CN105646701B - Recombinant human endostatin albumen of different aminoacids structure and its preparation method and application - Google Patents
Recombinant human endostatin albumen of different aminoacids structure and its preparation method and application Download PDFInfo
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- CN105646701B CN105646701B CN201610189012.2A CN201610189012A CN105646701B CN 105646701 B CN105646701 B CN 105646701B CN 201610189012 A CN201610189012 A CN 201610189012A CN 105646701 B CN105646701 B CN 105646701B
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- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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Abstract
The invention discloses recombinant human endostatin albumen of different aminoacids structure and its preparation method and application, the amino acid sequence of recombinant human endostatin albumen 1 is as shown in SEQ ID NO.1, and the amino acid sequence of recombinant human endostatin albumen 2 is as shown in SEQ ID NO.2.Two kinds of recombinant proteins of the invention have the activity for inhibiting the increment of the vascular endothelial cell under basic fibroblast growth factor (bFGF) induction activity and inhibiting chick chorioallantoic membrane (CAM) angiogenesis, and it is easier to enter in vascular endothelial cell and chick chorioallantoic membrane vascular endothelial cell, albumen wears film effect and the structural stability of N-terminal is all significantly increased, and have the function of directly inhibiting growth of tumour cell, the effect for preferably neovascular endothelium cell being inhibited to generate can be played, it can be used in treating various diseases caused by new vessels generate, including retinopathy caused by entity tumor and diabetes and rheumatoid arthritis.
Description
Technical field
The invention belongs to genetic engineering fields, and in particular to have with gene engineering method preparation and inhibit endothelial cell growth
Activity or inhibit growth of tumour cell recombinant human endostatin albumen of a variety of different aminoacids structures and preparation method thereof and
Using.
Background technique
1997, the culture solution of discovery mouse nemendothelioma (EOMA) cell line such as Harvard University O ' Reilly can press down
The proliferation of vascular endothelial cell processed.By isolating and purifying to obtain a kind of new protein, it is named as the Endostatin of mouse, later
Translate into vascellum esoderma inhibin (abbreviation Endostatin).On September 10th, 1997, Xu Genxing etc. has applied for the hair of human endostatin
Bright patent (Zl97107112.8), acquisition on January 10th, 2001 invention patent mandate, this is that Endostatin obtains earliest in the world
One of patent of authorization.It is human collagen octadecyl because of 1503-2055 by Bacillus coli expression Human endostatin gene
The protein active fragment of expression, 184 amino acid, molecular weight 20KD.Have 85.33% with mouse Endostatin amino acid sequence
Homology has completed the clinical research of three phases at present.The Endostatin of 184 amino acid of U.S.'s Yeast expression is earliest in the world
Into clinical research, in June, 1999, FDA ratifies EntreMed clinical trial application, and the starting of I phase clinic rests on the II phase at present
In clinical research, main reason is that the problems such as antibody generates high probability, indication selection and single medicine application method is subcutaneously injected.
The Endostatin of international first approval production is 192 amino acid similar to kind (rhEndostatin), is increased in the N-terminal of Endostatin
183 amino acid of 9 amino acid such as Met Gly Gly Ser His His His His His (His) and Endostatin connection
Close the endostatin protein for forming 192 amino acid, Bacillus coli expression.Patent (the number of patent application of Li Xiaoxin etc.
201410102963.2) it is related to the Endostatin different aminoacids structure of endothelium chalone mutant and polyethylene glycol (PEG) modification
The Endostatin recombinant protein of mutation.The patent (number of patent application 200410013621.X) of Liu Xinghan etc. is related to changing endothelium suppression
Plain amino acid structure enhances anti-tumor activity.The patent (number of patent application 201210149201.9) of Wang Fengshan etc. is related to the mankind
Immunodeficiency virus Trans-activating transduction albumen Tat (Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg)
The fusion protein being formed together with Endostatin.The patent (number of patent application 200810025406.X) of Yao Wenbing etc. be related to containing
The endothelium chalone mutant and its derivative of unnatural amino acid.
Summary of the invention
The first purpose of the invention is to provide a variety of recombinant human endostatin albumen, it, which has, inhibits endothelial cell growth
Activity, but amino acid sequence and structure and have been approved by patent or ratify clinical research endostatin protein it is all different.
A second object of the present invention is to provide the gene engineering preparation methods of both endostatin proteins, wherein distinguishing
It include three kinds of escherichia coli prokaryotic expression, Yeast expression and CHO eukaryotic expression different genes engineering expression-forms.
Another object of the present invention is to provide the application of above-mentioned recombinant human endostatin albumen.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of recombinant human endostatin albumen, the recombinant human endostatin albumen are recombinant human endostatin albumen 1, recombination
Any one in human endostatin albumen 2 and recombinant human endostatin 2- human serum albumin fusion proteins, the recombined human
The amino acid sequence of endostatin protein 1 is as shown in SEQ ID NO.1；The amino acid sequence of the recombinant human endostatin albumen 2
Column are as shown in SEQ ID NO.2；The amino acid sequence such as SEQ of the recombinant human endostatin 2- human serum albumin fusion proteins
Shown in ID NO.5.
The encoding gene of above-mentioned recombinant human endostatin albumen, the encoding gene have one of following nucleotide sequence:
(1) nucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.6 in sequence table, wherein
SEQ ID NO.3 is the nucleotides sequence of encoding amino acid sequence recombinant human endostatin albumen 1 as shown in SEQ ID NO.1
Column, SEQ ID NO.4 are the nucleotide of encoding amino acid sequence recombinant human endostatin albumen 2 as shown in SEQ ID NO.2
Sequence, SEQ ID NO.6 are encoding amino acid sequence recombinant human endostatin 2- human seralbumin egg as shown in SEQ ID NO.5
The nucleotide sequence of white fusion protein；
(2) in polynucleotide the amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.5
Nucleotide.
Include the recombinant expression carrier of above-mentioned recombinant human endostatin protein coding gene, transgenic cell line and turns base
Because of recombinant bacterium.
For expressing the recombinant expression carrier of above-mentioned recombinant human endostatin albumen, it is to prepare using following methods:
The recombinant expression carrier pET9c-En1 of the recombinant human endostatin albumen 1 uses following steps to prepare: with SEQ
Endostatin nucleotides sequence shown in ID NO.7 is classified as template, with the primer (1) as shown in SEQ ID NO.8 and such as SEQ ID
Primer shown in NO.9 (2) is that primer carries out PCR amplification, and pcr amplification product and pET9c carrier are used NdeI and BamHI respectively
Digestion simultaneously is attached to obtain recombinant plasmid pET9c-En1；
The recombinant expression carrier pET9c-En2 of the recombinant human endostatin albumen 2 uses following steps to prepare: with above-mentioned
Recombinant plasmid pET9c-En1 be template, respectively with the primer (3) as shown in SEQ ID NO.10 and such as SEQ ID NO.12 institute
The primer (5) shown, with the primer (4) as shown in SEQ ID NO.11 and the primer (5) as shown in SEQ ID NO.12, with such as
Primer (6) shown in SEQ ID NO.13 and the primer (7) as shown in SEQ ID NO.14 are that primer carries out PCR amplification acquisition
Pcr amplification product 1 is being template with the pcr amplification product 1 of acquisition, with the primer (3) as shown in SEQ ID NO.10 and such as
Primer (2) shown in SEQ ID NO.9 carries out PCR amplification and obtains pcr amplification product 2, by pcr amplification product 2 and pET9c carrier
It with NdeI and BamHI digestion and is attached to obtain recombinant plasmid pET9c-En2 respectively；
The recombinant expression carrier pcDNA3.1-En1 and the recombinant human endostatin of the recombinant human endostatin albumen 1
The recombinant expression carrier pcDNA3.1-En2 of albumen 2 uses following steps to prepare: respectively with recombinant plasmid pET9c-En1 and recombination
Plasmid pET9c-En2 is template, with the primer (9) as shown in SEQ ID NO.16 and the primer as shown in SEQ ID NO.17
(10) PCR amplification being carried out for primer and obtaining pcr amplification product, pcr amplification product and pcDNA3.1 carrier are used into HindIII respectively
With XhoI digestion and be attached and respectively obtain recombinant plasmid pcDNA3.1-En1 and recombinant plasmid pcDNA3.1-En2；
The recombinant expression carrier of the recombinant human endostatin 2- human serum albumin fusion proteins uses following steps system
It is standby: the recombinant human endostatin 2- human serum albumin fusion proteins encoding gene as shown in SEQ ID NO.6 is passed through into EcoRI
It is connected on shuttle plasmid pPICZ α A with the restriction enzyme site of NotI and obtains recombinant human endostatin 2- human serum albumins fusion egg
White recombinant expression carrier.
The preparation method of each recombinant human endostatin albumen of the invention includes the following steps:
The recombinant human endostatin albumen 1 and recombinant human endostatin albumen 2 the preparation method comprises the following steps: will be such as SEQ ID
The encoding gene of recombinant human endostatin albumen 1 shown in NO.3 and the recombinant human endostatin egg as shown in SEQ ID NO.4
White 2 encoding gene is cloned into pET9c or pCDNA3.1 genophore by round pcr respectively, passes through Escherichia coli table respectively
Reach or by hamster ovary cell (Chinese Hamster Ovary, CHO) carry out eukaryocyte solubility expression, purifying or
Purifying obtains active recombinant human endostatin albumen 1 and recombinant human endostatin albumen 2 after renaturation；
The recombinant human endostatin 2- human serum albumin fusion proteins the preparation method comprises the following steps: will be such as SEQ ID
The C-terminal of recombinant human endostatin albumen 2 shown in NO.2 passes through human serum albumins in flexible joint GGGGSGGGGS connection
Amino acid sequence, and its encoding gene is cloned into pPICZ α A genophore, it is recombinated by Pichia pastoris solubility expression
Human endostatin 2- human serum albumin fusion proteins.
Above-mentioned recombinant human endostatin albumen inhibits endothelial cell growth activity in preparation and inhibits growth of tumour cell
Drug in application.
The preparation method of recombinant human endostatin albumen of the invention includes the following steps:
(1) using Endostatin nucleotide sequence in the Zl97107112.8 patent of invention as shown in SEQ ID NO.7 as
Template increases 9 arginic nucleotide sequences in Endostatin N-terminal with PCR method
(CGCCGCCGCCGCCGCCGCCGCCGCCGC) nucleotide for meeting the recombinant human endostatin albumen 1 of SEQ ID NO.3 is obtained
Then sequence passes through the PCR method of 2 point mutation, obtain the core for meeting the recombinant human endostatin albumen 2 of SEQ ID NO.4
Nucleotide sequence；
(2) under DNA ligase effect respectively with coli expression carrier pMAL-c2 and pET9c genophore or
The connection of pCDNA3.1 expression vector, obtains different 4 kinds of prokaryotic expression plasmids or 2 kinds of eukaryon expression plasmids；
(3) protokaryon is carried out with above-mentioned 4 kinds different prokaryotic expression plasmid conversion bacillus coli DH 5 alphas, BL21 expression bacterium respectively
Expression carries out eukaryotic expression into Chinese hamster ovary celI with above-mentioned 2 kinds of different eukaryotic expression plasmids respectively；
(4) above-mentioned 6 kinds of expression products carry out separation and purification of protein respectively；
(5) purifying protein of above-mentioned 4 kinds of prokaryotic expressions carries out inhibition basic fibroblast growth factor (bFGF) induction
Under vascular endothelial cell increment activity and inhibit chick chorioallantoic membrane (CAM) angiogenesis activity, above-mentioned 2 kinds of eukaryon tables
The purifying protein reached directly carries out inhibiting growth of tumour cell and promotes the activity research of apoptosis of tumor cells effect.
(6) C-terminal of recombinant human endostatin albumen 2 is further connected into upper people's blood by GGGGSGGGGS flexible joint
Pure protein sequence forms recombinant human endostatin 2- human serum albumin fusion proteins, the amino acid sequence of the fusion protein
Meet SEQ ID NO.5, nucleotide sequence meets SEQ ID NO.6.
The present invention is with Endostatin nucleosides in the Zl97107112.8 patent of invention of the Xu Genxing as shown in SEQ ID NO.7
Acid sequence is template, joined continuous 9 arginic nucleotide sequences behind the ATG of N-terminal
(CGCCGCCGCCGCCGCCGCCGCCGCCGC), PCR is then carried out, both ends is obtained with different restriction enzyme sites and meets SEQ
Nucleotide sequence fragment involved in ID NO.3, pcr amplification product use respectively after NdeI and BamHI digestion with pass through same sample prescription
The pET9c carrier of formula processing connects under T4DNA connection enzyme effect together, is transformed into e.colistraindh5α, screening obtains
Positive colony obtains recombinant plasmid pET9c-En1；Or pcr amplification product is used respectively after EcoRI and BamHI digestion with warp
The pMAL-c2 carrier for crossing the same manner processing connects under T4DNA connection enzyme effect together, is transformed into bacillus coli DH 5 alpha bacterium
Strain, screening obtain positive colony, obtain recombinant plasmid pMAL-c2-En1.
Template is classified as using the nucleotides sequence in pET9c-En1 Endostatin, it, will by nucleotide sequence point mutation technology
The amino acid P point mutation of 135 of the recombinant human endostatin albumen 1 as shown in SEQ ID NO.3 is at A in pET9c-En1
(P135A), further by nucleotide sequence point mutation technology, after the 33rd amino acid of recombinant human endostatin albumen 1
RGIRGAD point mutation obtain N-terminal at RGDRGD, after 2 point mutation and carry 9 arginic both ends with different digestion positions
Point meets nucleotide sequence fragment involved in SEQ ID NO.4, after pcr amplification product uses NdeI and BamHI digestion respectively
It is connected under T4DNA connection enzyme effect together with the pET9c carrier by the same manner processing, is transformed into bacillus coli DH 5 alpha bacterium
Strain, screening obtain positive colony, obtain recombinant plasmid pET9c-En2；Or pcr amplification product is used into EcoRI and BamHI respectively
It is connected under T4DNA connection enzyme effect together with the pMAL-c2 carrier by the same manner processing after digestion, is transformed into large intestine bar
Bacterium DH5 α bacterial strain, screening obtain positive colony, obtain recombinant plasmid pMAL-c2-En2.
The recombinant plasmid that above-mentioned 4 kinds are extracted is transformed into respectively in competent E.coli BL21 (DE3), and is carried out respectively
Bacillus coli expression collects expression bacterium, purifies the destination protein recombinant human endostatin 1 and 2 or recombined human MBP expressed
(maltose-binding protein)-Endostatin 1 and 2,96% or more purity.
Compared with above-mentioned escherichia expression system, mammalian cell Chinese hamster ovary cell (Chinese hamster ovary celI) is expressed to mesh
Albumen posttranslational modification, in terms of bioactivity, stability, solubility, immunogenicity and biological circulating half-life in vivo
It is advantageous, therefore pET9c-En1 and pET9c-En2 plasmid is made into template respectively and carries out PCR, pcr amplification product is used respectively
Connect under T4DNA connection enzyme effect together with the pcDNA3.1 carrier by the same manner processing after HindIII and XhoI digestion
Connect, be transformed into e.colistraindh5α, screening obtains positive colony, therefrom respectively obtain recombinant plasmid pcDNA3.1-En1 and
pcDNA3.1-En2.Recombinant plasmid pcDNA3.1-En1 and pcDNA3.1-En2 after extraction is transfected into Chinese hamster ovary celI respectively,
Using G418 resistance screening, obtain positive expression cell strain, collect cell and supernatant after expanding culture, after clasmatosis with it is upper
Clear liquid merge purifying obtains two kinds of different aminoacids structures eukaryotic expression recombinant human endostatin 1 and 2, purity 96% with
On.
The present invention obtains 6 kinds of different Endostatins by said gene engineering method, respectively from pMAL-c2-En1 plasmid
1 fusion protein of soluble M BP- Endostatin and MBP- obtained with pMAL-c2-En2 plasmid by Bacillus coli expression purifying
2 fusion protein of Endostatin；It is multiple by passing through after Bacillus coli expression from pET9c-En1 plasmid and pET9c-En2 plasmid respectively
Property purifying obtain 2 albumen of 1 albumen of prokaryotic expression Endostatin and Endostatin, amino acid structure, which respectively corresponds, to be equal to
SEQ ID NO.1 and SEQ ID NO.2, nucleotide sequence, which respectively corresponds, is equal to SEQ ID NO.3 and SEQ ID NO.4.
By 1 albumen of eukaryotic expression Endostatin that obtains of purifying and interior after pcDNA3.1-En1 and pcDNA3.1-En2 plasmid expression
2 albumen of skin chalone, amino acid structure, which respectively corresponds, is equal to SEQ ID NO.1 and SEQ ID NO.2, nucleotide series pair
SEQ ID NO.3 and SEQ ID NO.4 should be equal to.
The recombinant human endostatin albumen 1 and 2 of two kinds of different aminoacids structures of the invention be respectively 193 amino acid and
192 amino acid are different from above-mentioned patent and have been approved by amino acid knot involved in clinical Endostatin as drug
Structure.Recombinant human endostatin albumen 1 in the present invention compares with recombinant human endostatin albumen 23 amino acid differences (figure
1)；For Recombinant Endostatin albumen 1 compared with the amino acid of Endostatin in patent Zl97107112.8, N-terminal has 10 amino
Sour different, only 183 amino acid are identical, and molecular weight of albumen is also different, respectively 22KD and 20KD (Fig. 2)；Recombinate endothelium suppression
Fibroin 2 has 13 amino acid differences (Fig. 3) compared with the amino acid of Endostatin (rhEndostatin) drug listed；Recombination
Endostatin protein 1 has the difference of 13 amino acid compared with the Endostatin of the AF184060 (gi:6013264) in gene pool
Different, wherein N-terminal has 10 amino acid variant and has 3 amino acid also variant (Fig. 4) in 183 amino acid below.
(Standker L, Schrader M, Kansa SM, the et al.1997.Isolation and such as Standker
characterization of the circulating form of human endostatin.FEBS Lett,420:
It 129-133) is separated to the human endostatin that N-terminal lacks 170 amino acid of 12 amino acid from human blood, and proves
This Endostatin Human Umbilical Vein Endothelial Cells do not have inhibiting effect, this may be that one of Endostatin metabolic breakdown is inactive
By-product.Also demonstrating N-terminal completely is that the important active structure of Endostatin guarantees, therefore is similar to Endostatin (rhEndostatin)
The patent that N-terminal increases modified amino acid or Wang Fengshan etc. with 6His increases the modified amino acid of Tat in Endostatin N-terminal
It is the innovation that a kind of Endostatin changes structure, the invention patent then increases 9 poly arginines in Endostatin N-terminal, and in endothelium
2 point mutation have been carried out in chalone amino acid sequence and have changed structure, to significantly improve the stability and activity of Endostatin.
Two kinds of recombinant proteins of the invention are different from the amino acid sequence of patent No. Zl97107112.8, but remain weight
Group human endostatin inhibits vascular endothelial cell increment activity and suppression under basic fibroblast growth factor (bFGF) induction
The activity of chick chorioallantoic membrane (CAM) angiogenesis processed, and be easier to enter vascular endothelial cell and chicken embryo villus allantois
In film vascular endothelial cell, albumen wears film effect and the structural stability of N-terminal is all considerably better than in the recombination of Zl97107112.8
Skin chalone is good, and has the function of directly inhibiting growth of tumour cell, can play and preferably inhibit neovascular endothelium thin
The effect that born of the same parents generate can be used in treating various diseases caused by new vessels generate, including entity tumor and diabetes cause
Retinopathy and rheumatoid arthritis.
Beneficial effects of the present invention:
The present invention is by 1 fusion protein of soluble M BP- Endostatin and 2 fusion protein of MBP- Endostatin of prokaryotic expression
Come using the strain of bovine adrenal microvascular endothelial cells (bovine adrenal capillary endothelial cell, BCE)
Detect albumen inhibition of endothelial cell proliferation activity；By 2 egg of Endostatin 1 and Endostatin by renaturation process of prokaryotic expression
It is white to be tested by chick chorioallantoic membrane (CAM) to observe angiogenesis suppression action；By the soluble endothelial of CHO eukaryotic expression
Chalone 1 and 2 albumen of Endostatin directly observe the life to human breast cancer cell line Bcap-37 cell by inhibiting tumour cells test
Long inhibiting rate and on apoptosis of tumor cells influence.As a result illustrate that above-mentioned 4 kinds of Bacillus coli expression methods obtain different aminoacids knot
The Endostatin of structure all has the function of significantly inhibiting vascular endothelial cell growth, and the Endostatin of eukaryotic expression is directly to swollen
Tumor cell growth has inhibiting effect.The Endostatin recombinant protein of two kinds of different aminoacids structures provided by the invention may conduct
A kind of potential drug more more advantageous than the Endostatin of Zl97107112.8 patent of invention.
The present invention obtains the recombinant human endostatin 2- human serum albumins of Yeast expression by said gene engineering method
Fusion protein, the expansion application for extending half-life period for Endostatin provide the gene engineering yeast expression of potential drug.
Detailed description of the invention
Fig. 1 is the recombinant human endostatin albumen 1 (on, 193 amino acid) and recombinant human endothelial of different aminoacids structure
The amino acid of chalone albumen 2 (under, 192 amino acid) compares
Fig. 2 is recombinant human endostatin albumen 1 (on, 193 amino acid) and Endostatin in patent Zl97107112.8
The amino acid of (under, 184 amino acid) compares
Fig. 3 is recombinant human endostatin albumen 2 compared with the amino acid of Endostatin (rhEndostatin) drug listed
Fig. 4 be recombination endostatin protein 1 (on, 193 amino acid) in gene pool AF184060 (gi:
6013264) amino acid of Endostatin compares
Fig. 5 is the endostatin protein electrophoretogram that 4 kinds of Prokaryotic expression, purifications and 2 kinds of eukaryotic expressions purify
Wherein, M mark, 1 be the recombinant human endostatin 1,2 of Bacillus coli expression is the recombined human of Bacillus coli expression
Endostatin 2,3 be eukaryotic expression recombinant human endostatin Isosorbide-5-Nitrae be eukaryotic expression recombinant human endostatin 2,5 be large intestine bar
The recombined human MBP- endostatin protein 1,6 of bacterium expression is the recombined human MBP- endostatin protein 2 of Bacillus coli expression
Fig. 6 is the growth that recombined human MBP- endostatin protein 1 and 2 inhibits BCE cell under various concentration, respectively with yin
Property control MBP albumen and the recombinant human endostatin albumen of positive control patent Zl97107112.8 compare.
Fig. 7 is the chicken embryo villus allantois under recombinant human endostatin 1 and 2 inhibits basic fibroblast growth factor to induce
Film (CAM) angiogenesis
Fig. 8 is that the recombinant human endostatin 1 and 2 of eukaryotic expression purifying inhibits the growth of MCF-7 cell, special with positive control
The recombinant human endostatin albumen of the Bacillus coli expression purifying of sharp Zl97107112.8 compares.
Fig. 9 is the recombinant human endostatin 2- human serum albumin fusion proteins of Yeast expression
Specific embodiment
Further citing description is made to the present invention with embodiment below.In the examples below that, using prokaryotic expression carrier
In the Endostatin 1 and 2 or Endostatin fusion protein 1 and 2 of the different aminoacids structure that e. coli expression purifies,
Prove that the recombinant human endostatin of these prokaryotic expressions after purification has the characteristic for inhibiting endothelial cell growth.Pass through eukaryon table
Up to carrier, it is transfected into Chinese hamster ovary celI, it was demonstrated that it can also be expressed in eukaryocyte, the different aminoacids knot that expression and purification obtains
The recombinant human endostatin 1 and 2 of the eukaryotic expression purifying of structure, which has, directly to be inhibited growth of tumour cell and tumour cell is promoted to wither
The activity died.Recombinant human endostatin 2 can form fusion protein with human serum albumins by flexible joint, and can be in ferment
Matrix reaches.To provide these three common gene engineering expression sides of Bacillus coli expression, Yeast expression, CHO eukaryotic expression
Method.
These embodiments are only to illustrate, and the invention is not limited in any way.
The genetic engineering recombinant protein and preparation method thereof of 1 recombinant plasmid pET9c-En1 of embodiment
This patent shows the amino acid sequence of coding prokaryotic expression Endostatin 1 and the hair of nucleotide sequence and approved
The comparison of bright patent or the Endostatin as drug approved clinical research.
PCR primer sequence in genetic engineering recombinant protein of recombinant plasmid pET9c-En1 and preparation method thereof is as follows:
Primer (1): ATTCATATGCGCCGCCGCCGCCGCCGCCGCCGCCGCCACAGCCACCGCGACTTCCA G (SEQ
ID NO.8)
Primer (2): GCCGGATCCCTACTTGGAGGCAGTCATGAAGCT (SEQ ID NO.9)
The primer of PCR reaction is synthesized by Shanghai Sangon company, with the Xu Genxing as shown in SEQ ID NO.7
Endostatin nucleotides sequence is classified as template in Zl97107112.8 patent of invention, and above-mentioned 2 kinds of primers and routine PCR reaction examination is added
Agent, PCR reaction condition are 94 DEG C of denaturation 4min, then 94 DEG C of denaturation 1min, 56 DEG C of renaturation 1min, 72 DEG C of extension 1min, 30
Circulation, last 72 DEG C of extensions 10min.1% agarose gel electrophoresis of PCR product.It is cut in the UV lamp containing 579bp or so
The Agarose plug of DNA fragmentation recycles target DNA with gel DNA QIAquick Gel Extraction Kit, all by the pET9c carrier and PCR fragment of purifying
With NdeI and BamHI double digestion.Product after digestion by 1.5% agarose gel electrophoresis, return after digestion by segment needed for recycling
Receive plasmid vector and connect with PCR product segment and react as follows: 1 μ L of carrier (pET9c) is inserted into 7 μ L of PCR fragment, 10 μ L of distilled water,
10 × connection, 2 μ L, T4DNA ligase of buffer, 0.8 μ L.In 22 DEG C of static 1hr after above-mentioned reaction system mixing, 65 DEG C are placed into
It is incubated for 10min in water-bath and inactivates ligase.Then competence E.coli DH will be prepared5αConnection reaction product 10 is added in 100 μ L
μ L mixes postposition 30min, 42 DEG C of heat shock 90s on ice.The 800 μ L of common LB culture medium of antibiotic-free is added in every pipe, and 37 DEG C incubate
Educate 1hr.3000rpm is centrifuged 2min, discards 800 μ L supernatants, thallus is mixed gently in remaining liquid.With sterile elbow glass
50 μ L bacterium solutions are laid on LB agar (1.5% agar) plate containing 50 μ g/mL kanamycins by glass paving bacterium device, 37 DEG C of inversion cultures
Overnight.With the positive colony on sterile toothpick picking plate, it is inoculated into the LB liquid medium containing 50 μ g/mL kanamycins and expands
Increase, 37 DEG C of shaken overnights.The 1.5mL bacterium solution of shaken overnight is taken, 10000g is centrifuged 2min, abandons supernatant.Reagent is extracted according to plasmid
Box specification extracts recombinant plasmid.Recombinant plasmid digestion identification, sequencing.
By sequencing meet the pET9c-En1 plasmid of SEQ ID NO.3 to be transformed into competence E.coli BL21 (DE3) big
In enterobacteria, 6hr is expressed with IPTG Induction of bacterial, the bacterial suspension before collecting induction is as control.Then 12% is carried out
SDS-PAGE electrophoresis.The bacterium each 1.5mL, 5000rpm for taking induction and not inducing are centrifuged 3min, are washed twice with PBS, abandon supernatant,
100 μ L sample-loading buffers are added, Ultrasonic Pulverization bacterium takes above-mentioned processed sample loading, PAGE gel electrophoresis is carried out,
Prove destination protein expression.Then with the Endostatin of Western blot identification expression.By the endostatin protein of expression into
Row SDS-PAGE electrophoresis.It transduces to nitrocellulose filter.Nitrocellulose filter cuts destination protein band after being dyed with Ponceaux.
It is decolourized with PBS (0.1M, pH7.0), 1% skimmed milk power (PBS matches) room temperature closes 30min.The diluted rabbit-anti of 1:1000 is added
Human endostatin polyclonal antibody, 37 DEG C of incubation 2hr.It is washed 3 times with 1% skimmed milk power (PBS matches), washes 5min every time.It is added
Goat anti-rabbit igg-HRP secondary antibody, 37 DEG C of incubation 1hr.It is washed 5 times with 1% skimmed milk power (PBS matches), washes 10min every time.0.05M
Tris-HCl (pH 7.4) is washed twice, washes 5min every time.0.05%DAB and 0.03%H2O2Mixed liquor carries out colour developing identification.
Expand scale fermentation, 37 DEG C of cultures to OD according to above-mentioned condition600=0.4-0.6 is added IPTG (0.5mmol/L),
Continue culture to OD600> 1.2 receive bacterium, and the buffer of 20mmol/L Tris-HCl. and 2mmol/L EDTA pH8.0 is added after broken bacterium
Bacterium is washed, centrifugation 7000rpm × 10min abandons supernatant, and 6mol/L guanidine hydrochloride and 1% mercaptoethanol containing 20% is added
Tris-HCl lysate, centrifugation 12000rpm × 30min take supernatant, inclusion body primary extract are obtained, with 20mmol/L Tris-
HCl, pH8.0 dilution, destination protein precipitating wash inclusion body with the buffer, are added and contain 6mol/L by being centrifuged foreigh protein removing
The Tris-HCl buffer of guanidine hydrochloride and 1% mercaptoethanol is slowly dropped into renaturation solution (L- containing 0.4mol/L with 1:50 volume ratio
Arg, 1mmol/L reduced glutathione, the 20mmol/L NaAC-HAC, pH5.0 of 0.1mmol/L oxidized form of glutathione)
In, 1h is stirred, 4 DEG C of renaturation take supernatant measurement protein concentration to determine renaturation effect, it dialyses in 20mmol/L NaAC-HAC,
After pH5.0, anion exchange (DEAE-Sepharose Fast Flow) and cation exchange resin (SP- is respectively adopted
Sepharose FF) separation, then recombination of the purity through 96% or more electroresis appraisal is obtained by the separation of Sephadex G-25 gel
Human endostatin albumen 1.
The genetic engineering recombinant protein and preparation method thereof of 2 recombinant plasmid pET9c-En2 of embodiment
This patent show coding prokaryotic expression recombinant human endostatin albumen 2 amino acid sequence and nucleotide sequence with
Embodiment 1 and compared with the patent of invention of approved or Endostatin as drug approved clinical research.
PCR primer sequence in genetic engineering recombinant protein of recombinant plasmid pET9c-En2 and preparation method thereof is as follows:
Primer (3) CATATGCGCCGCCGCCGCCGCCGCCGCCGCCGCCACAGCCACCGCGACTTCC (SEQ ID
NO.10)
Primer (4) GCGGCATGCGGGGCGATCGCGGGGACTTCCAGTGCTTCC (SEQ ID NO.11)
Primer (5) GGAAGCACTGGAAGTCCCCGCGATCGCCCCGCATGCCGC (SEQ ID NO.12)
Primer (6) GTGGCATGGCTCGGACGCAAACGGGCGCAGGCTG (SEQ ID NO.13)
Primer (7) CAGCCTGCGCCCGTTTGCGTCCGAGCCATGCCAC (SEQ ID NO.14)
The primer of PCR reaction is synthesized by Shanghai Sangon company, with the Endostatin of the pET9c-En1 in embodiment 1
Nucleotides sequence is classified as template, respectively with primer (3) and primer (5), with primer (4) and primer (5), with primer (6) and primer (7)
It being separately added into routine PCR reaction reagent and carries out PCR reaction, reaction condition is 95 DEG C of denaturation 5min, then 95 DEG C of denaturation 20s, 65
DEG C renaturation 20s, 72 DEG C of extension 30s, 35 circulations, last 72 DEG C of extensions 7min.1% agarose gel electrophoresis of PCR product.It uses again
Primer (3) and primer (2) do template with above-mentioned PCR product and carry out overall length PCR reaction, and reaction condition is 95 DEG C of denaturation 5min, so
95 DEG C of denaturation 20s afterwards, 70 DEG C of renaturation 20s, 72 DEG C of extension 40s, 35 recycle, last 72 DEG C of extensions 10min.PCR product is obtained,
The PCR for meeting SEQ ID NO.4 sequence that purifying obtains 2 point mutation containing 9 poly- essence acid after 1% agarose gel electrophoresis is produced
Object, PCR product pass through 1% agarose gel electrophoresis.The Agarose plug containing 576bp or so DNA fragmentation is cut in the UV lamp,
Target DNA is recycled with gel DNA QIAquick Gel Extraction Kit, clone obtains pET9c-En2 plasmid according to the method for embodiment 1, passes through reality
The method expression and purification for applying example 1 obtains the large intestine bar for meeting SEQ ID NO.2 sequence containing 9 poly arginines and 2 point mutation
2 albumen of recombinant human endostatin of bacterium expression, purity is through 96% or more electroresis appraisal.
The genetic engineering recombinant protein and preparation method thereof of embodiment 3 recombinant plasmid pMAL-c2-En1 and pMAL-c2-En2
Primer (8) CCGGAATTCCGCCGCCGCCGCCGCCGCCGCCGCCGCCAC (SEQ ID NO.15)
According to the method for embodiment 1, respectively in the Endostatin nucleotide sequence and pET9c-En2 of pET9c-En1
Skin chalone nucleotides sequence is classified as template, and routine PCR reaction reagent is added with primer (8) and primer (2) respectively and carries out PCR, respectively
The PCR product of acquisition and the pMAL-c2 carrier of purifying are used into EcoRI and BamHI digestion respectively, according to the method for embodiment 1 gram
Grand acquisition pMAL-c2-En1 and pMAL-c2-En2 plasmid.By empty carrier pMAL-c2 and recombinant plasmid pMAL-c2-En1 and
PMAL-c2-En2 is transformed into respectively in E.coli BL21 (DE3), is inoculated in the LB culture solution containing 100 μ g/mL penicillin,
OD is grown to it600When for 0.4-0.6, IPTG (final concentration of 0.4mM) inducing expression 4hr is added, 5000rpm is collected by centrifugation
Then thallus, the bacterium solution before collecting induction carry out SDS-PAGE electrophoresis and observe expression as control.The thallus of inducing expression
Be dissolved in lysis buffer (0.1M PBS, 0.5M NaCl, 0.25%Tween-20,10mM 2 mercapto ethanol, 10mM EDTA,
10mM EGTA), in ultrasonication on ice bath, 15000g is centrifuged 20min, and supernatant is in 100:1 ratio by supernatant and the affine layer of MBP
4 DEG C of column of analysis combine overnight, are washed with 50 times of column volume lysis buffers and 1 times of column volume 0.1M PBS (pH 7.2), then use 10mM
Maltose (preparation of 0.1M PBS, pH 7.2) elute respectively lower empty carrier MBP albumen or recombinant plasmid pMAL-c2-En1 and
The MBP- endostatin protein 1 and MBP- endostatin protein 2 of pMAL-c2-En2 expression, ultraviolet specrophotometer measurement elution
The protein content of liquid, then recombination MBP egg of the purity through 96% or more electroresis appraisal is obtained by the separation of Sephadex G-25 gel
White, recombined human MBP- endostatin protein 1 and recombined human MBP- endostatin protein 2.
The genetic engineering recombinant protein of embodiment 4 recombinant plasmid pcDNA3.1-En1 and pcDNA3.1-En2 and its preparation side
Method
Primer (9) CCCAAGCTTCGCCGCCGCCGCCGCCGCCGCCGCCGCCAC (SEQ ID NO.16)
Primer (10) CCGCTCGAGCTTGGAGGCAGTCATGAAGCT (SEQ ID NO.17)
Routine PCR reaction reagent is added using primer (9) and (10), respectively in pET9c-En1 and pET9c-En2 plasmid
Endostatin nucleotides sequence be classified as template carry out PCR, the PCR product of acquisition and the pcDNA3.1 carrier of purifying are used respectively
HindIII and XhoI digestion, clone obtains pcDNA3.1-En1 and pcDNA3.1-En2 plasmid according to the method for embodiment 1.It is logical
Plasmid is extracted after crossing sequencing identification, with 2 × 105/ mL Chinese hamster ovary celI is inoculated in 6 orifice plates, containing 10% calf serum and mould
The PRMI-1640 culture medium of element, streptomysin, in 37 DEG C of 5%CO2It cultivates in cell incubator to 50% cell coverage rate, presses
According to product description by 2,000 15 μ l of Lipofectamine respectively with 2 μ g pcDNA3.1-En1 and pcDNA3.1-En2 plasmids
After the culture solution that serum-free antibiotic-free is added in mixing adds up to 100 μ l culture 45min, 800 μ l serum-free antibiotic-frees are added
After culture solution, 37 DEG C of 5%CO25h is cultivated, 1ml is added, and the fetal calf serum antibiotic-free culture solution containing 20% continues to cultivate 48h, uses
0.9g/L G418 carry out pressurization screening stable clone, obtain positive colony cell after by identification after subculture.Using transfection
The CHO expression cell strain of pcDNA3.1-En1 and pcDNA3.1-En2 plasmid low serum or free serum culture 4d on a large scale, point
Not Shou Ji supernatant cell cell ultrasonication with the content of ELISA measurement Endostatin 1 and Endostatin 2, is as a result existed
Content not equal Endostatin is detected in supernatant clasmatosis supernatant.Then merge supernatant respectively, according to Ni-NTA
Beads column specification uses nickel column affinity purification, respectively with the buffering containing 20,50,100,250,500mmol/L imidazole
Liquid elution, purifying protein pass through the separation of Sephadex G-25 gel again and obtain eukaryon table of the purity through 96% or more electroresis appraisal
Up to recombinant human endostatin albumen 1 and recombinant human endostatin albumen 2.
5 recombined human MBP- endostatin protein 1 of embodiment and recombined human MBP- endostatin protein 2 inhibit endothelial cell raw
It is long
By the strain of bovine adrenal microvascular endothelial cells (bovine adrenal capillary endothelial cell,
BCE) cell inoculation 37 DEG C of cultures in the sterilizing culture bottle for being covered with one layer of 1.5% gelatin (preparation of Hanks liquid), it is good to choose growth
Good cell is inoculated in 24 well culture plates with the concentration in 12500/hole, and culture solution is DMEM (containing 10% calf serum), and 37 DEG C,
10%CO2R for 24 hours is cultivated, culture solution is sucked, the recombined human MBP- endostatin protein 1 of various concentration is contained in the change hole 0.25mL/
Or the culture solution of recombined human MBP- endostatin protein, final concentration of protein be respectively 0ng/mL, 250ng/mL, 500ng/mL,
750ng/mL, 1000ng/mL, 2000ng/mL, 4000ng/mL, after cultivating 20min, every hole adds 0.25mL containing bFGF's
DMEM complete medium, the final concentration of 1ng/ml of bFGF, 37 DEG C, 10%CO2It is incubated for 72hr, sucks supernatant, every hole 0.5mL
0.05% pancreatin (EDTA containing 10mM) vitellophag is suspended in Hematall device (Fisher Scientific product), presses
Description of product method, rolling counters forward of using tricks.The result shows that compared with negative control MBP protein control group, recombined human MBP- endothelium
Chalone albumen 1 or recombined human MBP- endostatin protein 2 have apparent inhibition effect to the growth of BCE cell under various concentration
The inhibitory effect of fruit, high dose becomes apparent from, and the inhibitory effect of recombined human MBP- endostatin protein 1 is similar to the special of positive control
The recombinant human endostatin albumen of sharp Zl97107112.8 provided by JiangSu WuZhong Medicine Group Co., Ltd, recombined human MBP-
The inhibitory effect of endostatin protein 2 is better than recombined human MBP- endostatin protein 1 (Fig. 6)
The recombinant human endostatin albumen 1 and recombinant human endostatin albumen 2 of 6 Bacillus coli expression of embodiment inhibit chicken embryo
Chorioallantoic membrane (CAM) vascular endothelial cell growth
It takes and qualified egg is examined to be incubated for 8-9 days in incubator, mark the position of foetus under candler, the position at rich blood vessel
External application iodine disinfection is prepared into chicken embryo villus allantois artifical-air cell between shell membrane and chorioallantoic membrane, places methylcellulose saucer, point
Not Jia Ru in Bacillus coli expression recombinant human endostatin 1 and recombinant human endostatin 2 protein 12 μ L, concentration 1.5mg/
ML, and be respectively negative control and positive control with the physiological saline of equal volume and bFGF.Recombinant human endostatin 1 and recombination
The dosage of 2 albumen of human endostatin is respectively 25 μ g/, and positive control bFGF 1ng/ is only.It 37 DEG C, dissects after incubation 3d
CAM film is taken, take a picture and makes sample, and counts vascular strip number.The result shows that the first order vessel number of saline control group is
34.5 ± 2.8, second level blood vessel number is 84.6 ± 3.6；The first order vessel number of bFGF positive controls is 64.2 ± 3.8, second level blood
Pipe number is 101.5 ± 3.8；The first order vessel number of recombinant human endostatin 1 be 23.5 ± 6.1, second level blood vessel number be 59.3 ±
12.2；The first order vessel number of recombinant human endostatin 2 is 18.3 ± 2.8, and second level blood vessel number is 39.9 ± 10.1, is illustrated and physiology
Saline control group and bFGF positive controls compare, recombinant human endostatin 1 and 2 can inhibit basic fibroblast growth because
Chick chorioallantoic membrane (CAM) angiogenesis under son induction, and the inhibitory effect of recombinant human endostatin 2 is more preferable (Fig. 7).
The recombinant human endostatin 1 and 2 albumen of recombinant human endostatin of 7 eukaryotic expression of embodiment inhibit growth of tumour cell
With promotion apoptosis of tumor cells effect
MCF-7 Human Breast Cancer Cells are placed in 37 DEG C of incubators in culture bottle with RPMI-1640 culture solution culture,
It is passed through 5%CO2(relative humidity 95%).With trypsin digestion, cell is pressed 1 × 103/ hole is inoculated in 96 well culture plates,
After cultivating 6h, culture solution is sucked, every hole is separately added into the recombinant human endostatin of the eukaryotic expression of RPMI-1640 doubling dilution
1 and 2 protein 10 of recombinant human endostatin, 0 μ L, each concentration set 3 parallel holes, and negative control hole adds 100 μ L of culture solution.Continue
After cultivating 48h, original fluid is discarded, every hole is separately added into the recombined human of the eukaryotic expression of RPMI-1640 doubling dilution again
0 μ L of Endostatin 1 and 2 protein 10 of recombinant human endostatin continues after cultivating 48h, sucks culture solution, 0.5g/L is added in every hole
The 20 μ L of PBS solution of MTT, is further cultured for 4h, sucks MTT solution in hole, and 150 μ L, 100rpm vibration of dimethyl sulfoxide is added in every hole
Swinging dissolves it sufficiently, measures absorbance in 490nm with microplate reader.The result shows that 1 He of recombinant human endostatin of eukaryotic expression
2 albumen of recombinant human endostatin has apparent inhibition growth to MCF-7 cell.The result shows that recombinant human endostatin 1
The growth of MCF-7 Human Breast Cancer Cells, eukaryotic expression can directly be inhibited under a certain concentration with 2 albumen of recombinant human endostatin
Recombinant human endostatin 1 and 2 albumen of recombinant human endostatin under 200 μ g/mL concentration levels to MCF-7 Human Breast Cancer Cells
Growth inhibition ratio respectively reach 35.6% and 51.3% inhibiting rate, positive control is using patent Zl97107112.8 by river
The recombinant human endostatin albumen (Fig. 8) that Su Wuzhong Pharmaceutical Group Co., Ltd provides, experimental result is shown, recombinant human endothelial suppression
The effect of fibroin 1 is close with positive control, and the significant effect of recombinant human endostatin albumen 2 is higher than positive control.
MCF-7 cell is as above cultivated, with trypsin digestion, cell is pressed 5 × 103/ hole is inoculated in 24 well culture plates
In, after cultivating 48h, culture solution is sucked, every hole is added with the recombinant human endostatin of the eukaryotic expression of RPMI-1640 doubling dilution
1 and each 1mL of 2 albumen of recombinant human endostatin, each concentration set 3 parallel holes, and negative control hole adds culture solution 1mL.Continue to train
After supporting 3h, culture solution is sucked, with trypsin digestion and cell is collected by centrifugation, with propidium iodide (PI), AnnexinV is to cell
It is marked, the case where with flow cytomery Apoptosis.The result shows that recombinant human endostatin 1 and recombinant human endothelial
2 albumen of chalone can directly inhibit MCF-7 Human Breast Cancer Cells under a certain concentration, and promote the early apoptosis of tumour cell
Rate is respectively 6.05% and 7.88%.Illustrate that the Endostatin of above-mentioned different structure directly inhibits MCF-7 Human Breast Cancer Cells
Growth and inducing apoptosis of tumour cell.
The genetic engineering preparation side of the recombinant human endostatin 2- human serum albumin fusion proteins of 8 Yeast expression of embodiment
Method
According to method for synthesizing gene, the Endostatin 2 for meeting SEQ ID NO.2 is added into flexible joint GGGGSGGGGS,
The 25-609 amino acid sequence of the human serum albumins amino acid sequence in GenBank CAA23754.1 is connected again, is added up to
The recombinant human endostatin 2- human serum albumin fusion proteins amino acid sequence for meeting SEQ ID NO.5 of 787 amino acid,
The amino acid sequence is converted into nucleotide sequence, and optimizes to form 2361 cores according to Yeast expression hobby codon
The recombinant human endostatin 2- human serum albumin fusion proteins nucleotide sequence for meeting SEQ ID NO.6 of thuja acid, synthesis
Nucleotide sequence is connected on shuttle plasmid pPICZ α A by the restriction enzyme site of EcoRI and NotI, is identified and is sequenced by digestion
After confirmation, the expression plasmid of extraction carries out linearization process, with I digestion expression plasmid of Sal, is tried with the quick glue recycling of DNA fragmentation
The expression plasmid of agent box purified linear transduces into Pichia pastoris GS115 competent cell, takes 1ml salmon sperm dna boiling water bath
5min is boiled, competence saccharomycete is centrifuged by rapid ice bath to prepare single-stranded monomer DNA, remaining LiCl solution is sucked, by following
Sequence is added: 50%PEG 3350 240ul, 1mol/L LiCl 36ul, 2mg/ml single-stranded salmon sperm dna 25ul, 5~10ug
Linearization plasmid DNA 50ul is mixed, and 30 DEG C of water-baths are incubated for 30min, 42 DEG C of water-bath heat shock 20~25min, 6000~
Yeast thallus is collected by centrifugation in 8000rpm, and yeast is resuspended in 1ml YPD culture medium, and 30 DEG C of shaking tables are incubated for, and after 2h, take 200ul bacterium solution
100 μ g/ml bleomycin culture medium flat plates are laid on, are cultivated 2~3 days in 30 DEG C, single bacterium colony is picked from the plate and carries out PCR mirror
It is fixed.By the positive transformant of screening in the triangular flask of the 50mL containing 5mL BMGY fluid nutrient medium, 30 DEG C, 250rpm oscillation
Culture, takes the culture solution 1ml of each bacterium in the 100ml triangular flask containing 15ml BMMY, 30 DEG C, 250rpm shaken cultivation is for 24 hours.
Per 100% methanol that 150 μ L are added for 24 hours in each culture bottle, 30 DEG C, 250rpm shaken cultivation collects 2d in each triangular flask
With the expression supernatant of 3d, -20 DEG C are saved for use.It expresses albumen to identify by SDS-PAGE electrophoresis and Western Blot, with exempting from
Epidemic disease turbidimetry for Determination expresses protein concentration, draws standard curve.Recombinant human endostatin-the human seralbumin for taking Yeast expression to generate
Albumen and its control medium are detected under the conditions of 510nm with 8 μ L with the sensitizing latex of 130nm partial size, according to standard song
Line obtains the concentration for the recombinant human endostatin 2- human serum albumins that Yeast expression generates.Then expand training according to the method described above
Expression is supported, expression supernatant is soluble protein, and anion exchange (DEAE-Sepharose Fast Flow) and sun is respectively adopted
Ion exchange resin (SP-Sepharose FF) separation, then acquisition purity is separated by Sephadex G-25 gel and is reflected through electrophoresis
Fixed 96% or more recombinant human endostatin 2- human serum albumin fusion proteins.This is provided for the expansion application of Endostatin
The method of the gene engineering yeast expression and other eukaryotic expression Endostatin soluble fusion proteins of potential drug.
Claims (6)
1. a kind of recombinant human endostatin albumen, it is characterised in that: the recombinant human endostatin albumen is recombinant human endostatin
Any one in albumen 1, recombinant human endostatin albumen 2 and recombinant human endostatin 2- human serum albumin fusion proteins,
The amino acid sequence of the recombinant human endostatin albumen 1 is as shown in SEQ ID NO.1, the recombinant human endostatin albumen 2
Amino acid sequence as shown in SEQ ID NO.2, the ammonia of the recombinant human endostatin 2- human serum albumin fusion proteins
Base acid sequence is as shown in SEQ ID NO.5.
2. the encoding gene of recombinant human endostatin albumen described in claim 1, it is characterised in that: the encoding gene has following
One of nucleotide sequence:
(1) nucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.6 in sequence table, wherein SEQ
ID NO.3 is the nucleotide sequence of encoding amino acid sequence recombinant human endostatin albumen 1 as shown in SEQ ID NO.1, SEQ
ID NO.4 is the nucleotide sequence of encoding amino acid sequence recombinant human endostatin albumen 2 as shown in SEQ ID NO.2, SEQ
ID NO.6 is that encoding amino acid sequence recombinant human endostatin 2- human serum albumins as shown in SEQ ID NO.5 merges egg
White nucleotide sequence；
(2) in polynucleotide the amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.5 nucleosides
Acid.
3. including that the recombinant expression carrier of recombinant human endostatin protein coding gene described in claim 2, transgenosis are thin
Born of the same parents system and transgenosis recombinant bacterium.
4. the recombinant expression carrier for expressing recombinant human endostatin albumen as described in claim 1, it is characterised in that:
The recombinant expression carrier of the recombinant human endostatin albumen 1 uses following steps to prepare: shown in SEQ ID NO.7
Endostatin nucleotides sequence is classified as template, draws with the primer (1) as shown in SEQ ID NO.8 and as shown in SEQ ID NO.9
Object (2) is that primer carries out PCR amplification, and pcr amplification product and pET9c carrier with NdeI and BamHI digestion and are connected respectively
It connects to obtain recombinant plasmid pET9c-En1；
The recombinant expression carrier of the recombinant human endostatin albumen 2 uses following steps to prepare: with above-mentioned recombinant plasmid
PET9c-En1 is template, respectively with the primer (3) as shown in SEQ ID NO.10 and the primer as shown in SEQ ID NO.12
(5), with the primer (4) as shown in SEQ ID NO.11 and the primer (5) as shown in SEQ ID NO.12, with such as SEQ ID
Primer shown in NO.13 (6) and the primer (7) as shown in SEQ ID NO.14 are that primer carries out PCR amplification acquisition PCR amplification production
Object 1 is being template with the pcr amplification product 1 of acquisition, with the primer (3) as shown in SEQ ID NO.10 and such as SEQ ID NO.9
Shown in primer (2) carry out PCR amplification obtain pcr amplification product 2, pcr amplification product 2 and pET9c carrier are used into NdeI respectively
With BamHI digestion and be attached to obtain recombinant plasmid pET9c-En2；
The recombinant expression of the recombinant expression carrier of the recombinant human endostatin albumen 1 and the recombinant human endostatin albumen 2
Carrier uses following steps to prepare: respectively using recombinant plasmid pET9c-En1 and recombinant plasmid pET9c-En2 as template, with such as SEQ
Primer shown in ID NO.16 (9) and the primer (10) as shown in SEQ ID NO.17 are that primer carries out PCR amplification acquisition PCR expansion
Increase production object, pcr amplification product and pcDNA3.1 carrier with HindIII and XhoI digestion and are attached respectively and respectively obtain weight
Group plasmid pcDNA3.1-En1 and recombinant plasmid pcDNA3.1-En2；
The recombinant expression carrier of the recombinant human endostatin 2- human serum albumin fusion proteins is prepared using following steps: will
The recombinant human endostatin 2- human serum albumin fusion proteins encoding gene as shown in SEQ ID NO.6 by EcoRI and
The restriction enzyme site of NotI is connected on shuttle plasmid pPICZ α A and obtains recombinant human endostatin 2- human serum albumin fusion proteins
Recombinant expression carrier.
5. the preparation method of recombinant human endostatin albumen as described in claim 1, it is characterised in that:
The recombinant human endostatin albumen 1 and recombinant human endostatin albumen 2 the preparation method comprises the following steps: will be such as SEQ ID NO.3
Shown in recombinant human endostatin albumen 1 encoding gene and the recombinant human endostatin albumen 2 as shown in SEQ ID NO.4
Encoding gene is cloned into pET9c or pCDNA3.1 genophore by round pcr respectively, passes through Bacillus coli expression or logical respectively
It crosses hamster ovary cell CHO and carries out eukaryocyte solubility expression, purify and obtained in active recombined human after purifying or renaturation
Skin chalone albumen 1 and recombinant human endostatin albumen 2；
The recombinant human endostatin 2- human serum albumin fusion proteins the preparation method comprises the following steps: will be such as SEQ ID NO.2 institute
The C-terminal of the recombinant human endostatin albumen 2 shown passes through human serum albumins amino acid in flexible joint GGGGSGGGGS connection
Sequence, and its encoding gene is cloned into pPICZ α A genophore, recombinant human endothelial is obtained by Pichia pastoris solubility expression
Chalone 2- human serum albumin fusion proteins.
6. recombinant human endostatin albumen described in claim 1 inhibits endothelial cell growth activity in preparation and inhibits by new life
Application in the drug of growth of tumour cell caused by angiogenesis.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1177005A (en) * | 1997-09-10 | 1998-03-25 | 徐根兴 | Method for gene clone of inhibitory factor for hyperplasia of inner blood vessel cells in human body's real tumor, and using same to anti tumer blood vessel regeneration |
| CN101002946A (en) * | 2006-01-20 | 2007-07-25 | 清华大学 | Medicine for treating tumor, and its application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1177005A (en) * | 1997-09-10 | 1998-03-25 | 徐根兴 | Method for gene clone of inhibitory factor for hyperplasia of inner blood vessel cells in human body's real tumor, and using same to anti tumer blood vessel regeneration |
| CN101002946A (en) * | 2006-01-20 | 2007-07-25 | 清华大学 | Medicine for treating tumor, and its application |
Non-Patent Citations (1)
| Title |
|---|
| RGD-modified endostatin fragments showed an antitumor effect through antiangiogenesis;Pu CY et al.;《Anticancer Drugs》;20120930;第23卷(第8期);788-802 |
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