CN105646701A - Recombinant human endostatin protein with different amino acid structures, method for preparing recombinant human endostatin protein and application thereof - Google Patents
Recombinant human endostatin protein with different amino acid structures, method for preparing recombinant human endostatin protein and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant human endostatin protein with different amino acid structures, a method for preparing the recombinant human endostatin protein and application thereof. Amino acid sequences of a recombinant human endostatin protein 1 are shown as SEQ ID NO.1, and amino acid sequences of a recombinant human endostatin protein 2 are shown as SEQ ID NO.2. The recombinant human endostatin protein, the method and the application have the advantages that the two types of recombinant proteins are active in the aspects of inhibiting the proliferation activity of vascular endothelial cells under the induction actions of basic fibroblast growth factors (bFGF) and inhibiting the angiogenesis activity of chick chorioallantoic membranes (CAM) and are easy to enter the vascular endothelial cells and vascular endothelial cells of the chick chorioallantoic membranes, and transmembrane effects of the recombinant human endostatin protein and the structural stability of N ends can be obviously improved; effects of directly inhibiting tumor cell growth can be realized, effects of inhibiting generation of neonatal vascular endothelial cells can be effectively realized, and the recombinant human endostatin protein can be used for treating various diseases, including solid tumor, retinal diseases due to diabetes mellitus and rheumatoid arthritis, due to angiogenesis.
Description
Technical field
The invention belongs to genetic engineering field, be specifically related to have with gene engineering method preparation suppress endothelial cell growth activity or the pQE30/en pPIC9K/en's albumen of multiple different aminoacids structure suppressing growth of tumour cell and its preparation method and application.
Background technology
1997, Harvard University O ' Reilly etc. found that the culture fluid of mice hemangioendothelioma (EOMA) cell line can suppress the propagation of vascular endothelial cell. Obtain a kind of new protein, the Endostatin of called after Mus by separating purification, translate into vascellum esoderma inhibin (abbreviation Endostatin) later. On JIUYUE 10th, 1997, Xu Genxing etc. has applied for the patent of invention (Zl97107112.8) of human endostatin, and January 10 calendar year 2001 obtains invention patent mandate, and this is Endostatin one of patent obtaining mandate the earliest in the world. The Human endostatin gene by escherichia coli expression, is human collagen octadecyl is because of the 1503-2055 protein active fragment expressed, 184 aminoacid, molecular weight 20KD. Homology with mouse Endostatin aminoacid sequence has 85.33%, is complete the clinical research of three phases at present. 184 amino acid whose Endostatins of U.S.'s yeast expression enter clinical research in the world the earliest, in June, 1999, FDA ratifies EntreMed clinical experiment application, I phase clinic starts, rest at present in II phase clinical research, main reason is that antibody produces the problems such as probability height, indication selection and subcutaneous injection list medicine application method. The similar kind of the Endostatin (rhEndostatin) that international first approval produces is 192 aminoacid, N end at Endostatin adds 9 aminoacid such as MetGlyGlySerHisHisHisHisHis (His), it is jointly formed 192 amino acid whose endostatin proteins, escherichia coli expression with the 183 of Endostatin aminoacid. The Endostatin recombiant protein of the Endostatin different aminoacids structural mutation that the patent (number of patent application 201410102963.2) of Li Xiaoxin etc. relates to endothelium chalone mutant and Polyethylene Glycol (PEG) is modified.The patent (number of patent application 200410013621.X) of Liu Xinghan etc. relates to changing Endostatin amino acid structure, strengthens anti-tumor activity. The fusion protein that the patent (number of patent application 201210149201.9) of Wang Fengshan etc. relates to HIV (human immunodeficiency virus) Trans-activating transduction albumen Tat (TyrGlyArgLysLysArgArgGlnArgArgArg) and Endostatin collectively forms. The patent (number of patent application 200810025406.X) of Yao Wenbing etc. relates to the endothelium chalone mutant containing alpha-non-natural amino acid and derivant thereof.
Summary of the invention
First purpose of the present invention is to provide multiple pQE30/en pPIC9K/en's albumen, and it has the activity suppressing endothelial cell growth, but aminoacid sequence and structure are all different from the endostatin protein having been approved by patent or approval clinical research.
Second purpose of the present invention is to provide the gene engineering preparation method of both endostatin proteins, wherein includes escherichia coli prokaryotic expression, yeast expression and three kinds of different genes engineering expression-forms of CHO eukaryotic expression respectively.
A further object of the present invention is in that to provide the application of above-mentioned pQE30/en pPIC9K/en's albumen.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of pQE30/en pPIC9K/en's albumen, this pQE30/en pPIC9K/en's albumen is any one in pQE30/en pPIC9K/en's albumen 1, pQE30/en pPIC9K/en's albumen 2 and pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins, and the aminoacid sequence of described pQE30/en pPIC9K/en's albumen 1 is such as shown in SEQIDNO.1; The aminoacid sequence of described pQE30/en pPIC9K/en's albumen 2 is such as shown in SEQIDNO.2; The aminoacid sequence of described pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins is such as shown in SEQIDNO.5.
The encoding gene of above-mentioned pQE30/en pPIC9K/en's albumen, this encoding gene has one of following nucleotide sequence:
(1) nucleotide sequence shown in SEQ ID NO.3, SEQIDNO.4 and SEQIDNO.6, wherein SEQIDNO.3 is the nucleotide sequence of the encoding amino acid sequence such as pQE30/en pPIC9K/en's albumen 1 shown in SEQIDNO.1, SEQIDNO.4 is the nucleotide sequence of the encoding amino acid sequence such as pQE30/en pPIC9K/en's albumen 2 shown in SEQIDNO.2, and SEQIDNO.6 is the nucleotide sequence of the encoding amino acid sequence such as pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins shown in SEQIDNO.5;
(2) in polynucleotide as shown in SEQIDNO.1, SEQIDNO.2 and SEQIDNO.5 the nucleotide of aminoacid sequence.
Include the recombinant expression carrier of above-mentioned pQE30/en pPIC9K/en's protein coding gene, transgenic cell line and transgenic recombinant bacterium.
For expressing the recombinant expression carrier of above-mentioned pQE30/en pPIC9K/en's albumen, it is in that to adopt following methods to prepare:
The recombinant expression carrier pET9c-En1 of described pQE30/en pPIC9K/en's albumen 1 adopts following steps to prepare: arrange as template with the Endostatin nucleotides sequence shown in SEQIDNO.7, pcr amplification is carried out for primer, by pcr amplification product and pET9c carrier respectively with NdeI and BamHI enzyme action and be attached obtaining recombiant plasmid pET9c-En1 with the such as primer (1) shown in SEQIDNO.8 and the primer (2) as shown in SEQIDNO.9;
The recombinant expression carrier pET9c-En2 of described pQE30/en pPIC9K/en's albumen 2 adopts following steps to prepare: with above-mentioned recombiant plasmid pET9c-En1 for template, respectively with the such as primer (3) shown in SEQIDNO.10 and the primer (5) as shown in SEQIDNO.12, with the such as primer (4) shown in SEQIDNO.11 and the primer (5) as shown in SEQIDNO.12, carry out pcr amplification with the such as primer (6) shown in SEQIDNO.13 and the primer (7) as shown in SEQIDNO.14 for primer and obtain pcr amplification product 1, at the pcr amplification product 1 to obtain for template, carry out pcr amplification with the such as primer (3) shown in SEQIDNO.10 and the primer (2) as shown in SEQIDNO.9 and obtain pcr amplification product 2, by pcr amplification product 2 and pET9c carrier respectively with NdeI and BamHI enzyme action and be attached obtaining recombiant plasmid pET9c-En2,
The recombinant expression carrier pcDNA3.1-En1 of described pQE30/en pPIC9K/en's albumen 1 and recombinant expression carrier pcDNA3.1-En2 of described pQE30/en pPIC9K/en's albumen 2 adopts following steps to prepare: respectively with recombiant plasmid pET9c-En1 and recombiant plasmid pET9c-En2 for template, carry out pcr amplification with the such as primer (9) shown in SEQIDNO.16 and the primer (10) as shown in SEQIDNO.17 for primer and obtain pcr amplification product, by pcr amplification product and pcDNA3.1 carrier respectively with HindIII and XhoI enzyme action and be attached respectively obtaining recombiant plasmid pcDNA3.1-En1 and recombiant plasmid pcDNA3.1-En2,
The recombinant expression carrier of described pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins adopts following steps to prepare: the such as pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins encoding gene shown in SEQIDNO.6 is connected to the recombinant expression carrier obtaining pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins on shuttle plasmid pPICZ �� A by the restriction enzyme site of EcoRI and NotI.
The preparation method of each pQE30/en pPIC9K/en's albumen of the present invention comprises the steps:
The preparation method of described pQE30/en pPIC9K/en's albumen 1 and pQE30/en pPIC9K/en's albumen 2 is: by round pcr, the encoding gene of the encoding gene of the such as pQE30/en pPIC9K/en's albumen 1 shown in SEQIDNO.3 and the pQE30/en pPIC9K/en's albumen 2 as shown in SEQIDNO.4 is cloned into pET9c or pCDNA3.1 genophore respectively, respectively through escherichia coli expression or by hamster ovary cell (ChineseHamsterOvary, CHO) eukaryotic cell solubility expression is carried out, after purification or renaturation, purification obtains activated pQE30/en pPIC9K/en's albumen 1 and pQE30/en pPIC9K/en's albumen 2,
The preparation method of described pQE30/en pPIC9K/en 2 human serum albumin fusion proteins is: by the C-terminal of the such as pQE30/en pPIC9K/en's albumen 2 shown in SEQIDNO.2 by human serum albumin's aminoacid sequence in flexible joint GGGGSGGGGS connection, and its encoding gene is cloned into pPICZ �� A genophore, obtain pQE30/en pPIC9K/en 2 human serum albumin fusion proteins by Pichia sp. solubility expression.
Above-mentioned pQE30/en pPIC9K/en's albumen suppresses the application in the medicine of endothelial cell growth activity and suppression growth of tumour cell in preparation.
The preparation method of pQE30/en pPIC9K/en's albumen of the present invention comprises the steps:
(1) using Endostatin nucleotide sequence in the such as Zl97107112.8 patent of invention shown in SEQIDNO.7 as template, add 9 arginic nucleotide sequences (CGCCGCCGCCGCCGCCGCCGCCGCCGC) by PCR method at Endostatin N-terminal and obtain the nucleotide sequence of the pQE30/en pPIC9K/en's albumen 1 meeting SEQIDNO.3, then pass through the PCR method of 2 point mutation, it is thus achieved that meet the nucleotide sequence of pQE30/en pPIC9K/en's albumen 2 of SEQIDNO.4;
(2) connect with coli expression carrier pMAL-c2 and pET9c genophore or pCDNA3.1 expression vector respectively under DNA ligase effect, obtain different 4 kind prokaryotic expression plasmid or 2 kinds of eukaryon expression plasmids;
(3) express bacterium and carry out prokaryotic expression with above-mentioned prokaryotic expression plasmid conversion bacillus coli DH 5 alpha 4 kinds different, BL21 or carry out eukaryotic expression with in above-mentioned eukaryotic expression plasmids 2 kinds different to Chinese hamster ovary celI respectively 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 suppressing the vascular endothelial cell increment activity under basic fibroblast growth factor (bFGF) induction and the activity suppressing chick chorioallantoic membrane (CAM) angiogenesis, and the purifying protein of above-mentioned 2 kinds of eukaryotic expressions is made directly and suppresses growth of tumour cell and promote the activity research of apoptosis of tumor cells effect.
(6) further the C-terminal of pQE30/en pPIC9K/en's albumen 2 is connected upper human serum albumin's sequence by GGGGSGGGGS flexible joint, form pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins, the aminoacid sequence of this fusion protein meets SEQIDNO.5, and nucleotide sequence meets SEQIDNO.6.
The present invention arranges as template with Endostatin nucleotides sequence in the Zl97107112.8 patent of invention of the such as Xu Genxing shown in SEQIDNO.7, continuous 9 arginic nucleotide sequences (CGCCGCCGCCGCCGCCGCCGCCGCCGC) are added after the ATG of N-terminal, then PCR is carried out, obtain the two ends nucleotide sequence fragment met involved by SEQIDNO.3 with different restriction enzyme sites, pcr amplification product connects together with the pET9c carrier processed through the same manner with after NdeI and BamHI enzyme action respectively under T4DNA ligase effect, it is transformed into e.colistraindh5��, screening obtains positive colony, obtain recombiant plasmid pET9c-En1, or pcr amplification product is connected together with the pMAL-c2 carrier processed through the same manner with after EcoRI and BamHI enzyme action respectively under T4DNA ligase effect, it is transformed into e.colistraindh5��, screening obtains positive colony, obtains recombiant plasmid pMAL-c2-En1.
The nucleotides sequence in pET9c-En1 Endostatin is adopted to be classified as template, by nucleotide sequence point mutation technology, the aminoacid P point mutation of 135 of pQE30/en pPIC9K/en's albumen 1 as shown in SEQIDNO.3 in pET9c-En1 is become A (P135A), further by nucleotide sequence point mutation technology, RGIRGAD point mutation after the 33rd of pQE30/en pPIC9K/en's albumen 1 aminoacid is become RGDRGD, obtain N end after 2 point mutation and carry 9 arginic two ends nucleotide sequence fragment met involved by SEQIDNO.4 with different restriction enzyme sites, pcr amplification product connects together with the pET9c carrier processed through the same manner with after NdeI and BamHI enzyme action respectively under T4DNA ligase effect, it is transformed into e.colistraindh5��, screening obtains positive colony, obtain recombiant plasmid pET9c-En2, or pcr amplification product is connected together with the pMAL-c2 carrier processed through the same manner with after EcoRI and BamHI enzyme action respectively under T4DNA ligase effect, it is transformed into e.colistraindh5��, screening obtains positive colony, obtains recombiant plasmid pMAL-c2-En2.
Above-mentioned 4 kinds of recombiant plasmid extracted are transformed in competence e. coli bl21 (DE3) respectively, and carry out escherichia coli expression respectively, collect and express bacterium, purification obtains the destination protein pQE30/en pPIC9K/en 1 that expresses and 2 or recombined human MBP (maltose-binding protein)-Endostatin 1 and 2, purity more than 96%.
Compare with above-mentioned escherichia expression system, mammalian cell Chinese hamster ovary cell (Chinese hamster ovary celI) expresses the post translational modification to destination protein, at biological activity, stability, solubility, immunogenicity and biological circulating half-life in vivo aspect have superiority, therefore respectively pET9c-En1 and pET9c-En2 plasmid is carried out PCR as template, pcr amplification product connects together with the pcDNA3.1 carrier processed through the same manner with after HindIII and XhoI enzyme action respectively under T4DNA ligase effect, it is transformed into e.colistraindh5��, screening obtains positive colony, therefrom respectively obtain recombiant plasmid pcDNA3.1-En1 and pcDNA3.1-En2.Recombiant plasmid pcDNA3.1-En1 and pcDNA3.1-En2 after extracting is transfected in Chinese hamster ovary celI respectively, adopt G418 resistance screening, obtain positive expression cell strain, cell and supernatant is collected after amplification culture, merge the pQE30/en pPIC9K/en 1 and 2 that purification obtains the eukaryotic expression of two kinds of different aminoacids structures, purity more than 96% with supernatant after cell breakage.
The present invention obtains 6 kinds different Endostatin by said gene engineering method, soluble M BP-Endostatin 1 fusion protein obtained from pMAL-c2-En1 plasmid and pMAL-c2-En2 plasmid by escherichia coli expression purification respectively and MBP-Endostatin 2 fusion protein; Respectively from pET9c-En1 plasmid and pET9c-En2 plasmid by prokaryotic expression Endostatin 1 albumen obtained by Purification after escherichia coli expression and Endostatin 2 albumen, its amino acid structure correspondence respectively is equal to SEQIDNO.1 and SEQIDNO.2, and its nucleotide sequence correspondence respectively is equal to SEQIDNO.3 and SEQIDNO.4. Eukaryotic expression Endostatin 1 albumen obtained by purification after pcDNA3.1-En1 and pcDNA3.1-En2 plasmid expression and Endostatin 2 albumen, its amino acid structure correspondence respectively is equal to SEQIDNO.1 and SEQIDNO.2, and its nucleotide series correspondence is equal to SEQIDNO.3 and SEQIDNO.4.
PQE30/en pPIC9K/en's albumen 1 and 2 of two kinds of different aminoacids structures of the present invention respectively 193 aminoacid and 192 aminoacid, be all different from above-mentioned patent and have been approved by the amino acid structure involved by Endostatin of clinic as medicine. PQE30/en pPIC9K/en's albumen 1 in the present invention compares with pQE30/en pPIC9K/en's albumen 23 aminoacid different (Fig. 1); Recombinant Endostatin albumen 1 compares with the aminoacid of Endostatin in patent Zl97107112.8, and N-terminal has 10 aminoacid differences, and only 183 aminoacid are identical, and molecular weight of albumen is also different, respectively 22KD and 20KD (Fig. 2); Recombinant Endostatin albumen 2 compares with the aminoacid of Endostatin (rhEndostatin) medicine listed, and has 13 aminoacid differences (Fig. 3); Recombinant Endostatin albumen 1 compares with the Endostatin of the AF184060 (gi:6013264) in gene bank 13 amino acid whose differences, and wherein N end has 10 aminoacid variant and has 3 aminoacid also variant (Fig. 4) in 183 aminoacid below. (the StandkerL such as Standker, SchraderM, KansaSM, etal.1997.Isolationandcharacterizationofthecirculatingfo rmofhumanendostatin.FEBSLett, from human blood, 420:129-133) it is separated to N-terminal 12 amino acid whose 170 amino acid whose human endostatins of disappearance, and proving that this Endostatin Human Umbilical Vein Endothelial Cells does not have inhibitory action, this one being probably in Endostatin metabolic breakdown does not have activated by-product. Also demonstrating N-terminal is completely that the important active structure of Endostatin ensures, therefore being similar to Endostatin (rhEndostatin) N end, to increase the modified amino acid that the patent with the modified amino acid of 6His or Wang Fengshan etc. adds Tat at Endostatin N end be the innovation that a kind of Endostatin changes structure, patent of the present invention then adds 9 poly arginines at Endostatin N end, and in Endostatin aminoacid sequence, carried out 2 point mutation change structure, thus significantly improve stability and the activity of Endostatin.
Two kinds of recombiant proteins of the present invention are different from the aminoacid sequence of patent No. Zl97107112.8, but remain pQE30/en pPIC9K/en suppress the vascular endothelial cell increment activity under basic fibroblast growth factor (bFGF) induction and suppress the activity of chick chorioallantoic membrane (CAM) angiogenesis, and it is easier in intravasation endotheliocyte and chick chorioallantoic membrane vascular endothelial cell, albumen wears the structural stability of film effect and N end, and to be all considerably better than the Recombinant Endostatin of Zl97107112.8 good, and there is the effect directly suppressing growth of tumour cell, the effect better suppressing neovascular endothelium Hemapoiesis can be played, can be used in treatment new vessels and generate the various diseases caused, the retinopathy caused including entity tumor and diabetes and rheumatoid arthritis.
Beneficial effects of the present invention:
Soluble M BP-Endostatin 1 fusion protein and MBP-Endostatin 2 fusion protein of prokaryotic expression are adopted bovine adrenal capillary endothelium strain (bovineadrenalcapillaryendothelialcell, BCE) to detect albumen inhibition of endothelial cell proliferation activity by the present invention; The Endostatin 1 through renaturation process of prokaryotic expression and Endostatin 2 albumen are observed angiogenesis suppression action by chick chorioallantoic membrane (CAM) test; The soluble endothelial chalone 1 of CHO eukaryotic expression and Endostatin 2 albumen are observed the growth inhibition ratio on human breast cancer cell line Bcap-37 cell and on apoptosis of tumor cells impact either directly through inhibiting tumour cells test. Result illustrates that the Endostatin that above-mentioned 4 kinds of escherichia coli expression methods obtain different aminoacids structure all has the effect significantly suppressing vascular endothelial cell growth, and growth of tumour cell is directly had inhibitory action by the Endostatin of eukaryotic expression. The Endostatin recombiant protein of two kinds of different aminoacids structures provided by the invention is possibly as a kind of potential medicine having more advantage than the Endostatin of Zl97107112.8 patent of invention.
The present invention obtains pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins of yeast expression by said gene engineering method, and the expansive approach extending the half-life for Endostatin provides the gene engineering yeast expression of potential drug.
Accompanying drawing explanation
The aminoacid that Fig. 1 is pQE30/en pPIC9K/en's albumen 1 (on, 193 aminoacid) and pQE30/en pPIC9K/en's albumen 2 (under, 192 aminoacid) of different aminoacids structure compares
Fig. 2 is pQE30/en pPIC9K/en's albumen 1 (on, 193 aminoacid) and the aminoacid of Endostatin in patent Zl97107112.8 (under, 184 aminoacid) compares
Fig. 3 is that pQE30/en pPIC9K/en's albumen 2 compares with the aminoacid of Endostatin (rhEndostatin) medicine listed
Fig. 4 compares with the aminoacid of the Endostatin of the AF184060 (gi:6013264) in gene bank for restructuring endostatin protein 1 (on, 193 aminoacid)
Fig. 5 is the endostatin protein electrophoretogram of 4 kinds of Prokaryotic expression, purifications and 2 kinds of eukaryotic expression purification
Wherein, M is mark, 1 is the pQE30/en pPIC9K/en 1 of escherichia coli expression, 2 is the pQE30/en pPIC9K/en 2 of escherichia coli expression, 3 is the pQE30/en pPIC9K/en 1 of eukaryotic expression, 4 is the recombined human MBP-endostatin protein 2 that the recombined human MBP-endostatin protein that pQE30/en pPIC9K/en 2,5 is escherichia coli expression 1,6 is escherichia coli expression of eukaryotic expression
Fig. 6 is the growth that recombined human MBP-endostatin protein 1 and 2 suppresses BCE cell under variable concentrations, compares with pQE30/en pPIC9K/en's albumen of negative control MBP albumen and positive control patent Zl97107112.8 respectively.
Fig. 7 is chick chorioallantoic membrane (CAM) angiogenesis that pQE30/en pPIC9K/en 1 and 2 suppresses under basic fibroblast growth factor induction
Fig. 8 is the growth of the pQE30/en pPIC9K/en 1 and 2 suppression MCF-7 cell of eukaryotic expression purification, and the pQE30/en pPIC9K/en's albumen purified with the escherichia coli expression of positive control patent Zl97107112.8 compares.
Fig. 9 is pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins of yeast expression
Detailed description of the invention
The present invention illustrated further description by embodiment below. In the examples below that, adopt the Endostatin 1 of the different aminoacids structure that prokaryotic expression carrier obtains at e. coli expression purification and 2 or Endostatin fusion protein 1 and 2, it was demonstrated that the pQE30/en pPIC9K/en of the prokaryotic expression after these purification has the characteristic suppressing endothelial cell growth.Pass through carrier for expression of eukaryon, it is transfected into Chinese hamster ovary celI, proving that it can also express in eukaryotic cell, the pQE30/en pPIC9K/en 1 and 2 of the eukaryotic expression purification of the different aminoacids structure that expression and purification obtains has the activity directly suppressing growth of tumour cell and promotion apoptosis of tumor cells. PQE30/en pPIC9K/en 2 can form fusion protein with human serum albumin by flexible joint, and can at yeast expression. Thus providing the gene engineering expression method that escherichia coli expression, yeast expression, CHO eukaryotic expression these three are conventional.
These embodiments are only citing, do not limit the present invention in any form.
Genetic engineering recombiant protein of embodiment 1 recombiant plasmid pET9c-En1 and preparation method thereof
This patent shows the aminoacid sequence of coding prokaryotic expression Endostatin 1 and the comparison of the patent of invention of nucleotide sequence and approved or the Endostatin as medicine approved clinical research.
PCR primer sequence in genetic engineering recombiant protein of recombiant plasmid pET9c-En1 and preparation method thereof is as follows:
Primer (1): ATTCATATGCGCCGCCGCCGCCGCCGCCGCCGCCGCCACAGCCACCGCGACTTCCA G (SEQIDNO.8)
Primer (2): GCCGGATCCCTACTTGGAGGCAGTCATGAAGCT (SEQIDNO.9)
The primer of PCR reaction is synthesized by Shanghai Sangon company, to arrange as template with Endostatin nucleotides sequence in the such as emerging Zl97107112.8 patent of invention of Xu's root shown in SEQIDNO.7, add above-mentioned 2 kinds of primers and Standard PCR reaction reagent, PCR reaction condition is 94 DEG C of degeneration 4min, then 94 DEG C of degeneration 1min, 56 DEG C of renaturation 1min, 72 DEG C extend 1min, 30 circulations, last 72 DEG C extend 10min. PCR primer 1% agarose gel electrophoresis. Under uviol lamp, cut the Agarose plug containing about 579bp DNA fragmentation, reclaim test kit with gel DNA and reclaim target DNA, the pET9c carrier of purification and PCR fragment are all used NdeI and BamHI double digestion. Product after enzyme action passes through 1.5% agarose gel electrophoresis, reclaim required fragment, reclaim plasmid vector after enzyme action and PCR primer fragment coupled reaction is as follows: carrier (pET9c) 1 �� L, insert PCR fragment 7 �� L, distilled water 10 �� L, 10 �� connect buffer 2 �� L, T4DNA ligase 0.8 �� L. At 22 DEG C of static 1hr after the mixing of above-mentioned reaction system, place into and 65 DEG C of water-baths are hatched 10min inactivation ligase. Then will preparation competence E.coliDH5��100 �� L add coupled reaction product 10 �� L, mixes rearmounted 30min on ice, 42 DEG C of heat shock 90s. Often pipe adds the common LB culture medium 800 �� L of antibiotic-free, hatches 1hr for 37 DEG C. 3000rpm is centrifuged 2min, discards 800 �� L of supernatant, is mixed gently in remaining liquid by thalline. Spread bacterium device with aseptic elbow glass and 50 �� L bacterium solution are laid on LB agar (1.5% agar) flat board containing 50 �� g/mL kanamycin, be inverted overnight incubation for 37 DEG C. With the positive colony on sterile toothpick picking flat board, it is inoculated in the LB fluid medium containing 50 �� g/mL kanamycin and expands, 37 DEG C of shaken overnight. Taking the 1.5mL bacterium solution of shaken overnight, 10000g is centrifuged 2min, abandons supernatant. Recombiant plasmid is extracted according to plasmid extraction kit description. Recombiant plasmid enzyme action is identified, order-checking.
By order-checking meet SEQIDNO.3 pET9c-En1 plasmid be transformed in competence E.coliBL21 (DE3) escherichia coli, with IPTG Induction of bacterial express 6hr, collect induction before bacterial suspension as comparison.Then 12%SDS-PAGE electrophoresis is carried out. Take induction and the centrifugal 3min of antibacterial each 1.5mL, 5000rpm not induced, wash twice with PBS, abandon supernatant, add 100 �� L sample-loading buffers, Ultrasonic Pulverization antibacterial, take the above-mentioned sample loading processed, carry out PAGE gel electrophoresis, it was demonstrated that destination protein is expressed. Then the Endostatin expressed is identified with Westernblot. The endostatin protein of expression is carried out SDS-PAGE electrophoresis. Nitrocellulose filter is arrived in transduction. Nitrocellulose filter cuts destination protein band after dyeing with Ponceaux. Decolouring with PBS (0.1M, pH7.0), defatted milk powder (PBS joins) room temperature of 1% closes 30min. Add the anti-human Endostatin polyclonal antibody of rabbit of 1:1000 dilution, hatch 2hr for 37 DEG C. Defatted milk powder (PBS joins) with 1% is washed 3 times, washes 5min every time. Add goat anti-rabbit igg-HRP two to resist, hatch 1hr for 37 DEG C. Defatted milk powder (PBS joins) with 1% is washed 5 times, washes 10min every time. 0.05MTris-HCl (pH7.4) washes twice, washes 5min every time. 0.05%DAB and 0.03%H2O2Mixed liquor carries out colour developing to be identified.
Expanding scale fermentation according to above-mentioned condition, 37 DEG C are cultured to OD600=0.4-0.6, adds IPTG (0.5mmol/L), continues to be cultured to OD600> 1.2 receipts bacterium, the buffer solution antibacterial of 20mmol/LTris-HCl. and 2mmol/LEDTApH8.0 is added after broken bacterium, centrifugal 7000rpm �� 10min abandons supernatant, add the 6mol/L guanidine hydrochloride containing 20% and the Tris-HCl lysate of 1% mercaptoethanol, centrifugal 12000rpm �� 30min takes supernatant, obtain inclusion body primary extract, with 20mmol/LTris-HCl, pH8.0 dilutes, destination protein precipitates, by centrifugal foreigh protein removing, with this buffer solution inclusion body, add the Tris-HCl buffer containing 6mol/L guanidine hydrochloride and 1% mercaptoethanol, with 1:50 volume ratio, it is slowly dropped into renaturation solution (containing 0.4mol/LL-Arg, 1mmol/L reduced glutathion, the 20mmol/LNaAC-HAC of 0.1mmol/L oxidized form of glutathione, pH5.0) in, stirring 1h, 4 DEG C of renaturation, take supernatant and measure protein concentration to determine renaturation effect, dialyse in 20mmol/LNaAC-HAC, after pH5.0, it is respectively adopted anion exchange (DEAE-SepharoseFastFlow) and cation exchange resin (SP-SepharoseFF) separates, the pQE30/en pPIC9K/en's albumen 1 obtaining purity through electroresis appraisal more than 96% is separated again through SephadexG-25 gel.
Genetic engineering recombiant protein of embodiment 2 recombiant plasmid pET9c-En2 and preparation method thereof
This patent shows the aminoacid sequence and nucleotide sequence and embodiment 1 and the comparison with the patent of invention of approved or the Endostatin as medicine approved clinical research that encode prokaryotic expression pQE30/en pPIC9K/en's albumen 2.
PCR primer sequence in genetic engineering recombiant protein of recombiant plasmid pET9c-En2 and preparation method thereof is as follows:
Primer (3) CATATGCGCCGCCGCCGCCGCCGCCGCCGCCGCCACAGCCACCGCGACTTCC (SEQIDNO.10)
Primer (4) GCGGCATGCGGGGCGATCGCGGGGACTTCCAGTGCTTCC (SEQIDNO.11)
Primer (5) GGAAGCACTGGAAGTCCCCGCGATCGCCCCGCATGCCGC (SEQIDNO.12)
Primer (6) GTGGCATGGCTCGGACGCAAACGGGCGCAGGCTG (SEQIDNO.13)
Primer (7) CAGCCTGCGCCCGTTTGCGTCCGAGCCATGCCAC (SEQIDNO.14)
The primer of PCR reaction is synthesized by Shanghai Sangon company, arrange as template with the nucleotides sequence of the Endostatin of the pET9c-En1 in embodiment 1, respectively with primer (3) and primer (5), with primer (4) and primer (5), it is separately added into Standard PCR reaction reagent with primer (6) and primer (7) and carries out PCR reaction, reaction condition is 95 DEG C of degeneration 5min, then 95 DEG C of degeneration 20s, 65 DEG C of renaturation 20s, 72 DEG C extend 30s, 35 circulations, last 72 DEG C extend 7min.PCR primer 1% agarose gel electrophoresis. Doing template with primer (3) and primer (2) with above-mentioned PCR primer again and carry out total length PCR reaction, reaction condition is 95 DEG C of degeneration 5min, then 95 DEG C of degeneration 20s, 70 DEG C of renaturation 20s, 72 DEG C extend 40s, 35 circulations, and last 72 DEG C extend 10min. Obtaining PCR primer, after 1% agarose gel electrophoresis, purification obtains the PCR primer meeting SEQIDNO.4 sequence of 2 point mutation containing 9 poly-essence acid, and PCR primer is through 1% agarose gel electrophoresis. The Agarose plug containing about 576bp DNA fragmentation is cut under uviol lamp, reclaim test kit with gel DNA and reclaim target DNA, clone according to the method for embodiment 1 and obtain pET9c-En2 plasmid, obtaining pQE30/en pPIC9K/en 2 albumen containing 9 poly arginines and the escherichia coli expression meeting SEQIDNO.2 sequence of 2 point mutation by the method expression and purification of embodiment 1, purity is through electroresis appraisal more than 96%.
Genetic engineering recombiant protein of embodiment 3 recombiant plasmid pMAL-c2-En1 and pMAL-c2-En2 and preparation method thereof
Primer (8) CCGGAATTCCGCCGCCGCCGCCGCCGCCGCCGCCGCCAC (SEQIDNO.15)
Method according to embodiment 1, arrange as template with the Endostatin nucleotide sequence of pET9c-En1 and the Endostatin nucleotides sequence of pET9c-En2 respectively, add Standard PCR reaction reagent with primer (8) and primer (2) respectively and carry out PCR, respectively by the pMAL-c2 carrier of the PCR primer obtained and purification respectively with EcoRI and BamHI enzyme action, clone according to the method for embodiment 1 and obtain pMAL-c2-En1 and pMAL-c2-En2 plasmid. Empty carrier pMAL-c2 and recombiant plasmid pMAL-c2-En1 and pMAL-c2-En2 is transformed in E.coliBL21 (DE3) respectively, is inoculated in the LB culture fluid containing 100 �� g/mL penicillins, treats that it grows to OD600During for 0.4-0.6, adding the centrifugal thalline of collecting of IPTG (final concentration of 0.4mM) abduction delivering 4hr, 5000rpm, the bacterium solution before collection induction, as comparison, then carries out SDS-PAGE electrophoresis observation expression. the thalline of abduction delivering is dissolved in lysis buffer (0.1MPBS, 0.5MNaCl, 0.25%Tween-20, 10mM2-mercaptoethanol, 10mMEDTA, 10mMEGTA), ultrasonication on ice bath, 15000g is centrifuged 20min, supernatant is combined overnight with MBP affinity column 4 DEG C by supernatant in 100:1 ratio, wash with 50 times of column volume lysis buffers and 1 times of column volume 0.1MPBS (pH7.2), again with 10mM maltose (0.1MPBS, pH7.2 prepares) distinguish the MBP albumen of empty carrier under eluting or the MBP-endostatin protein 1 of recombiant plasmid pMAL-c2-En1 and pMAL-c2-En2 expression and MBP-endostatin protein 2, ultraviolet spectrophotometer measures the protein content of eluent, the restructuring MBP albumen obtaining purity through electroresis appraisal more than 96% is separated again through SephadexG-25 gel, recombined human MBP-endostatin protein 1 and recombined human MBP-endostatin protein 2.
Genetic engineering recombiant protein of embodiment 4 recombiant plasmid pcDNA3.1-En1 and pcDNA3.1-En2 and preparation method thereof
Primer (9) CCCAAGCTTCGCCGCCGCCGCCGCCGCCGCCGCCGCCAC (SEQIDNO.16)
Primer (10) CCGCTCGAGCTTGGAGGCAGTCATGAAGCT (SEQIDNO.17)
Primer (9) and (10) is adopted to add Standard PCR reaction reagent, arrange with the Endostatin nucleotides sequence in pET9c-En1 and pET9c-En2 plasmid respectively and carry out PCR for template, the PCR primer obtained and the pcDNA3.1 carrier of purification with HindIII and XhoI enzyme action, are cloned according to the method for embodiment 1 and are obtained pcDNA3.1-En1 and pcDNA3.1-En2 plasmid respectively.Plasmid is extracted, with 2 �� 10 after identifying by checking order5/ mLCHO cell is inoculated on 6 orifice plates, calf serum containing 10% and the PRMI-1640 culture medium of penicillin, streptomycin, at 37 DEG C of 5%CO2Cell culture incubator is cultured to the cell coverage rate of 50%; according to product description, Lipofectamine200015 �� l being mixed with 2 �� gpcDNA3.1-En1 and pcDNA3.1-En2 plasmid culture fluid respectively that add serum-free antibiotic-free adds up to 100 �� l to cultivate after 45min; after adding the culture fluid of 800 �� l serum-free antibiotic-frees, 37 DEG C of 5%CO2Cultivate 5h, add 1ml containing the hyclone antibiotic-free culture fluid continuation cultivation 48h of 20%, carry out pressurization screening stable clone with 0.9g/LG418, it is thus achieved that after positive colony cell, after identifying, turn culture. Adopt the extensive low serum of CHO expression cell line or the serum-free culture 4d that have transfected pcDNA3.1-En1 and pcDNA3.1-En2 plasmid, collect upper cleer and peaceful cell respectively, by cell ultrasonication, measure the content of Endostatin 1 and Endostatin 2 with ELISA, result all detects the Endostatin that content does not wait in upper cleer and peaceful cell breakage supernatant. Then supernatant is merged respectively, nickel post affinity purification is adopted according to Ni-NTAbeads post description, respectively with containing 20,50,100,250, the buffer solution elution of 500mmol/Limidazole, purifying protein separates again through SephadexG-25 gel and obtains purity through eukaryotic expression pQE30/en pPIC9K/en's albumen 1 of electroresis appraisal more than 96% and pQE30/en pPIC9K/en's albumen 2.
Embodiment 5 recombined human MBP-endostatin protein 1 and recombined human MBP-endostatin protein 2 suppress endothelial cell growth
By bovine adrenal capillary endothelium strain (bovineadrenalcapillaryendothelialcell, BCE) cell is inoculated in 37 DEG C of cultivations in the sterilizing culture bottle being covered with one layer of 1.5% gelatin (preparation of Hanks liquid), choose well-grown cell and be inoculated in 24 well culture plates with the concentration in 12500/hole, culture fluid is DMEM (containing 10% calf serum), 37 DEG C, 10%CO2Cultivate 24hr, suck culture fluid, change to the culture fluid of recombined human MBP-the endostatin protein 1 or recombined human MBP-endostatin protein containing variable concentrations in 0.25mL/ hole, final concentration of protein is 0ng/mL, 250ng/mL, 500ng/mL, 750ng/mL, 1000ng/mL, 2000ng/mL respectively, 4000ng/mL, after cultivating 20min, every hole adds the 0.25mL DMEM complete medium containing bFGF, the final concentration of 1ng/ml of bFGF, 37 DEG C, 10%CO2Hatch 72hr, suck supernatant, every hole 0.5mL0.05% pancreatin (containing 10mMEDTA) peptic cell, be suspended in Hematall device (FisherScientific product), by description of product method, use rolling counters forward. Result shows, compare with negative control MBP protein control group, the growth of BCE cell is all had obvious inhibition by recombined human MBP-endostatin protein 1 or recombined human MBP-endostatin protein 2 under variable concentrations, the inhibition of high dose becomes apparent from, the inhibition of recombined human MBP-endostatin protein 1 is similar to the pQE30/en pPIC9K/en's albumen provided by JiangSu WuZhong Medicine Group Co., Ltd of the patent Zl97107112.8 of positive control, and the inhibition of recombined human MBP-endostatin protein 2 is better than recombined human MBP-endostatin protein 1 (Fig. 6)
PQE30/en pPIC9K/en's albumen 1 of embodiment 6 escherichia coli expression and pQE30/en pPIC9K/en's albumen 2 suppress chick chorioallantoic membrane (CAM) vascular endothelial cell growth
Take the qualified egg of inspection to hatch in incubator 8-9 days, the labelling fetus position under candler, in rich blood vessel place position external iodine disinfection, prepare into Embryo Gallus domesticus fine hair allantois artifical-air cell shell membrane and allantois are intermembranous, place methylcellulose saucer, being separately added into pQE30/en pPIC9K/en 1 and the pQE30/en pPIC9K/en 2 protein 12 �� L of interior escherichia coli expression, concentration is 1.5mg/mL, and with the normal saline of equal volume and bFGF respectively negative control and positive control.Only, positive control bFGF1ng/ is only for the dosage of pQE30/en pPIC9K/en 1 and pQE30/en pPIC9K/en 2 albumen respectively 25 �� g/. 37 DEG C, after incubation 3d, solution takes CAM film, takes a picture and makes specimen, and counting vascular strip number. It is shown that the first order vessel number of saline control group is 34.5 �� 2.8, two grades of blood vessel numbers are 84.6 �� 3.6; The first order vessel number of bFGF positive controls is 64.2 �� 3.8, and two grades of blood vessel numbers are 101.5 �� 3.8; The first order vessel number of pQE30/en pPIC9K/en 1 is 23.5 �� 6.1, and two grades of blood vessel numbers are 59.3 �� 12.2; The first order vessel number of pQE30/en pPIC9K/en 2 is 18.3 �� 2.8, two grades of blood vessel numbers are 39.9 �� 10.1, illustrate to compare with saline control group and bFGF positive controls, pQE30/en pPIC9K/en 1 and 2 can suppress chick chorioallantoic membrane (CAM) angiogenesis under basic fibroblast growth factor induction and the inhibition better (Fig. 7) of pQE30/en pPIC9K/en 2.
The pQE30/en pPIC9K/en 1 of embodiment 7 eukaryotic expression and pQE30/en pPIC9K/en 2 albumen suppress growth of tumour cell and promote apoptosis of tumor cells effect
MCF-7 Human Breast Cancer Cells is cultivated in culture bottle with RPMI-1640 culture fluid, is placed in 37 DEG C of incubators, passes into 5%CO2(relative humidity is 95%). With trypsinization, by cell by 1 �� 103/ hole is inoculated in 96 well culture plates, after cultivating 6h, sucking culture fluid, every hole is separately added into pQE30/en pPIC9K/en 1 and the pQE30/en pPIC9K/en 2 protein 10 0 �� L of the eukaryotic expression with RPMI-1640 doubling dilution, each concentration sets 3 parallel holes, and negative control hole adds culture fluid 100 �� L. after continuing cultivation 48h, discard original fluid, again every hole is separately added into pQE30/en pPIC9K/en 1 and the pQE30/en pPIC9K/en 2 protein 10 0 �� L of the eukaryotic expression with RPMI-1640 doubling dilution, after continuing cultivation 48h, sucks culture fluid, every hole adds the PBS solution 20 �� L of 0.5g/LMTT, it is further cultured for 4h, sucks MTT solution in hole, every hole adds dimethyl sulfoxide 150 �� L, 100rpm vibration makes it fully dissolve, and measures absorbance by microplate reader at 490nm. it is shown that MCF-7 cell is had obvious Developing restraint effect by the pQE30/en pPIC9K/en 1 of eukaryotic expression and pQE30/en pPIC9K/en 2 albumen. result shows, pQE30/en pPIC9K/en 1 and pQE30/en pPIC9K/en 2 albumen can directly suppress the growth of MCF-7 Human Breast Cancer Cells under finite concentration, the growth inhibition ratio of MCF-7 Human Breast Cancer Cells is respectively reached the suppression ratio of 35.6% and 51.3% by the pQE30/en pPIC9K/en 1 of eukaryotic expression and pQE30/en pPIC9K/en 2 albumen under 200 �� g/mL concentration levels, positive control adopts the pQE30/en pPIC9K/en's albumen (Fig. 8) provided by JiangSu WuZhong Medicine Group Co., Ltd of patent Zl97107112.8, experimental result shows, the effect of pQE30/en pPIC9K/en's albumen 1 is close with positive control, the effect of pQE30/en pPIC9K/en's albumen 2 is significantly higher than positive control.
MCF-7 cell is as above cultivated, with trypsinization, by cell by 5 �� 103/ hole is inoculated in 24 well culture plates, after cultivating 48h, sucking culture fluid, every hole adds pQE30/en pPIC9K/en 1 and each 1mL of pQE30/en pPIC9K/en 2 albumen of the eukaryotic expression with RPMI-1640 doubling dilution, each concentration sets 3 parallel holes, and negative control hole adds culture fluid 1mL. After continuing cultivation 3h, sucking culture fluid, with trypsinization centrifugal collecting cell, with iodate the third ingot (PI), cell is carried out labelling by AnnexinV, by the apoptotic situation of flow cytomery.It is shown that pQE30/en pPIC9K/en 1 and pQE30/en pPIC9K/en 2 albumen can directly suppress MCF-7 Human Breast Cancer Cells under finite concentration, and promote the early apoptosis rate of tumor cell respectively 6.05% and 7.88%. Illustrate that the Endostatin of above-mentioned different structure directly suppresses growth and the inducing apoptosis of tumour cell of MCF-7 Human Breast Cancer Cells.
The gene engineering preparation method of pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins of embodiment 8 yeast expression
According to method for synthesizing gene, the Endostatin 2 meeting SEQIDNO.2 is added flexible joint GGGGSGGGGS, connect the 25-609 aminoacid sequence of human serum albumin's aminoacid sequence in GenBankCAA23754.1 again, add up to 787 amino acid whose pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins aminoacid sequences meeting SEQIDNO.5, convert this aminoacid sequence to nucleotide sequence, and like codon according to yeast expression and be optimized the pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins nucleotide sequence meeting SEQIDNO.6 forming 2361 nucleotide, the nucleotide sequence of synthesis is connected on shuttle plasmid pPICZ �� A by the restriction enzyme site of EcoRI and NotI, identified by enzyme action and after confirmation of checking order, the expression plasmid extracted carries out linearization process, with Sal I enzyme action expression plasmid, the linearizing expression plasmid of kits is reclaimed with the quick glue of DNA fragmentation, transduction is in Pichia pastoris GS115 competent cell, take 1ml salmon sperm dna boiling water bath and boil 5min, rapid ice bath is to prepare strand monomer DNA, competence yeast is centrifuged, suck the LiCl solution of remnants, add in the following order: 50%PEG3350240ul, 1mol/LLiCl36ul, 2mg/ml strand salmon sperm dna 25ul, 5��10ug linearization plasmid DNA50ul, mixing, 30min is hatched in 30 DEG C of water-baths, 42 DEG C of water-bath heat shock 20��25min, 6000��8000rpm is centrifugal collects yeast thalline, resuspended yeast is in 1mlYPD culture medium, 30 DEG C of shaking tables are hatched, after 2h, take 200ul bacterium solution and be laid on 100 �� g/ml bleomycin culture medium flat plates, cultivate 2��3 days in 30 DEG C, from flat board, the single bacterium colony of picking carries out PCR qualification. by the positive transformant of screening in the triangular flask of the 50mL containing 5mLBMGY fluid medium, 30 DEG C, 250rpm shaken cultivation, take the culture fluid 1ml of each bacterium in the 100ml triangular flask containing 15mlBMMY, 30 DEG C, 250rpm shaken cultivation 24h. in each culture bottle, every 24h adds 100% methanol of 150 �� L, 30 DEG C, and 250rpm shaken cultivation collects the expression supernatant of 2d and 3d in each triangular flask, and-20 DEG C of preservations are stand-by. expressing protein is identified by SDS-PAGE electrophoresis and WesternBlot, measures expressing protein concentration, drawing standard curve with immunoturbidimetry. take pQE30/en pPIC9K/en-human serum albumin that yeast expression produces and the control medium sensitizing latex of 8 �� L 130nm particle diameters detects when 510nm, draw the concentration of pQE30/en pPIC9K/en 2-human serum albumin that yeast expression produces according to standard curve. then according to said method amplification culture is expressed, expression supernatant is soluble protein, it is respectively adopted anion exchange (DEAE-SepharoseFastFlow) and cation exchange resin (SP-SepharoseFF) separates, separate the pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins obtaining purity through electroresis appraisal more than 96% again through SephadexG-25 gel.This expansive approach being Endostatin provides the gene engineering yeast of potential drug and expresses the method with other eukaryotic expression Endostatin soluble fusion protein.
Claims (6)
1. pQE30/en pPIC9K/en's albumen, it is characterized in that: this pQE30/en pPIC9K/en's albumen is any one in pQE30/en pPIC9K/en's albumen 1, pQE30/en pPIC9K/en's albumen 2 and pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins, the aminoacid sequence of described pQE30/en pPIC9K/en's albumen 1 is such as shown in SEQIDNO.1, the aminoacid sequence of described pQE30/en pPIC9K/en's albumen 2 is such as shown in SEQIDNO.2, and the aminoacid sequence of described pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins is such as shown in SEQIDNO.5.
2. the encoding gene of pQE30/en pPIC9K/en's albumen described in claim 1, it is characterised in that: this encoding gene has one of following nucleotide sequence:
(1) nucleotide sequence shown in SEQ ID NO.3, SEQIDNO.4 and SEQIDNO.6, wherein SEQIDNO.3 is the nucleotide sequence of the encoding amino acid sequence such as pQE30/en pPIC9K/en's albumen 1 shown in SEQIDNO.1, SEQIDNO.4 is the nucleotide sequence of the encoding amino acid sequence such as pQE30/en pPIC9K/en's albumen 2 shown in SEQIDNO.2, and SEQIDNO.6 is the nucleotide sequence of the encoding amino acid sequence such as pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins shown in SEQIDNO.5;
(2) in polynucleotide as shown in SEQIDNO.1, SEQIDNO.2 and SEQIDNO.5 the nucleotide of aminoacid sequence.
3. include the recombinant expression carrier of pQE30/en pPIC9K/en's protein coding gene described in claim 2, transgenic cell line and transgenic recombinant bacterium.
4. for expressing the recombinant expression carrier of pQE30/en pPIC9K/en's albumen as claimed in claim 1, it is characterised in that:
The recombinant expression carrier pET9c-En1 of described pQE30/en pPIC9K/en's albumen 1 adopts following steps to prepare: arrange as template with the Endostatin nucleotides sequence shown in SEQIDNO.7, pcr amplification is carried out for primer, by pcr amplification product and pET9c carrier respectively with NdeI and BamHI enzyme action and be attached obtaining recombiant plasmid pET9c-En1 with the such as primer (1) shown in SEQIDNO.8 and the primer (2) as shown in SEQIDNO.9;
The recombinant expression carrier pET9c-En2 of described pQE30/en pPIC9K/en's albumen 2 adopts following steps to prepare: with above-mentioned recombiant plasmid pET9c-En1 for template, respectively with the such as primer (3) shown in SEQIDNO.10 and the primer (5) as shown in SEQIDNO.12, with the such as primer (4) shown in SEQIDNO.11 and the primer (5) as shown in SEQIDNO.12, carry out pcr amplification with the such as primer (6) shown in SEQIDNO.13 and the primer (7) as shown in SEQIDNO.14 for primer and obtain pcr amplification product 1, at the pcr amplification product 1 to obtain for template, carry out pcr amplification with the such as primer (3) shown in SEQIDNO.10 and the primer (2) as shown in SEQIDNO.9 and obtain pcr amplification product 2, by pcr amplification product 2 and pET9c carrier respectively with NdeI and BamHI enzyme action and be attached obtaining recombiant plasmid pET9c-En2,
The recombinant expression carrier pcDNA3.1-En1 of described pQE30/en pPIC9K/en's albumen 1 and recombinant expression carrier pcDNA3.1-En2 of described pQE30/en pPIC9K/en's albumen 2 adopts following steps to prepare: respectively with recombiant plasmid pET9c-En1 and recombiant plasmid pET9c-En2 for template, carry out pcr amplification with the such as primer (9) shown in SEQIDNO.16 and the primer (10) as shown in SEQIDNO.17 for primer and obtain pcr amplification product, by pcr amplification product and pcDNA3.1 carrier respectively with HindIII and XhoI enzyme action and be attached respectively obtaining recombiant plasmid pcDNA3.1-En1 and recombiant plasmid pcDNA3.1-En2,
The recombinant expression carrier of described pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins adopts following steps to prepare: the such as pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins encoding gene shown in SEQIDNO.6 is connected to the recombinant expression carrier obtaining pQE30/en pPIC9K/en's 2-human serum albumin fusion proteins on shuttle plasmid pPICZ �� A by the restriction enzyme site of EcoRI and NotI.
5. the preparation method of pQE30/en pPIC9K/en's albumen as claimed in claim 1, it is characterised in that:
The preparation method of described pQE30/en pPIC9K/en's albumen 1 and pQE30/en pPIC9K/en's albumen 2 is: by round pcr, the encoding gene of the encoding gene of the such as pQE30/en pPIC9K/en's albumen 1 shown in SEQIDNO.3 and the pQE30/en pPIC9K/en's albumen 2 as shown in SEQIDNO.4 is cloned into pET9c or pCDNA3.1 genophore respectively, carry out eukaryotic cell solubility expression respectively through escherichia coli expression or by hamster ovary cell CHO, purification or after renaturation purification obtain activated pQE30/en pPIC9K/en's albumen 1 and pQE30/en pPIC9K/en's albumen 2;
The preparation method of described pQE30/en pPIC9K/en 2 human serum albumin fusion proteins is: by the C-terminal of the such as pQE30/en pPIC9K/en's albumen 2 shown in SEQIDNO.2 by human serum albumin's aminoacid sequence in flexible joint GGGGSGGGGS connection, and its encoding gene is cloned into pPICZ �� A genophore, obtain pQE30/en pPIC9K/en 2 human serum albumin fusion proteins by Pichia sp. solubility expression.
6. the pQE30/en pPIC9K/en's albumen described in claim 1 suppresses the application in the medicine of endothelial cell growth activity and suppression growth of tumour cell in preparation.
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| CN113912739A (en) * | 2021-09-30 | 2022-01-11 | 哈尔滨医科大学 | Endostatin 33 peptide with anti-tumor activity and application thereof |
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| CN111995686A (en) * | 2019-05-27 | 2020-11-27 | 兰州大学 | Medicine with anti-angiogenesis activity and preparation method thereof |
| WO2020238924A1 (en) * | 2019-05-27 | 2020-12-03 | 兰州大学 | Drug with anti-angiogenesis activity and preparation method therefor |
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| CN113912739A (en) * | 2021-09-30 | 2022-01-11 | 哈尔滨医科大学 | Endostatin 33 peptide with anti-tumor activity and application thereof |
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