CN102321588A - Chinese hamster ovary genetic engineering cell line for performing high-level secretory expression on AcAPc2 - Google Patents
Chinese hamster ovary genetic engineering cell line for performing high-level secretory expression on AcAPc2 Download PDFInfo
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Abstract
The invention discloses a Chinese hamster ovary (CHO) genetic engineering cell line for performing high-level secretory expression on AcAPc2 and relates to a CHO cell line for performing high-level secretory expression on the AcAPc2. The invention aims to solve the problems that the expression level of an exogenous gene product expressed by using CHO is low at present and the industrialized production cannot be realized. The CHO genetic engineering cell line for performing high-level secretory expression on the AcAPc2 is obtained by the following step of: screening dihydrofolate reductase-deficient CHO cells which are taken as host cells, and amplifying to obtain the cell line for performing stable and high-level expression on the AcAPc2. The CHO cell line can perform high-level expression on the AcAPc2; the expression level can be up to 10mg/L.72h; and the CHO cell line can be industrially produced. The cell line is used for resisting blood coagulation and treating tumor, septicaemia and the like.
Description
Technical field
The present invention relates to the Chinese hamster ovary celI strain of a kind of efficient secretory expression AcAPc2.
Background technology
When the dog hookworm is sucked blood, a kind of material of its cephalic gland secretion with anticoagulant active, be known as the Ancylostoma caninum anticoagulant hemepeptide (anticoagulant peptide, AcAPs).This material essence is a kind of proteolytic ferment, has prolongation plasma prothrombin time (pt), suppresses blood coagulation and promotes Fibrinolytic effect.The existing at present AcAP5 of AcAPs, AcAP6, three kinds of recombinant proteins of AcAPc2.AcAPc2 (10KD) is the efficient special suppressor factor of Xa factor.It is reported that the antithrombotic effect of Xa suppressor factor is superior to thrombin inhibitors, AcAPs is very little to the platelet aggregation influence as the efficient special suppressor factor of Xa factor, causes hemorrhage dangerous little when being used for antithrombotic therapy.1998, report AcAPs such as Donnelly also had the effect that antitumor cell shifts in vivo.The anticoagulation of AcAPs, anti thrombotic action might become a kind of clinically new antithrombotics, antithrombotic and antineoplaston medicine.
The high expression level amount of at present expressing AcAPc2 with CHO is 6mg/L, and can't satisfy suitability for industrialized production.
Summary of the invention
The present invention is that will to solve present expression amount with CHO expression alien gene product low, and can't satisfy the problem of suitability for industrialized production, and the Chinese hamster ovary genetically engineered cell strain of efficient secretory expression AcAPc2 is provided.
The present invention efficiently secretes, expresses the Chinese hamster ovary genetically engineered cell strain of AcAPc2, utilizes Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell (CHO-dhfr
-) as host cell, after screening, amplification, stablized, efficiently expressed the cell strain of AcAPc2, expression amount is up to 10mg/L72h.
The construction process of the Chinese hamster ovary genetically engineered cell strain of efficient secretory expression AcAPc2 of the present invention, carry out according to the following steps:
One, purpose fragment double digestion: give birth to the synthetic AcAPc2 gene order that has signal peptide of worker company by Shanghai and be cloned on the T carrier, the T carrier is carried out double digestion with BamHI and NotI, reaction system is following:
The endonuclease reaction condition is 37 ℃ placed 3 hours, then enzyme was cut product and used 1.5% agarose gel electrophoresis, and the purpose fragment obtains inserting fragment with DNA GEL EXTRACTION KIT purifying and recovering;
Two, plasmid vector double digestion: with BamHI and NotI double digestion digested plasmid carrier pcDNA3.1/V5-His-C, reaction system is following:
The endonuclease reaction condition is 37 ℃ placed 3 hours, then enzyme was cut product and used 1.5% agarose gel electrophoresis, used the DNAGELEXTRACTIONKIT purifying and recovering again, the plasmid vector after obtaining enzyme and cutting;
Three, the connection of dna fragmentation, reaction system is following:
The ligation condition is that 16 ℃ of placements are spent the night, and obtains to connect product recombinant plasmid pcDNA3.1/V5-His-C-AcAPc2;
Four, cell cultures: with the DMEM culture medium culturing Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell CHO-dhfr that contains 100mL/L foetal calf serum, NAA, 1 * HT
-
Five, recombinant plasmid transfection CHO: the recombinant plasmid pcDNA3.1/V5-His-C-AcAPc2 that builds is mixed with plasmid pDCH1P11, use Lipofeetmine
TM2000Reagent cotransfection CHO-dhfr
-Cell carries out transfection in 24 orifice plates, when cell reaches 95% fusion; Substratum is replaced by the CHO-S-SFM II of antibiotic-free, serum-free, and the CHO-S-SFM II that contains 100mL/L FBS is changed in 500 μ L/ holes behind the transfection 6h; Continue to cultivate 48h, identify that with the ELISA test kit positive cells transfected went down to posterity by 1: 10, was replaced by selective medium behind the 24h cell attachment; There is positive colony to form after cultivating 14～16d; Method with directed digestion changes positive colony over to 24 orifice plates then, is passaged to 12 orifice plates again, through the ELISA kit measurement to positive colony in the 10 plant heights cell clone of expressing carry out enlarged culturing;
Six, the pressurization of MTX is cultivated: with MTX pressurization screening, concentration is followed successively by 2 * 10
-8Mmol/L, 1 * 10
-7Mmol/L and 5 * 10
-7Mmol/L, finally obtaining can be 5 * 10
-7The Chinese hamster ovary celI strain of normal growth among the MTX of mmol/L is the Chinese hamster ovary genetically engineered cell strain of efficient secretory expression AcAPc2.
The said original series that has the AcAPc2 gene order of signal peptide according to the mRNA sequence (Aceession:U30793) of dog hookworm among the Genbank (Ancylostoma caninum) of step 1; Remove the original signal peptide sequence, select CHO-dhft according to the characteristics of expression system simultaneously
-Preference codon.To transcribe with translation efficiency the highlyest in order making, to make expression product be secreted into the extracellular simultaneously, add Kozak sequence and signal peptide, add the site of restriction enzyme BamHI and NotI at the original series two ends respectively at original series 5 ' end.
The sequence of AcAPc2 gene that step 1 is said to have signal peptide is shown in SEQ ID NO:1.
The said gene order that has the signal peptide that adds in the AcAPc2 gene of signal peptide of step 1 is shown in SEQ ID NO:2.
The said aminoacid sequence that has the signal peptide that adds in the AcAPc2 gene of signal peptide of step 1 is shown in SEQ ID NO:6.
The aminoacid sequence of the expression product of the Chinese hamster ovary genetically engineered cell strain of efficient secretory expression AcAPc2 of the present invention is shown in SEQ ID NO:3.
The present invention selects CHO-dhfr for use
-Cell has the following advantages as host cell: CHO-dhfr
-Cell is applicable to the secreting, expressing and the interior expression of kytoplasm of range protein; Under the MTX selective pressure, the sequence of DHFR gene and these gene both sides can increase in a large number; Flexibility to substratum is strong, can use the culture medium culturing of serum-free; Chinese hamster ovary celI both can carry out adherent culture also can carry out suspension culture; Scale property production in large quantities, the cultivation amount can reach 5000L.
Chinese hamster ovary celI strain of the present invention can efficiently express AcAPc2, and expression amount is 8～10mg/L72h, and can suitability for industrialized production.AcAPc2 is through the fVIIa/TF mixture performance anticoagulation of anticoagulant approach, and rAcAPc2 has the long transformation period (greater than 50h) in human body.The transformation period length of rAcAPc2 is mainly relevant with the binding affinity of itself and fXa or fX; It can combine with high-affinity with people fX or fXa; Form stabilized complex (Kd=640pM), thereby particularly rAcAPc2 can combine to make the transformation period of rAcAPc2 in human body, to reach more than the 50h with the proenzyme fX of fXa.The thrombosis of rAcAPc2 behind prevention coronary artery reconstruction operations also is safely and effectively.RAcAPc2 also can be used for treating diseases such as tumour, septicemia, Ebola virus hemorrhagic fever.
Description of drawings
Fig. 1 is the sequencer map of recombinant plasmid dna in the embodiment one; Fig. 2 is the RT-PCR product electrophorogram of positive colony in the embodiment one.
Embodiment
Technical scheme of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the Chinese hamster ovary genetically engineered cell strain of this embodiment efficient secretory expression AcAPc2, utilize Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell (CHO-dhfr
-) as host cell, after screening, amplification, stablized, efficiently expressed the cell strain of AcAPc2, expression amount is up to 10mg/L72h.
The construction process of the Chinese hamster ovary genetically engineered cell strain of this embodiment efficient secretory expression AcAPc2 is specific as follows:
One, purpose fragment double digestion: give birth to the synthetic AcAPc2 gene order that has signal peptide of worker company by Shanghai and be cloned on the T carrier, the T carrier is carried out double digestion with BamHI and NotI, reaction system is following:
The endonuclease reaction condition: placed 3 hours for 37 ℃, enzyme is cut product use 1.5% agarose gel electrophoresis, the purpose fragment obtains inserting fragment with DNA GEL EXTRACTION KIT purifying and recovering.
Two, plasmid vector double digestion: with BamHI and NotI double digestion digested plasmid carrier pcDNA3.1/V5-His-C (available from Invitrogen company), reaction system is following:
Endonuclease reaction condition: placed 3 hours for 37 ℃, enzyme is cut product use 1.5% agarose gel electrophoresis, use DNA GEL EXTRACTION KIT purifying and recovering again, the plasmid vector after obtaining enzyme and cutting.
Three, the connection of dna fragmentation: reaction system is following:
The ligation condition: 16 ℃ of placements are spent the night; Ligation product transformed competence colibacillus cell E.coli Top10 (available from the favourable Science and Technology Ltd. in sky, Tianjin); Adopt the method for bacterium colony PCR to screen positive bacterium colony, and further extract recombinant plasmid dna and check order, sequencing result shows the mistake of no base; Do not change carrier and read frame, direction identical (like Fig. 1).Obtain recombinant plasmid pcDNA3.1/V5-His-C-AcAPc2.BamHI, NotI and dna ligase are available from NEB company.
Four, cell cultures:
Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell CHO-dhfr
-Grant by Harbin Medical University's Pathological Staff Room, cultivate with the DMEM substratum (available from GIBCO company) that contains 100mL/L foetal calf serum (FBS), NAA, 1 * HT.
Five, recombinant plasmid transfection CHO:
The recombinant plasmid pcDNA3.1/V5-His-C-AcAPc2 that builds is mixed with plasmid pDCH1P11, use Lipofeetmine
TM2000Reagent (available from Invitrogen company) cotransfection CHO-dhfr
-Cell carries out transfection in 24 orifice plates, when cell reaches 95% fusion; Substratum is replaced by the CHO-S-SFM II of antibiotic-free, serum-free; CHO-S-SFM II (not containing HT, the NAA) 1mL that contains 100mL/L FBS is changed in 500 μ L/ holes behind the transfection 6h, continue to cultivate 48h; Identify that with ELISA test kit (available from R&D company) positive cells transfected went down to posterity by 1: 10; Be replaced by selective medium (do not contain HT, NAA, but contain the G418 (available from the great Bioisystech Co., Ltd in Shanghai) of 0.75mg/L, the FBS of 100mL/L) behind the 24h cell attachment; There is positive colony to form (because of not containing HT in the substratum, so survivaling cell is the cell of successful cotransfection) after cultivating 14～16d; Method with directed digestion changes positive colony over to 24 orifice plates then, is passaged to 12 orifice plates again, through the ELISA kit measurement to positive colony in the 10 plant heights cell clone of expressing carry out enlarged culturing.
Six, the pressurization of MTX is cultivated: treat that cell grows up to individual layer, get the supernatant of cultivating 3d and detect with the ELISA test kit that with MTX (methotrexate) pressurization screening, concentration is followed successively by 2 * 10
-8Mmol/L, 1 * 10
-7Mmol/L and 5 * 10
-7Mmol/L, finally obtaining can be 5 * 10
-7The Chinese hamster ovary celI strain of normal growth among the MTX of mmol/L is the Chinese hamster ovary genetically engineered cell strain of efficient secretory expression AcAPc2.
The RT-PCR test of the positive colony of this embodiment; Concrete grammar is: collect the cell after the MTX pressurization is screened; Carrying out the extraction of cell total rna with TRIzol, is that primer carries out pcr amplification with P1 and P2 after the RNA reverse transcription, and the PCR reaction conditions is: 95 ℃ of preparatory sex change 5min; 94 ℃ of 30s, 51 ℃ of 30s, 72 ℃ of 30s, totally 30 circulations; 72 ℃ are finally extended 5min again.
Upstream primer P1:5 '-TAATACGACTCACTATAGGG-3 '
Downstream primer P2:5 '-TAGAAGGCACAGTCGAGG-3 '
Test-results is as shown in Figure 2, and swimming lane 1 is DNAmarker, swimming lane 2 positive clones' RT-PCR product, and swimming lane 3 is the cell of untransfected.To the Chinese hamster ovary celI of untransfected pcDNA3.1/V5-His-C-AcAPc2 and the pDCH1P11 no band in back that increases.Do not exist because Chinese hamster ovary celI self has the AcAPc2mRNA form, do not increased so non-transfected cells has respective segments.And amplify the fragment of 318bp in the cell strain of stable transfection, and show complete being incorporated in the Chinese hamster ovary celI of purpose fragment, transcribing of goal gene arranged.
The detection test of reorganization AcAPc2 protein expression level, concrete grammar is: respectively to 2 * 10
-8Mmol/L, 1 * 10
-7Mmol/L and 5 * 10
-7Cell strain after three different concns MTX pressurizations of mmol/L are cultivated is cultivated 72h, gets the nutrient solution supernatant afterwards, with ELISA kit measurement reorganization AcAPc2 protein content.Detected result is following:
| MTX concentration (mmol/L) | Reorganization AcAPc2 expressing quantity (mg/L) |
| MTX - | 0.1-0.3 |
| 2×10 -8 | 1.0-2.5 |
| 1×10 -7 | 3.0-5.0 |
| 5×10 -7 | 8.0-10.0 |
The experiment of anticoagulant protein AcAPc2 Chinese People's Anti-Japanese Military and Political College mouse thrombus; Concrete grammar is: get 40 of SD rats (the SD rat body weight is that 270-330g purchases the Experimental Animal Center in Second Affiliated Hospital of Harbin Medical Univ.); Be divided into 5 groups at random; Every group 8, dose groups and AcAPc2 high dose group among difference model control group, positive drug control group, AcAPc2 low dose group, the AcAPc2.Positive drug is selected heparin sodium injection for use, and (dosage is 1650U/kg; Heparin sodium injection is available from Shanghai No.1 Bio-Chemical Pharmacetical Industry Co., Ltd; The accurate word H31022051 of traditional Chinese medicines; Lot number: 091104), the dosage of dose groups and AcAPc2 high dose group among AcAPc2 low dose group, the AcAPc2 (5 times increase progressively) is respectively 40 μ g/kg, 200 μ g/kg and 1mg/kg.All by intravenous administration, behind intravenous administration, the carotid artery with separator well places YLS-14B animalcule thrombus to generate in the detection joint of appearance immediately, starts thrombus and generates appearance, with constant current galvanic current stimulation (1mA).The record probe is measured the circulation of blood and is conversed blood vessel blockage degree (timed interval of measurement is 4s, and with percentage table registration certificate, all processes continues 5min) through infrared scan.All data are represented with
; Adopt the SPSS17.0 statistical software to carry out statistical study, adopt one-way analysis of variance and SNK-q check.Wherein thrombus generation appearance is that YLS-14B animalcule thrombus generates appearance, prolongs development in science and technology ltd available from the Jinan benefit.
[n,
%] is as shown in the table for the effect of AcAPc2 Chinese People's Anti-Japanese Military and Political College mouse carotid atery thrombus:
| Group | n | Dosage | Carotid chocking-up degree (%) |
| Model control group | 8 | 0μg/kg | 100.00±0.00 |
| Positive drug control group | 8 | 1650U/kg | 49.56±41.68 |
| The AcAPc2 low dose group | 8 | 40μg/kg | 40.20±43.14 * |
| Dose groups among the AcAPc2 | 8 | 200μg/kg | 26.32±40.83 * |
| The AcAPc2 high dose group | 8 | 1mg/kg | 11.24±13.36 ** |
Compare with model control group:
*P＜0.05,
*P＜0.01.
The result shows: the carotid artery chocking-up degree of model control group and AcAPc2 are basic, normal, high, and 3 dose groups comparing differences all have statistical significance.
Embodiment two: what this embodiment and embodiment one were different is: the sequence of AcAPc2 gene that step 1 is said to have signal peptide is shown in SEQ ID NO:1.Other is identical with embodiment one.
This embodiment has the sequence of the AcAPc2 gene of signal peptide and is synthesized by the living worker in Shanghai company.
The said original series that has the AcAPc2 gene order of signal peptide according to the mRNA sequence (Aceession:U30793) of dog hookworm among the Genbank (Ancylostoma caninum) of step 1; Remove the original signal peptide sequence, select CHO-dhft according to the characteristics of expression system simultaneously
-Preference codon.To transcribe with translation efficiency the highlyest in order making, to make expression product be secreted into the extracellular simultaneously, add Kozak sequence and signal peptide, add the site of restriction enzyme BamHI and NotI at the original series two ends respectively at original series 5 ' end.
Embodiment three: what this embodiment and embodiment one were different is: the said gene order that has the signal peptide that adds in the AcAPc2 gene of signal peptide of step 1 is shown in SEQ ID NO:2.Other is identical with embodiment one.
Embodiment four: what this embodiment and embodiment one were different is: the said aminoacid sequence that has the signal peptide that adds in the AcAPc2 gene of signal peptide of step 1 is shown in SEQ ID NO:6.Other is identical with embodiment one.
Embodiment five: the aminoacid sequence of the expression product of the Chinese hamster ovary genetically engineered cell strain of this embodiment efficient secretory expression AcAPc2 is shown in SEQ ID NO:3.
Claims (5)
1. the Chinese hamster ovary genetically engineered cell strain of efficient secretory expression AcAPc2; It is characterized in that utilizing Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell as host cell; After screening, amplification, stablized, efficiently express the cell strain of AcAPc2, concrete grammar carries out according to the following steps:
One, purpose fragment double digestion: give birth to the synthetic AcAPc2 gene order that has signal peptide of worker company by Shanghai and be cloned on the T carrier, the T carrier is carried out double digestion with BamHI and NotI, reaction system is following:
The endonuclease reaction condition is 37 ℃ placed 3 hours, then enzyme was cut product and used 1.5% agarose gel electrophoresis, and the purpose fragment obtains inserting fragment with DNA GEL EXTRACTION KIT purifying and recovering;
Two, plasmid vector double digestion: with BamHI and NotI double digestion digested plasmid carrier pcDNA3.1/V5-His-C, reaction system is following:
The endonuclease reaction condition is 37 ℃ placed 3 hours, then enzyme was cut product and used 1.5% agarose gel electrophoresis, used DNA GEL EXTRACTION KIT purifying and recovering again, the plasmid vector after obtaining enzyme and cutting;
Three, the connection of dna fragmentation, reaction system is following:
The ligation condition is that 16 ℃ of placements are spent the night, and obtains to connect product recombinant plasmid pcDNA3.1/V5-His-C-AcAPc2;
Four, cell cultures: with the DMEM culture medium culturing Tetrahydrofolate dehydrogenase defective type Chinese hamster ovary cell CHO-dhfr that contains 100mL/L foetal calf serum, NAA, 1 * HT
-
Five, recombinant plasmid transfection CHO: the recombinant plasmid pcDNA3.1/V5-His-C-AcAPc2 that builds is mixed with plasmid pDCH1P 11, use Lipofeetmine
TM2000Reagent cotransfection CHO-dhfr
-Cell carries out transfection in 24 orifice plates, when cell reaches 95% fusion; Substratum is replaced by the CHO-S-SFM II of antibiotic-free, serum-free, and the CHO-S-SFM II that contains 100mL/L FBS is changed in 500 μ L/ holes behind the transfection 6h; Continue to cultivate 48h, identify that with the ELISA test kit positive cells transfected went down to posterity by 1: 10, was replaced by selective medium behind the 24h cell attachment; There is positive colony to form after cultivating 14～16d; Method with directed digestion changes positive colony over to 24 orifice plates then, is passaged to 12 orifice plates again, through the ELISA kit measurement to positive colony in the 10 plant heights cell clone of expressing carry out enlarged culturing;
Six, the pressurization of MTX is cultivated: with MTX pressurization screening, concentration is followed successively by 2 * 10
-8Mmol/L, 1 * 10
-7Mmol/L and 5 * 10
-7Mmol/L, finally obtaining can be 5 * 10
-7The Chinese hamster ovary celI strain of normal growth among the MTX of mmol/L is the Chinese hamster ovary genetically engineered cell strain of efficient secretory expression AcAPc2.
2. the Chinese hamster ovary genetically engineered cell strain of efficient secretory expression AcAPc2 according to claim 1, the sequence that it is characterized in that the said AcAPc2 gene that has a signal peptide of step 1 is shown in SEQ ID NO:1.
3. the Chinese hamster ovary genetically engineered cell strain of efficient secretory expression AcAPc2 according to claim 1 is characterized in that the said gene order that has the signal peptide that adds in the AcAPc2 gene of signal peptide of step 1 is shown in SEQ ID NO:2.
4. the Chinese hamster ovary genetically engineered cell strain of efficient secretory expression AcAPc2 according to claim 1 is characterized in that the said aminoacid sequence that has the signal peptide that adds in the AcAPc2 gene of signal peptide of step 1 is shown in SEQ ID NO:6.
5. the aminoacid sequence of the expression product of the Chinese hamster ovary genetically engineered cell strain of the said efficient secretory expression AcAPc2 of claim 1 is shown in SEQ ID NO:3.
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| CN103013899A (en) * | 2012-11-22 | 2013-04-03 | 黑龙江大学 | OGP-AcAP2 fusion protein transgenic engineering strain |
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| CN103031267B (en) * | 2012-12-31 | 2014-06-25 | 黑龙江大学 | Calcitonin gene-related peptide and AcAPc2 fusion protein production gene engineering strain and construction method thereof |
| CN103031267A (en) * | 2012-12-31 | 2013-04-10 | 黑龙江大学 | Calcitonin gene-related peptide and AcAPc2 fusion protein production gene engineering strain and construction method thereof |
| CN103013902B (en) * | 2012-12-31 | 2014-08-06 | 黑龙江大学 | Calcitonin gene-related peptide (CGRP) and ancylostoma caninum anticoagulant peptide (AcAP5) fusion protein gene engineering strain and construction method thereof |
| CN103031266A (en) * | 2012-12-31 | 2013-04-10 | 黑龙江大学 | msCT-AcAPc2 fusion protein transgenic engineering strain |
| CN103013902A (en) * | 2012-12-31 | 2013-04-03 | 黑龙江大学 | Calcitonin gene-related peptide (CGRP) and ancylostoma caninum anticoagulant peptide (AcAP5) fusion protein gene engineering strain and construction method thereof |
| CN106318970A (en) * | 2016-08-18 | 2017-01-11 | 广东东阳光药业有限公司 | High-throughput screening method for CHO-Dhfr expression system cell strains |
| CN106318970B (en) * | 2016-08-18 | 2019-06-25 | 广东东阳光药业有限公司 | A kind of high-throughput screening method of CHO-Dhfr expression system cell strain |
| CN106442967A (en) * | 2016-09-27 | 2017-02-22 | 杭州鸿运华宁生物医药工程有限公司 | Method for detecting affinity of monoclonal antibodies of transmembrane proteins |
| CN106442967B (en) * | 2016-09-27 | 2018-05-15 | 鸿运华宁（杭州）生物医药有限公司 | A kind of method for detecting transmembrane protein monoclonal antibody affinity |
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