CN102775492A - High-activity human parathyroid hormone (1-34) mutant protein and activity detecting method thereof - Google Patents
High-activity human parathyroid hormone (1-34) mutant protein and activity detecting method thereof Download PDFInfo
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- CN102775492A CN102775492A CN2012100013197A CN201210001319A CN102775492A CN 102775492 A CN102775492 A CN 102775492A CN 2012100013197 A CN2012100013197 A CN 2012100013197A CN 201210001319 A CN201210001319 A CN 201210001319A CN 102775492 A CN102775492 A CN 102775492A
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Abstract
The invention provides a 'high-activity human parathyroid hormone (1-34) mutant protein and an activity detecting method of the high-activity human parathyroid hormone (1-34) mutant protein', belonging to the field of protein drug. The invention discovers the high-activity human parathyroid hormone (1-34) mutant protein by replacing the 25th, 26th and 27th amino acid of the natural human parathyroid hormone (1-34). The invention further builds a quick and stable activity detecting method, so that the activity of one or various protein(s) or polypeptide drug(s) can be detected within 3-10 days, and can be compared with that of the standards of the human parathyroid hormone. The activity detecting result shows that the activity of the human parathyroid hormone (1-34) mutant protein is better than that of the standards, so that the osteoporosis curing drug which is higher in curative effect and less in side effects can be prepared.
Description
Technical field
A kind of highly active human parathyroid hormone (1-34) mutain and activity test method thereof belong to the pharmaceutical grade protein field
Background technology
Osteoporosis is a kind of common bone metabolism disease, shows as bone resorption and has surpassed bone forming, thereby cause the bone amount to reduce, and bone fragility increases.Osteoporosis is occupied larger proportion in menopausal women, and increasingly serious along with the world population aging, seeks new more effective osteoporosis prevention and medicine and seems and be even more important.At present, the clinical treatment medication of osteoporosis is main to have two types, is respectively bone resorption inhibitor and bone formation-promoter.
Human parathyroid hormone (PTH) is considered to the short anabolic medicine of bone of unique ability at present, and its characteristics that are superior to other medicine for treating osteoporosis are to rebuild the bone trabecula system.Research shows that low dose of PTH promptly has obvious osteogenesis, and its effect possibly realize calcium and phosphatic the absorption again with the adjusting uriniferous tubules through regulating bone metabolism.
FDA approval teriparatide (rhPTH (1-34)) in 2002 listing is mainly used in the treatment osteoporosis in postmenopausal women.Another parathyroid hormone agents rhPTH (1-84) is also in listing in 2006.Domestic PTH is studied started late, and the PTH medicine that at present enters into the clinical study stage has injection recombinant human parathyroid hormone (1-34), and the research focus concentrates on the various two mutants of PTH fusion rotein and PTH.
Protein or polypeptide are carried out single or a plurality of amino acid whose displacements, may make proteinic structure change, and produce the change of a series of physics and biochemical property (solubleness, stability, to avidity of substrate and acceptor etc.).Therefore natural human Rat parathyroid hormone 1-34 (1-34) is carried out amino acid whose displacement, it is stronger to be hopeful to obtain physiologically active, the osteoporosis therapy medicine that spinoff is littler.The activity determination method that the present invention sets up can fast and stable ground detects the external activity of human parathyroid hormone (1-34) mutain, and the technology that relates to comprises fluorescence real-time quantitative PCR, and scleroblast and osteoclast are cultivated etc. altogether.
Summary of the invention
A content of the present invention is through comparison of different plant species homologous sequence and preliminary computer simulation molecular docking; Three amino acid R25K26K27 to natural human parathyroid hormone (1-34) carry out the displacement of Q25E26L27; And entrust the biochemical (Shanghai) Co., Ltd. of gill to carry out the complete synthesis of polypeptide, polypeptide purity is more than 95%.
Another content of the present invention is to have set up a kind of active method of detection mutain of ability fast and stable.
Rat parathyroid hormone 1-34 (PTH) is the single chain polypeptide hormone that is contained 84 amino-acid residues by the principal cell excretory, is to regulate one of calcium, the of paramount importance peptide hormone of phosphorus metabolism in the organism.Rat parathyroid hormone 1-34 aminoterminal 1-34 fragment has the ability and the biological activity of full molecule PTH and receptors bind, therefore is widely used in studying structure and the function of PTH.
The mechanism of action and activity research to PTH and analogue thereof start from the eighties in last century.Present known PTH can through with matrix/scleroblast on g protein coupled receptor-Rat parathyroid hormone 1-34/parathyroid hormone-related protein acceptor 1 (PTHR1) combine, stimulate these cell expressings M-CSF and NF κ B-ligand (RANKL).Research shows that the mouse of said gene disappearance can lose the ability that forms osteoclast, and during vitro culture, exists even without matrix/scleroblast, and above-mentioned two kinds of albumen also can stimulate the formation of osteoclast.Prove that with experiment in vitro the formation of osteoclast is directly proportional with the RANKL expression of gene, and does not have tangible proportional relation with M-CSF in the body.RANKL can through with hematopoiesis precursor osteoclast on the RANK receptors bind, stimulate its differentiation to become sophisticated osteoclast.When RANKL played a role, a kind of bait acceptor-osteoprotegerin (OPG) of solubility can hinder it and play a role.Many researchs show that PTH acts on former being commissioned to train when foster matrix/scleroblast or rat, the stimulation RANKL expression of gene that it can continue and suppress the OPG expression of gene.Probably the formation that influences osteoclast through the ratio of the joint RANKL/OPG that withers when therefore, PTH plays a role.
Phosphoric acid esterase be a kind of can hydrolysed fat family and aromatic phosphoric ester, thereby discharge phosphatic enzyme.Required pH can be divided into acid phosphatase and SEAP again when Phosphoric acid esterase played a role according to it.During acid phosphatase is present in various cells and organizes, like prostate gland, liver,kidney,spleen, red corpuscle, thrombocyte and osteoclast.Nineteen fifty-nine, the Burstone report, osteoclast can demonstrate the intensive activity of acid phosphatase, and scleroblast then demonstrates alkaline phosphatase activities.A lot of reports afterwards show that osteoclast still can show acid phosphates activity under the situation that tartrate exists.Therefore anti-tartaic acid Phosphoric acid esterase (TARCP) is extensively applied to the evaluation and the analysis of osteoclast now as the mark of an osteoclast.PTH can through with matrix/scleroblast on g protein coupled receptor-Rat parathyroid hormone 1-34/parathyroid hormone-related protein acceptor 1 (PTHR1) combine; Stimulate these cell expressings NF κ B-ligand (RANKL); RANKL can through with hematopoiesis precursor osteoclast on the RANK receptors bind, stimulate its differentiation to become sophisticated osteoclast.And osteoclast can show anti-tartaic acid Phosphoric acid esterase (TARCP) activity and be colored, and therefore stimulates the differentiation and the sophisticated ability of osteoclast through coloration result ability side light PTH.
The external activity detection method of human parathyroid hormone (1-34) mutain that the present invention sets up; Can in 3-10 days, carry out the activity detection to one or more protein or polypeptide drug; And compare with the human parathyroid hormone standard substance, be a kind of detection of active detection method of fast and stable.
Description of drawings
Fig. 1: quantitative real time PCR Instrument detects the real-time fluorescence situation
Fig. 2: PTH (1-34)-(RKK-QEL) is to the influence of UAMS-32P cell rna KL/OPG genetic transcription
The X axle is represented group: vehicle group, PTH (1-34) positive controls, PTH (1-34)-(RKK-QEL) group, little peptide (LQDVHNF) group human serum albumin (HSA) group.
The Y axle is represented mRNA relative expression quantity (with respect to the Rps27 gene)
Fig. 3: osteoclast forms experiment (TRACP dyeing)
1:vehicle group (3 every group multiple holes), 2:PTH (1-34) positive controls, 3:PTH (1-34)-(RKK-QEL) group, 4: little peptide (LQDVHNF) group, 5:HSA group
Fig. 4: osteoclast forms experiment (red cell count under the mirror)
The X axle is represented group: vehicle group, PTH (1-34) positive controls, HSA group, PTH (1-34)-(RKK-QEL) group, little peptide (LQDVHNF) group.
The Y axle is represented every group of red cell count in average every hole (3 every group multiple holes)
Fig. 5: PTH (1-34)-(RKK-QEL) is to the influence of former mouse femur marrow attached cell RANKL/OPG genetic transcription of being commissioned to train foster
The X axle is represented group: vehicle group, PTH (1-34) positive controls, HSA group, PTH (1-34)-(RKK-QEL) group, little peptide (LQDVHNF) group.
The Y axle is represented mRNA relative expression quantity (with respect to the Rps27 gene), establishes 3 multiple holes for every group
Embodiment
Embodiment 1:
PTH (1-34)-(RKK-QEL) and little peptide (aminoacid sequence: LQDVHNF) entrust the biochemical (Shanghai) Co., Ltd. of gill synthetic, purity＞95%; Test with standard control human parathyroid hormone (1-34) available from sigma company; Human serum albumin (HSA) is prepared and purifying by the laboratory voluntarily, and the host bacterium is pichia spp GS115, purity＞90%.
Embodiment 2:PTH (1-34)-(RKK-QEL) is to the influence of UAMS-32P cell rna KL/OPG genetic transcription
(1) clone and culture condition
Clone: UAMS-32P cell (stable transfection PTHR1)
Substratum: α-MEM substratum+10% foetal calf serum
Culture condition: 37 ℃ of incubator for tissue cultures, 5%CO
2
Cell grows to about 80% and converges the time-division bottle and go down to posterity, and grows to about 80% again and experimentizes when converging.
(2) bed board of UAMS-32P cell is cultivated and dosing
6 orifice plates are by 10
5/ porocyte bed board grows to about 80% and changes liquid when converging, and change 6h dosing drug dose behind the liquid: the administration group is all by 10
-7The mol/L dosing
Group: vehicle group
PTH (1-34) positive controls
PTH (1-34)-(RKK-QEL) group
Little peptide (LQDVHNF) group
The HSA group
(3) extraction of the total mRNA of UAMS-32P cell
The cell dosing discards substratum after cultivating 24h, presses the test kit explanation and extracts total RNA (Takara).Get part RNA sample and measure the OD value in 260 with the 280nm place, estimate RNA purity and carry out quantitative with ultraviolet spectrophotometer.
(4) reverse transcription becomes cDNA first chain
Control every group of equivalent RNA (1-1.5 μ g), reverse transcription is cDNA first chain.20 μ L systems are adopted in reverse transcription, and system is following:
, as template template is suitably diluted with the cDNA of above-mentioned reverse transcription, be used for real-time fluorescence quantitative RT-PCR.
(5) real-time fluorescence quantitative PCR detects the mRNA transcriptional level of RANKL and OPG
The vehicle group is established in experiment, PTH (1-34) positive controls, and the HSA group, PTH (1-34) (RKK-QEL) organizes and little peptide (LQDVNFH) is organized.Every group is detected Rps27 respectively, RANKL, and OPG gene transcription situation, every group of each gene established 3 parallel multiple holes.
3 footwork PCR:50 ℃, 2min; 95 ℃, 10min; 95 ℃, 15s, 60 ℃, 1min; 40 circulations.Quantitative real time PCR Instrument detects the real-time fluorescence situation.
Activity detection/the osteoclast of embodiment 3:PTH (1-34)-(RKK-QEL) in external scleroblast and osteoclast co-culture system forms experiment (TRACP staining)
Day1：
(1) acquisition of mouse femur medullary cell and cultivation
Under aseptic condition, take out 3-6 week mouse femur, and the flushing medullary space, the medullary cell that centrifugal collection washes.
The resuspended medullary cell of substratum, use diameter as the petridish of 10cm at 37 ℃, 5%CO
2Cultivate 2d under the condition.
(2) UAMS-32P cell (stable transfection PTHR1) grows to about 80% when converging, and pair cell digests.Cell is spread 24 orifice plates by 2000/ hole, and 37 ℃, 5%CO
2Cultivate 2d under the condition.
Day3：
The foundation of mouse femur marrow suspension cell and UAMS-32P co-culture of cells system
(1) the medullary cell nutrient solution supernatant in the collection petridish merges supernatant and centrifugal collecting cell.Carry out resuspendedly with the substratum pair cell of certain volume, blood counting chamber carries out cell counting.
(2) the nutrient solution supernatant of the UAMS-32P cell of Day1 bed board is removed in suction, and above-mentioned mouse femur marrow suspension cell is pressed 16 * 10
4/ hole bed board uses substratum to mend to volume of culture and is 1mL.After the dosing 37 ℃, 5%CO
2Cultivate under the condition.
Test and establish the vehicle group, the positive group of PTH (1-34), the HSA group, PTH (1-34) (RKK-QEL) organizes and little peptide (LQDVNFH) is organized.Establish 3 parallel multiple holes for every group.Administration concentration is 10-7mol/L.
Day5：
Cell in 24 orifice plates is partly changed liquid, inhale and remove the 0.5mL substratum, using fresh culture to supply 1mL and supply medicine is 10 to keep drug level
-7Mol/L.
Day7：
Repeat the 5th day operation, pair cell partly changes liquid.
Day9：
The cell culture fluid of 24 orifice plates is removed in suction, and with soft the washing once of PBS.
Attached cell is carried out TRACP (Phosphoric acid esterase of anti-the tartaic acid) dyeing (the TRACP/ALP staining kit is available from Takara), and concrete steps are following:
250 μ L Fixation solution fixed cells are added in the every hole of 24 orifice plates, and room temperature is placed 5min.
Every hole is removed after adding 2mL sterilized water dilution stationary liquid, and the 2mL sterile water wash is added once in every again hole, removes liquid.
Prepare and contain 10% tartaric ACP (acid phosphatase) substrate solution, 250 μ L are added in the every hole of 24 orifice plates, cover plate and are placed on 37 ℃ and hatch 15-45min.
Remove supernatant, with aseptic washing 3 times with color development stopping.
Microscopically calculates in every hole by the cell count of empurple (seeing accompanying drawing 4), and take pictures (seeing accompanying drawing 3).
Embodiment 4:PTH (1-34)-(RKK-QEL) is to the influence of former mouse femur marrow attached cell RANKL/OPG genetic transcription of being commissioned to train foster
With the digestion of the attached cell in the petridish among the Day3 among the embodiment 3, suitably count after the dilution with substratum.
Press (2) among the embodiment 2, (3), (4), the operation in (5) step, detect the influence of PTH (1-34)-(RKK-QEL) former mouse femur marrow attached cell RANKL/OPG genetic transcription of being commissioned to train foster.
Claims (14)
1. the aminoacid sequence of natural human Rat parathyroid hormone 1-34 (1-34) is SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF.
2. claim human parathyroid hormone (1-34) mutain has carried out the albumen of sudden change for the R25K26K27 to natural human Rat parathyroid hormone 1-34 (1-34).
3. be that the R25 of natural human Rat parathyroid hormone 1-34 (1-34) sports Q according to the human parathyroid hormone described in the claim 2 (1-34) mutain; K26 sports Q or E, and K27 sports the albumen of E or L, and wherein comparatively preferably R25 sports Q; K26 sports E, and K27 sports L.
4. be artificial chemosynthesis or recombinant expressed albumen according to the human parathyroid hormone described in the claim 3 (1-34) mutain.
5. according to claim 4 recombinant expressed for being the prokaryotic expression system of representative with intestinal bacteria or being the eukaryotic expression system and the mammalian cell expression system of representative with the yeast.
6. the cell that the external activity detection method of human parathyroid hormone according to claim 2 (1-34) mutain adopts is former be commissioned to train foster scleroblast or medullary cell, scleroblast system or scleroblast strain.
7. foster scleroblast or the medullary cell source of being commissioned to train, claim 6 Central Plains is mouse, comparatively preferably mouse femur medullary cell.
8. according to the scleroblast in the claim 6 system or scleroblast strain, the it is characterized in that transfection cell of PTH 1 receptor, comparatively preferably UAMS-32P clone.
9. the index of the active detection of the described human parathyroid hormone of claim 3 (1-34) mutain is the relative expression quantity of UAMS-32P cell specific gene mRNA and stimulates the mouse femur medullary cell to be differentiated to form the ability of osteoclast.
10. the special genes in according to Claim 8, it is characterized in that: coupling is unified into osteocyte, stroma cell and osteoclast differentiation, activation and bioactive major cytokine.
11. special genes in the claim 9, comparatively preferably protect ossein (osteoprotegerin, OPG), NF-κ B receptor activation factor part (receptor activator of nuclear factor κ B ligand, RANKL) 2 genes.
12. the relative expression quantity of mRNA in the claim 8, its detection method are reverse transcription PCR and real-time fluorescence quantitative PCR.Real-time fluorescence quantitative PCR comparatively preferably wherein.
13. it is to estimate through the dyeing to osteoclast that claim 9 moderate stimulation mouse femur medullary cell is differentiated to form the ability of osteoclast, the comparatively preferably Phosphoric acid esterase of anti-tartaic acid the (TRACP) dyeing.
14. claim 2 is used in the treatment osteoporosis agents with 3 described human parathyroid hormone (1-34) mutains.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586382A (en) * | 2014-10-21 | 2016-05-18 | 周亚伟 | Quality evaluation method of traditional Chinese medicines for resisting osteoporosis |
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|---|---|---|---|---|
| US5382658A (en) * | 1992-04-03 | 1995-01-17 | Allelix Biopharmaceuticals Inc. | Stability-enhanced variants of parathyroid hormone |
| CN1408865A (en) * | 2001-09-25 | 2003-04-09 | 中国人民解放军军事医学科学院生物工程研究所 | Oligonucleotide of coding human parathyroid hormone and its high efficiency expression method |
| CN1465703A (en) * | 2002-06-04 | 2004-01-07 | 北京双鹭药业股份有限公司 | Synthesis, expression, preparation and application for human parathyroid hormone gene mutant |
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2012
- 2012-01-05 CN CN2012100013197A patent/CN102775492A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5382658A (en) * | 1992-04-03 | 1995-01-17 | Allelix Biopharmaceuticals Inc. | Stability-enhanced variants of parathyroid hormone |
| CN1408865A (en) * | 2001-09-25 | 2003-04-09 | 中国人民解放军军事医学科学院生物工程研究所 | Oligonucleotide of coding human parathyroid hormone and its high efficiency expression method |
| CN1465703A (en) * | 2002-06-04 | 2004-01-07 | 北京双鹭药业股份有限公司 | Synthesis, expression, preparation and application for human parathyroid hormone gene mutant |
Non-Patent Citations (4)
| Title |
|---|
| 《Molecular and Cellular Endocrinology》 20000225 John F. Reidhaar-Olson等 Active variants of human parathyroid hormone (1-34) with multiple amino acid substitutions 135-147页 1-14 第160卷, * |
| 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 20021002 Qiang Fu等 Parathyroid Hormone Stimulates Receptor Activator of NFkappaB Ligand and Inhibits Osteoprotegerin Expression via Protein Kinase A Activation of cAMP-response Element-binding Protein 48868-48875页 1-14 第277卷, 第50期 * |
| JOHN F. REIDHAAR-OLSON等: "Active variants of human parathyroid hormone (1–34) with multiple amino acid substitutions", 《MOLECULAR AND CELLULAR ENDOCRINOLOGY》, vol. 160, 25 February 2000 (2000-02-25), pages 135 - 147 * |
| QIANG FU等: "Parathyroid Hormone Stimulates Receptor Activator of NFκB Ligand and Inhibits Osteoprotegerin Expression via Protein Kinase A Activation of cAMP-response Element-binding Protein", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》, vol. 277, no. 50, 2 October 2002 (2002-10-02), pages 48868 - 48875 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105586382A (en) * | 2014-10-21 | 2016-05-18 | 周亚伟 | Quality evaluation method of traditional Chinese medicines for resisting osteoporosis |
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