single underscore partly is the 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 partly 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 control group tumor weight are relatively.

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 based on the analytical method of protein three-dimensional structure, but all there is limitation in any method, and is wherein the highest based on the Epitope prediction accuracy of protein three-dimensional structure.At present, generally be to unite the epitope that the analytical procedure of using two or more is come predicted protein matter, and determine finally an epitope whether also must verify by experiment by real epitope.

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

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

Table 1 people VEGF Antigen Epitope Prediction

Numbering	Zero position	Final position	The epitope peptide section	The 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

In conjunction with the crystalline structure (1FLT) of people VEGF in the PDB database (http://www.rcsb.org/pdb/home/home.do) and its receptor protein complex body as can be known, No. 4 candidate's epi-positions " PHQGQHI " in the upper table (SEQ ID NO:2) 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 might be exactly the epitope of people VEGF.

In order to verify the whether real epitope of people VEGF of " PHQGQ " sequence, with " 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 the 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), determine that therefrom its weight chain variable region amino acid sequence is shown in SEQ ID NO:4, (such as Fig. 1, two line parts are the FR2 district, italicized item is the CDR3 district), and with the basic aminoacid sequence of this sequence as the mouse single domain antibody.

Single domain antibody basis, mouse source sequence among Fig. 1 is carried out the camelization transformation, wherein the 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 the CDR3 district, sequence after replacement is finished (is seen accompanying drawing 2, wherein shown in SEQ ID NO:5, the amino acid of italicized item is the improved amino acid of FR2, and two line partly 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 the downstream thereon simultaneously, obtain full length sequence shown in SEQ ID NO:6 (such as Fig. 3, underscore partly is respectively BamHI and Xho I restriction enzyme site), it is synthetic and be cloned in the pUCE carrier called after pUCE-VE1 that SEQ ID NO:6 sequence is transferred to the luxuriant industry bio tech ltd of Beijing English.

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

Under 37 ℃ of conditions, enzyme was cut 6 hours.Then, reclaim the goal gene fragment by electrophoresis.

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

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

The goal gene fragment that reclaims is connected pET24a expression vector large fragment connects 16 ℃ of water-baths and spend the night with recovery, system is as follows:

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

With the recombinant bacterial strain behind the abduction delivering, place the ice bath supersound process, condition is electric current 350mA, pulse 5 seconds, interval 4 seconds, ultrasonic 30 minutes altogether, then with the thalline after ultrasonic in 12000r/min, 4 ℃ are centrifugal 15 minutes, difference collecting precipitation and supernatant, and carry out 12.5% SDS-polyacrylamide gel electrophoresis, electrophoresis result shows that 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, at last, 12000r/min, centrifugal 15 minutes under 4 ℃ of conditions, collecting precipitation is inclusion body.

The inclusion body that washing is good is weighed, and adds the ratio of 20ml 8mol/L urea (being dissolved among the 20mM Tris-HCl) with the inclusion body cracking in every gram inclusion body.Next day, with the lysate centrifugal 15min of 10000r/min at room temperature, collect the cracking supernatant.

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

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

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

With the sample after the renaturation, according to 5 of the female Balb/c mouse in ages in ordinary method immunity 6～8 week.Basic skills is: for the first time immunity is that the target protein of 100 μ g adds the fully emulsified pneumoretroperitoneum injection of isopyknic Fu Shi 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, from tail vein blood, detect serum antibody titer by the ELISA method after one week.

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 ℃ of refrigerators, next day, bovine serum albumin with 1% seals, 37 ℃ were sealed 1 hour, used the Tris salt buffer (TBST solution) of 0.1%Tween 20 to wash 5 times, then added immune serum and the corresponding preimmune serum of 100 times of dilutions, 37 ℃ of incubations 1 hour, then, use TBST solution washing 5 times, the sheep anti mouse two that adds afterwards horseradish peroxidase-labeled is anti-, 37 ℃ of incubations 1 hour, use again the 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, and termination reaction is surveyed CD ₄₅₀

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

For further No. 4 candidate's epi-positions " PHQGQHI " of checking take " PHQGQ " as the core real epitope of people VEGF whether, No. 4 candidate's epi-positions are illustrated in the CDR3 district of the single domain antibody of a kind of camel, aminoacid sequence (accompanying drawing 5 shown in SEQ ID NO:7 of the protein that forms, wherein two line partly are 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, then 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 the luxuriant industry bio tech ltd of Beijing English and two ends obtain full length sequence shown in SEQ ID NO:8, (accompanying drawing 6, underscore partly is respectively BamHI and Xho I restriction enzyme site) and be cloned in the pUCE carrier called after pUCE-VE2.Because therefore the single domain antibody of camel need not to carry out the camelization transformation for deriving from the variable region of heavy chain of heavy chain antibody fully.

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 ℃ of conditions, enzyme was cut 6 hours.Then, reclaim the large fragment of pET24a carrier by electrophoresis.

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

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

The engineering bacteria that screening is obtained places the ice bath supersound process, and condition is electric current 350mA, pulse 5 seconds, interval 4 seconds, ultrasonic 30 minutes altogether, then with the thalline after ultrasonic in 12000r/min, 4 ℃ 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 ℃ of conditions, centrifugal 15 minutes, collecting precipitation is inclusion body.

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

Purified product can carry out renaturation by dilution and dialysis, basic skills is that purified product is done 4 times of dilutions, protein concentration maintains about 100 μ g/ml after the dilution, diluent is put 4 ℃ of refrigerator overnight, second day is fully dialysed with 20mmol/L Tris-HCl (pH8.0) damping fluid, carries out concentration determination with BCA determination of protein concentration test kit more afterwards.

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

For the epi-position that guarantees to select can be simulated corresponding natural epi-position among the people VEGF better, we have increased the sequence of " PHQGQ " core sequence both sides, whole epitope sequences is " QIMRIKPHQGQHIGEM ", makes up people VEGF epiposition vaccine with this epitope sequences.

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

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

Employment VEGF epitope sequence " QIMRIKPHQGQHIGEM " is replaced " DRLKVEYYDSSGYYVSRFGA " aminoacid sequence in the CDR3 district in the accompanying drawing 8, thereby form a kind of aminoacid sequence of new protein, its aminoacid sequence is shown in SEQ ID NO:10, (such 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 shown in SEQ ID NO:11, (such as accompanying drawing 10, wherein underscore partly 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 the luxuriant industry bio tech ltd of Beijing English, and are connected in the pUCE carrier called after pUCE-VE3.

Full length sequence after synthetic is carried out double digestion with BamH I (Takara) and Xho I (Takara), and the enzyme system of cutting is:

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

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

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

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

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

With sample concentration, filtration sterilization after the renaturation, namely obtain people VEGF epiposition vaccine, and with BCA determination of protein concentration kit measurement protein concn.After the packing, 4 ℃ save backup.

Embodiment 3: people VEGF epiposition vaccine Analysis of Immunity Effect

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

Immunizing dose is 50 μ g/, and immune position is the abdominal cavity, and immune programme for children is the 0th, 14,21,35 day.The Fu Shi Freund's complete adjuvant is adopted in for the first time immunity, and freund 's incomplete adjuvant is adopted in second and third time immunity, and last immunity does not add adjuvant.Blood is got in rear 7 days of last immunity, detects immune effect of vaccine.

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

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

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

With the abundant 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, with the abundant balance of 20mmol/L phosphoric acid buffer (pH7.0), then, (pH2.7) gets off antibody elution with 0.1mol/L glycine-hydrochloride buffer, and the sample that elutes is transferred pH to 7.0 with 1mol/LTris.Be 1000 μ g/ml with the antibody concentration behind the BCA determination of protein concentration kit measurement purifying.

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

The result shows that the cell proliferation that adds merely people VEGF is better, and relatively poor with the group cell growth state in the antibody, the two has significant difference (accompanying drawing 12).This explanation, derive from antibody in the antiserum(antisera) really have in and the activity of people VEGF, the epitope of the really people VEGF of the epitope " QIMRIKPHQGQHIGEM " in the people VEGF epiposition vaccine also just has 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 the upstream primer, before initiator codon ATG, added the 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 with PCR method.The PCR condition is: in PCR instrument reaction 30 circulations, the condition of each circulation is: 94 ℃ 30 seconds, 55 ℃ 45 seconds, 72 ℃ 1 minute 30 seconds.After PCR finishes, the PCR product is carried out electrophoresis, obtain molecular size and be the band about 590bp.The PCR product is linked (pT-sVEGF) in the T carrier, show that through order-checking sequence is entirely true.

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

PT-sVEGF is carried out double digestion with BamH I (Takara) and EcoR I (Takara), and the enzyme system of cutting is:

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

The VEGF gene fragment that reclaims is connected pcDNA3.1 (+) carrier large fragment connects 16 ℃ of water-baths and spend the night with recovery, system is as follows:

10 μ l are connected 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 ℃ of water-baths, behind the heat shock 90s, fast centrifuge tube is transferred in the ice bath, made it to cool off 5 minutes, afterwards, to the LB substratum 700 μ l of every pipe adding antibiotic-free, 37 ℃ of concussions were cultivated 50 minutes; Afterwards, bacterium liquid is applied on the LB agar plate that contains the Amp resistance, is inverted plate overnight incubation in 37 ℃ of incubators, 3～5 clones of picking next day evaluation of checking order.

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

Use the 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 that increases gradually in the substratum.

Get 20 of the Balb/c mouse in 6～8 ages in week, be divided into two groups of A, B according to the Stochastic Equilibrium principle, 10 every group.The people VEGF epiposition vaccine immunity shown in SEQ ID NO:3 described in the embodiment 24 times of A group mouse, immunizing dose is 100 μ g/ time, the immunity position is the abdominal cavity, for the first time immunity is fully emulsified with vaccine with equal-volume Fu Shi Freund's complete adjuvant, second and third time immunity is fully emulsified with vaccine with the equal-volume freund 's incomplete adjuvant, and last immunity does not add adjuvant.The immunity cycle is the 1st, 14,21,28 day.The B group is immune with isopyknic physiological saline, and program is organized with A.Every right side of mice oxter inoculation 3 * 10 is given in rear 3 days of last immunity ⁶The hV-H22 cell, the preparation tumor model, after the inoculation the 10th day, available hand was touched tumour, in the time of the 20th day, kill animals is got tumour, measures 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 heavily is respectively 5.46g and 2.67g, vaccine reaches 51% to the inhibiting rate of tumour, illustrate that people VEGF epiposition vaccine really can induced animal produces the neutrality antibody for people VEGF, thereby in and the activity of the people VEGF that produces of tumour cell, and then the growth of inhibition tumour, this shows that people VEGF epiposition vaccine can be used for the treatment of noumenal tumour.

Obviously, above-described embodiment only is to be the example done of explanation clearly, and is not the 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 need not also can't give all embodiments exhaustive.And the apparent variation of being extended out thus or change still are among the protection domain of the invention.

Claims

1. the epitope of a human vascular endothelial growth factor is characterized in that: comprise the aminoacid sequence shown in SEQ ID NO:1, SEQ ID NO:2 or the SEQ ID NO:3.

2. the epitope of a human vascular endothelial growth factor, it is characterized in that: its aminoacid sequence is shown in SEQ ID NO:3.

3. albumen that includes the human vascular endothelial growth factor epitope, it is characterized in that, described albumen utilizes single domain antibody to be molecular skeleton, and the epitope of claim 1 or 2 described human vascular endothelial growth factor is showed in the CDR3 district of described single domain antibody.

4. albumen according to claim 3 is characterized in that, described albumen utilizes single domain antibody to be molecular skeleton, and replaces the CDR3 district of described single domain antibody with the epitope of claim 1 or 2 described human vascular endothelial growth factor.

5. albumen according to claim 3, the 9th, 10 and 12 the amino acid in the FR2 district of wherein said single domain antibody is replaced by respectively L-glutamic acid, arginine and glycine.

6. it is characterized in that according to claim 3 or 4 or 5 described albumen: described single domain antibody makes up from people's antibody, camel heavy chain antibody or murine antibody.

7. albumen according to claim 6, it is characterized in that: the aminoacid sequence of described albumen comprises the sequence shown in SEQ ID NO:5 or SEQ ID NO:7 or the SEQ ID NO:10.

8. a method for preparing the arbitrary described albumen of claim 3-7 is characterized in that, comprises the steps:

Or

(2) replace the CDR3 region amino acid sequence of described single domain antibody with the epitope sequence of claim 1 or 2 described human vascular endothelial growth factor, and the full length amino acid sequence after will replacing is converted to its gene coded sequence, synthetic full-length gene order, and this full-length gene order is cloned in the expression vector, obtain recombinant plasmid;

(3) with in the extremely corresponding host cell of the recombinant plasmid transformed that obtains, screening obtains positive colony, and the recombinant protein that this positive colony is expressed is the arbitrary described albumen that includes the human vascular endothelial growth factor epitope of claim 3-7.

9. a human vascular endothelial growth factor epiposition vaccine is characterized in that, described epiposition vaccine comprises the arbitrary described albumen of claim 3-7.

The arbitrary described albumen of claim 3-7 for the preparation of the treatment or the prevention solid tumor medicine in purposes.

11. purposes according to claim 10, wherein said medicine are vaccine.