CN1256431C - Recombined human parathyroid hormone gene, its expression carrier and preparing method of recombined human parathyroid protein - Google Patents
Recombined human parathyroid hormone gene, its expression carrier and preparing method of recombined human parathyroid protein Download PDFInfo
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Abstract
The present invention relates to an artificially synthesized gene (SEQ ID NO. 1) for expressing human parathyroid hormone proteins, a constructed recombinant expression carrier of the gene, and a host cell converted by the carrier. The present invention also relates to a method for preparing human parathyroid hormone proteins. The present invention successfully synthesizes the gene for expressing human parathyroid hormone proteins and establishes the method for producing recombination human parathyroid hormone proteins by the genetic engineering technique; human parathyroid proteins obtained by the method can obviously stimulate osteosarcoma cells cAMP and have high biological activity. The present invention create a foundation for the large-scale industrialization production of recombination human parathyroid proteins.
Description
Technical field
The present invention relates to the gene of the expressing human Rat parathyroid hormone 1-34 of synthetic, the recombinant expression vector of this gene of structure is by this carrier transformed host cells.
The invention still further relates to the proteic method of preparation recombinant human parathyroid hormone.
Background of invention
(parathyroid hormone is to contain 84 parathyroid secretory product of amino acid whose Mammals PTH) to Rat parathyroid hormone 1-34, is to regulate alcium and phosphor metabolization, keeps the major hormone of body calcium balance, and its periphery metabolism is mainly carried out in bone, kidney and liver.PTH is number and the vigor that increases osteoclast to the effect of bone on the one hand, promotes bone resorption, discharges calcium phosphorus and goes into blood, and PTH increases osteoblastic number on the other hand, and promotes scleroblast to discharge bone growth factor, thereby promotes bone forming.PTH also increases the heavily absorption of kidney nearby bleeding tubule to calcium ion, promotes the α hydroxylase activity, increases 25-(OH) D3 and is converted into 1 at kidney, 25-(OH) 2D3.
The secretion of the interior PTH of normal human is phasic property sometimes, is the dynamic (dynamical) polytropy of secretory product on the one hand, regulates blood calcium concentration with this, prevents the downward modulation of PTH acceptor to PTH avidity; Be PTH excretory high stability on the other hand, promptly secreting number of times every day is clocklike with each secretory volume, keeps the balance of normal bone amount and bone metabolism with this.The secretion of osteoporosis patient PTH does not have rule, causes the imbalance of bone forming and bone resorption, causes that bone loss and bone structure change.Thereby to osteoporosis people subcutaneous injection at intermittence PTH, the people causes the secretion of PTH in the body similar to the normal people, thereby reach the osteoporotic purpose of treatment.
Introduce extra amino acid at the proteic aminoterminal of PTH, the activity of PTH reduces greatly, and when the expression in escherichia coli foreign protein, often introduces methionine(Met) at aminoterminal; On the other hand, PTH gene 5 ' end has a possible translation initiation site, easily expresses the 8-84 amino acid fragment of non-activity when expression in escherichia coli; The easy oxidation of PTH loses activity on the one hand again.These reasons cause being difficult to obtain a large amount of activated PTH with the method for directly expressing in intestinal bacteria.By adopting the codon that is difficult for the formation secondary structure to be beneficial to translation initiation, utilize colibacillary endogenous methionine aminopeptidase (methionineaminopeptidase) to remove the aminoterminal methionine(Met) of expression product, can obtain activated PTH (US patent6146852 in conjunction with suitable purification process, Sung et.al., J.Bio.Chem.1991,266 (5): 2831-2835).
There is several different methods to overcome of the influence of the unfavorable secondary structure in translation initiation district to translation, such as by the sequence in the translation initiation district is transformed, but this transformation depends on not too accurate computer mRNA secondary structure prediction, therefore transforming the result is difficult to expect, and the bicistronic mRNA expression method is a kind of relatively effective means, and its principle is that effective translation of leading cistron can open follow-up intracistronic unfavorable secondary structure.The bicistronic mRNA expression method is exactly to introduce another coding that is easy to express (generally in 20 amino acid) section of DNA sequence than short amino acid sequence before the gene of coding target protein, disadvantageous secondary structure obtains the effective expression of goal gene to the influence of translation initiation in the goal gene thereby overcome.
Summary of the invention
The object of the present invention is to provide a kind of proteic gene of expressing human Rat parathyroid hormone 1-34 of synthetic, on design theory, it has utilized the bicistronic mRNA expression method on the one hand, selected the codon of difficult formation secondary structure on the other hand for use, thereby overcome of the influence of translation initiation region two-stage structure translation initiation.The gene order of coding PTH provided by the invention is all inequality with the sequence of present any coding PTH that has reported.
Another object of the present invention is to provide recombinant human parathyroid hormone expression carrier and this expression vector transformed host cells.
Another purpose of the present invention is to provide with genetic engineering technique and prepares the proteic method of recombinant human parathyroid hormone.
According to an aspect of the present invention, provide the recombinant human parathyroid hormone gene of the dna sequence dna that contains SEQ ID NO.:1, wherein, base 81 to 332 a coding human parathyroid hormone 1-84 aminoacid sequence (SEQ ID NO.3).Base 7 is to the base 57 coding one small peptide IVSEIQLMHNLGKHLTS as leading cistron (SEQ ID NO:2).Before this two fragment an initial codon ATG is arranged respectively, a terminator codon TAA is respectively arranged afterwards, base 64 to base 70 is an intestinal bacteria SD series.
In a preferred embodiment of the invention, by synthetic primer and template, behind the DNA chain extension reaction, the product of extension is carried out polymerase chain reaction again increase, the PCR reaction product transforms the DNA that obtains after ligation and enters intestinal bacteria E.Coli JM109 competent cell.After identifying, select positive colony.
According to a further aspect in the invention, provide the recombinant vectors that comprises SEQ ID NO:1 sequence.Wherein carrier can be with forms such as plasmid, virion and phages.In one embodiment of the invention, made up the carrier of plasmid form.
Can target DNA be inserted in the carrier by several different methods, generally speaking, dna sequence dna of the present invention can be inserted on the suitable restriction endonuclease sites by certain methods well known by persons skilled in the art.DNA in the expression vector operationally is connected with a suitable expression regulation sequence (promotor) to instruct the synthetic of mRNA, and expression vector also contains the ribosome bind site and the transcription terminator that are used for translation initiation.Expression vector contains the phenotypic character gene that is useful on the screening transformed host cell simultaneously.The plasmid or the virus that are used for construction of expression vector can obtain by commercial sources.
In the preferred embodiment of the present invention, by recombinant human parathyroid hormone gene (SEQ IDNO.1) being inserted restriction enzyme NdeI, the SalI site of plasmid pTHioHisA, be built into plasmid expression vector pTrcPTH, this carrier can go out to have the recombinant human parathyroid hormone albumen of high biologic activity at expression in escherichia coli
According to another aspect of the invention, by expression vector host transformed of the present invention, host cell can comprise prokaryotic cell prokaryocyte and eukaryotic cell, the prokaryotic cell prokaryocyte that wherein is used to transform can comprise various bacteriums, in a preferred embodiment of the invention, with the host cell of intestinal bacteria as carrier of the present invention, e. coli bl21 (DE3) more preferably.
In accordance with a further aspect of the present invention, utilize genetic engineering technique to prepare the proteic method of recombinant human parathyroid hormone, comprise: the gene of the recombinant human parathyroid hormone of 1) will encoding (SEQ ID NO.1) is operably connected to expression regulation sequence, construction of expression vector; 2) transform protokaryon or eukaryotic cell with this expression vector, form host cell; 3) under suitable culture condition, cultivate host cell of the present invention; 4) from nutrient solution, isolate and have the active protein of human parathyroid hormone; 5) purifying obtains recombinant human parathyroid hormone.
The present invention has successfully synthesized the proteic gene of expressing human Rat parathyroid hormone 1-34, make up this expression carrier and formed the host cell of expressing this gene, set up the employing genetic engineering technique and produced the proteic method of recombinant human parathyroid hormone, but the human parathyroid hormone albumen obvious stimulation osteosarcoma cell cAMP by this method obtains has higher biologic activity.The present invention is for recombinant human parathyroid hormone is proteic on a large scale, suitability for industrialized production is laid a good foundation.
Brief description of drawings
Fig. 1 is the collection of illustrative plates of recombinant plasmid pTrcPTH;
Show the encoding sequence of PTH, replicon pUCori, amicillin resistance (AMP
R) gene, TAC promotor, term terminator, the initiator codon of the element of leading cistron Lac: ATG=ATG, RBS=ribosome bind site, the sequence of Leader=coding IVSEIQLMHNLGKHLTS;
Fig. 2 is segmental nucleotide sequence of the bicistronic mRNA among the recombinant plasmid pTrcPTH and amino acid sequence corresponding;
Fig. 3 is the purification result in the example three; Among Fig. 3,
A illustrates the cation-exchange chromatography collection of illustrative plates;
B illustrates the hydrophobic chromatography collection of illustrative plates;
C illustrates the SDS-PAGE electrophoretogram, 18%SDS-PAGE:
Swimming lane 1: 5 hours bacterial proteins ferment;
Swimming lane 2: 9 hours bacterial proteins ferment;
Swimming lane 3: induce 1 hour bacterial protein;
Swimming lane 4: induce 1.5 hours bacterial proteins;
Swimming lane 7: molecular weight standard: 14KD, 20KD, 24KD, 29KD, 36KD, 45KD, 66KD;
Fig. 4 illustrates the biologic activity of reorganization PTH albumen to osteosarcoma cell.
Specific embodiment
Come further to set forth the present invention by the following examples, the experimental technique of unreceipted actual conditions among the embodiment, basically " molecular cloning: laboratory manual " all write (New YorkCold Spring Harbor Laboratory Press according to people such as Sambrook, 1989) condition described in, or the condition of advising according to business men.
Synthetic and the clone of embodiment one gene:
(1), the following primer of chemosynthesis:
CAG212F1(SEQ?ID?NO：4)：
5’CATATGATCGTAAGTGAAATACAGCTAATGCACAACCTGGGAAAACATCTGACTTCATAATAAAGGAGGA?3’
CAG212F2(SEQ?ID?NO：5)：
5’GACTTCATAATAAAGGAGGAAACAGCTATGTCAGTAFCAGAAATACAATTAATGCATAATTTAGGTAAGC?3’
CAG212F3(SEQ?ID?No：6)：
5’AATGCATAATTTAGGTAAGCACTTGAACTCTATGGAAAGAGTTGAATGGTTGAGAAAGAAGCTGCAGGAC?3’
CAG212R1(SEQ?ID?No：7)：
5’GTCGACTTACTGGGATTTAGCTTTAGTGAGTACATTCACATCGGCCTTGTCAGCCTCGCCA?3’
CAG212R2(SEQ?ID?NO：8)：
5’CGGCCTTGTCAGCCTCGCCAAGGGATTTTTCATGGCTCTCAACTAAGACATTGTCTTCCT?3’
CAG212R3(SEQ?ID?No：9)：
5’AACTAAGACATTGTCTTCCTTTTTACGTGGTCTTTGGGAACCAGCATCACGAGGCGCCAA?3’
CAG212R4(SEQ?ID?NO：10)：
5’CCAGCATCACGAGGCGCCAACGGAGCGCCCAGCGCAACGAAATTGTGAACGTCCTGCAGCTTCTTTCTCA?3’
2) DNA chain extension reaction.
The following reaction solution of modulation in the Microtue pipe:
CAG212F3(0.33ug/ul) 5ul
CAG212R4(0.33ug/ul) 5ul
10×TaKaPa?LA?Buffer 10ul
dNTP 16ul
H2O 63ul
Reaction solution 94 ℃ of insulations after 5 minutes, at 10 minutes internal cooling to 55 ℃, is added TaKaRa LATaq enzyme 1ul, 72 ℃ of insulations 5 minutes.
3), PCR reaction:
The following reaction solution of modulation in the Microtube pipe:
CAG212F1(0.33ug/ul) 1ul
CAG212R2(0.33ug/ul) 1ul
CAG212R1(0.33ug/ul) 1ul
CAG212R2(0.33ug/ul) 1ul
CAG212R3(0.33ug/ul) 1ul
DNA extends liquid 1ul
10×TaKaPa?LA?Buffer 10ul
TaKaRa Ex Taq enzyme 1ul
dNTP 10ul
H2O 73ul
The PCR reaction conditions is: 94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 30 seconds, 20 the circulation.
Reaction product is dissolved among the 100ulTE after with ethanol sedimentation.
4), ligation
The following reaction solution of modulation in the Microtube pipe:
PCR reaction product 2ul
Pmd18-T 1ul(50ng)
H2O 2ul
Ligation?Solution?I 5ul
16 ℃ of insulations are after 30 minutes, and full dose is converted into E.Coli JM109Competent Cell.
5), positive colony detects
Picking colony, identify for primer carries out PCR with RV-M (SEQ ID NO:11) and M13-47 (SEQ ID NO:12), picking can amplify the segmental bacterium colony in the 400bp left and right sides, after the incubated overnight, extract plasmid with conventional rapid plasmid extraction method, identify with NdeI and SalI double digestion, picking can cut out the segmental plasmid of 400bp by enzyme, called after pMD18PTH, be that primer carries out full-automatic sequencing and confirms that further sequencing result shows synthetic and cloned correct gene fragment (SEQ ID NO:1) with RV-M.
Embodiment two: the structure of the recombinant plasmid pTrcPTH of expressing human recombined parathyroid hormone.
With NdeI enzyme and SalI enzyme cutting pMD18PTH plasmid, reclaim the purpose fragment about 340bp.The plasmid pThioHisa that Invitrogen company sells, cut through NdeI and SalI enzyme, enzyme is cut product and is run agarose gel electrophoresis, electrophoretic separation goes out 360bp left and right sides fragment and 3.4kb left and right sides fragment, reclaim test kit with gel and reclaim 3.4kbp left and right sides fragment, it is connected with purpose fragment about above-mentioned 340bp, transformed into escherichia coli DH5a, transformant extracts plasmid after with the LB culture medium culturing, identify with NdeI and SalI double digestion again, identify that correct plasmid is that primer carries out complete sequence determination with TrxR (SEQ ID NO:13), sequencing result shows and has successfully made up recombinant expression plasmid pTrcPTH, its plasmid map such as Fig. 1, segmental nucleotide sequence of its bicistronic mRNA and amino acid sequence corresponding are as shown in Figure 2.
The preparation of embodiment three reorganization human parathyroid hormones
1) foundation of engineering bacteria
Select for use e. coli bl21 (DE3) to be the host bacterium, be equipped with competent cell with Lime Chloride, pTrcPTH is transformed into competent cell with plasmid, be coated in the LB (1%trypton that contains the 100ug/ml penbritin, 0.5%yeast extract, 1%NaCl, 1.5%agrose) on the flat board, the reorganization bacterium colony is with containing the LB substratum of 100ug/ml penbritin 30 ℃ of cultivations, after screening, set up engineering bacteria BL21 (DE3)/pTrcPTH of expressing human PTH, original strain is stored in-70 ℃ with the LB substratum that contains 15% glycerine, engineering bacteria is typical intestinal bacteria colony characteristics containing on the LB flat board of penbritin growth, atlas analysis of the every biography of this engineering bacteria 20 generations work, the result is consistent with original strain.
2) fermentation
Planting daughter bacteria cultivates:
BL21 (the DE3)/pTrcPTH inoculation of preserving is gone into the LB substratum that 200ml contains the 100ug/ml penbritin, shakes in the bottle in 30 ℃ at 1 liter and cultivates about 16 hours, and inoculation is gone in 3.5 liters of fermention mediums.
7 liters of fermentor cultivation:
Fermention medium R2 (3.5 liters):
(NH
4)
2HPO
4 2g/l
KH
2PO
4 6.75g/l
Citric?acid 0.85g/l
MgSO
4 1.8G/L
Glucose 20g/l
CaCl
2 0.054g/l
ZnCl
2 0.02g/l
Trace?metal 2ml/l
Vitamin complex liquid 1ml/l
Penbritin 0.2g/l
Trace?metal(1liter)：
Citric?acid 5g
CoCl
2.6H
2O 2g
MnCl
2.4H
2O 12g
CuSO
4.5H
2O 1.85g
H
3BO
3 2.5g
(NH
4)
6M
07.4H
2O?1.32g
FeCl
3.6H
2O 3.4g
Vitamin complex liquid (1 liter):
VITMAIN B1 20g
Vitamin B12 200mg
Vitamin B6 2.5g
Wei ShengsuB2 1.0g
Vitamin H 12.5mg
Fermentation parameter: 30 ℃ of culture temperature, pH is controlled at 6.8～7.2, and tank pressure is a normal pressure, air flow 2vvm, dissolved oxygen is controlled at more than 10% by control stir speed (S.S.) or logical pure oxygen.
When adding glucose and be cultured to the nectar degree and be the OD600=30 left and right sides (greatly about back 10 hours of inoculation) by stream, add inductor (4ml 0.5M IPTG), be warming up to 36 ℃, continue to cultivate, according to nutrient consumption situation supplemental acid hydrolysis casein food grade, glucose and yeast extract, stagnate until thalli growth, greatly about inducing 4 ℃ of centrifugal collection thalline back 2 hours.Expression of results is seen Fig. 3 C.
3) purifying
Thalline is pressed 1g/ml consumption extract (1N HCl/1%NaCl/1%TFA) extracting, extracting is once again after centrifugal, centrifugal, merge supernatant, transfer pH to 3.8 with 5N NaOH, five times of dilute with waters, last sample is to the SP post, the SP post uses buffer A l (50mM Formic acid Ph3.8) balance good in advance, then with buffer A 1 flushing baseline before detecting the approaching last sample of baseline, carry out gradient elution with buffer A 1 for the addition O-1N NaCl gradient that flows then, fraction collection elutriant (Fig. 3 A) confirms that with 18%SDS-PAGE each the fraction collection sample that contains target protein merges these collection samples, the 5N NaCl that adds one of 20 parts volumes, transfer pH to 8.0 with 5N NaOH, cross phenyl hydrophobic chromatography post, the phenyl post is used buffer A 2 (1M NH in advance
4AC, pH8.0) balance is good, with buffer A 2 flushings baseline before detecting the approaching last sample of baseline, uses 20mM NH then behind the end of the sample
4AC, the pH8.0 wash-out is collected elution peak (Fig. 3 B).The reorganization PTH albumen that purifying obtains, purity is greater than 95% (Fig. 3 C).
4) the biologic activity analysis of recombinant human parathyroid hormone
The adenosine cyclase of recombinant human parathyroid hormone is active measures (Rabbani et al., J.Bio.Chem.1988,263 (3): 1307-1313) with the osteosarcoma cell of vitro culture.Parathyroid hormone receptor on the osteosarcoma cell in conjunction with Rat parathyroid hormone 1-34 after adenosine cyclase in the activating cells, cause increasing of ring gland purine (cAMP).
Osteosarcoma cell MG-63 and MC3T3-EI cultivate based on 37 ℃ of cultivations with the high sugared DMEM that contains 15% foetal calf serum and 1.5mM glutamine, keep CO in the incubator
210%, humidity is 99%.With 1 * 10
3The every hole of individual cell is seeded on 24 well culture plates, continue to cultivate until covering with, remove substratum, with detecting substratum (DMEM/0.1%BSA/2mM 3-isobutyl-l-methylxanthine, 20ug/ml ascorbicacid) washing once, add the detection substratum that contains detected sample, cultivated 20-30 minute at 37 ℃, remove substratum then, add the 0.1NHCl lysing cell of ice precooling, press R﹠amp then; The cAMP detection kit explanation of Dsystem company is operated, and measures the content of cAMP.Compare with the control group that does not contain PTH, the experimental group obvious stimulation osteosarcoma cell that contains PTH produces cAMP (Fig. 4).
This shows that the recombinant human parathyroid hormone albumen for preparing by the inventive method has higher biologic activity.
The above description of this invention does not limit the present invention, and those skilled in the art can make various changes and distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the defined scope of claim of the present invention.
SEQUENCE?LISTING
＜110〉Kunming Yun Jian Pharmacy stock Co., Ltd
＜120〉recombinant human parathyroid hormone gene, its expression vector and the proteic preparation method of recombinant human parathyroid hormone
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Claims (7)
1, the nucleotide sequence of a kind of SEQ ID NO:1.
2, the recombinant vectors of dna sequence dna shown in a kind of SEQ ID NO:1, described carrier comprises dna sequence dna shown in the SEQID NO:1 and pTHioHisA carrier.
3, with the plasmid vector transformed host cells of dna sequence dna shown in a kind of SEQ ID NO:1, described plasmid vector comprises dna sequence dna shown in the SEQ ID NO:1 and pTHioHisA carrier, and described host cell is intestinal bacteria.
4, host cell according to claim 3 is characterized in that described intestinal bacteria are BL21 (DE3).
5, a kind of method for preparing recombinant human parathyroid hormone comprises following step:
1) nucleotide sequence with a kind of SEQ ID NO.1 is operably connected to expression regulation sequence, construction of expression vector;
2) transform protokaryon or eukaryotic cell with this expression vector, form host cell;
3) under suitable culture condition, cultivate host cell of the present invention;
4) from nutrient solution, isolate and have the active protein of human parathyroid hormone;
5) purifying obtains recombinant human parathyroid hormone.
6, method according to claim 5, wherein the described expression vector of step 1) is the plasmid vector that is made up by plasmid pTHioHisA and SEQ ID NO.1.
7, method according to claim 5, wherein step 2) described host cell is e. coli bl21 (DE3)/pTrcPTH.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 02127856 CN1256431C (en) | 2002-08-01 | 2002-08-01 | Recombined human parathyroid hormone gene, its expression carrier and preparing method of recombined human parathyroid protein |
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| CN 02127856 CN1256431C (en) | 2002-08-01 | 2002-08-01 | Recombined human parathyroid hormone gene, its expression carrier and preparing method of recombined human parathyroid protein |
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| CN1256431C true CN1256431C (en) | 2006-05-17 |
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| CN101555485B (en) * | 2009-05-15 | 2013-03-27 | 中国医学科学院医学生物学研究所 | Non-fusion expression of recombinant human parathyroid hormone (1-84) and large-scale preparation method |
| CN111876388A (en) * | 2020-07-24 | 2020-11-03 | 赛瑞诚(苏州)生物科技有限公司 | Bone and bone tissue targeted exosome and preparation method and application thereof |
| CN112143755A (en) * | 2020-10-19 | 2020-12-29 | 深圳市瑞普逊科技有限公司 | Human parathyroid hormone eukaryotic expression recombinant plasmid vector and construction method thereof |
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