CN102964451A - msCT-CTx fusion protein for treating osteoporosis and reliving pain and nucleic acid encoding fusion protein - Google Patents

msCT-CTx fusion protein for treating osteoporosis and reliving pain and nucleic acid encoding fusion protein Download PDF

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CN102964451A
CN102964451A CN2012105339535A CN201210533953A CN102964451A CN 102964451 A CN102964451 A CN 102964451A CN 2012105339535 A CN2012105339535 A CN 2012105339535A CN 201210533953 A CN201210533953 A CN 201210533953A CN 102964451 A CN102964451 A CN 102964451A
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msct
ctx
fusion protein
fusion rotein
osteoporosis
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CN102964451B (en
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余琼
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Heilongjiang University
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Heilongjiang University
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Abstract

The invention discloses an msCT-CTx fusion protein for treating osteoporosis and reliving pain and a nucleic acid encoding the fusion protein, relating to a fusion protein and a nucleic acid encoding the fusion protein. The defect that no medicaments for simultaneously effectively treating osteoporosis and reliving pain exist in the prior art can be overcome. The amino acid sequence of the msCT-CTx fusion protein for treating osteoporosis and reliving pain is as shown in SEQ ID NO: 2. The sequence of the nucleic acid of the fusion protein is as shown in SEQ ID NO: 1. The msCT-CTx fusion protein disclosed by the invention can be used for the field of medicines.

Description

Be used for the treatment of the msCT-CTx fusion rotein of osteoporosis and pain relieving and the nucleic acid of this fusion rotein of encoding
Technical field
The present invention relates to the nucleic acid of a kind of fusion rotein and this fusion rotein of encoding.
Background technology
The senile osteoporosis sickness rate is higher, and there are 200,000,000 sufferers of osteoporosis face in the whole world, and the women is more than the male sex.Standard according to the World Health Organization (WHO), healthy and nutrition survey (the NHANES III of American National, 1988 ~ 1994 years) result shows, osteoporosis has a strong impact on the life of elderly person quality, more than 50 years old among the crowd, 1/2 women, 1/5 the male sex osteoporotic fracture can occur in life theirs, in case patient experience osteoporotic fracture for the first time, the dangerous of secondary fracture obviously strengthened.Chinese Aged occupy first place, the world, and existing patients with osteoporosis 9,000 ten thousand accounts for 7.1% of total population.Along with the process of social senilization, the sickness rate of osteoporosis is in rising trend, expects 2050 and will be increased to 2.21 hundred million, and whole world osteoporotic fracture over half will occur in the Asia at that time, and the overwhelming majority is in China.There is the scholar that the number that occurs in year of 1995 ~ 1996 years U.S.'s osteoporosis, myocardial infarction, palsy and mammary cancer is investigated demonstration, annual generation osteoporotic fracture 1,500,000 times, wherein vertebral fracture is 700,000 times, Wrist fracture 200,000 times, Hip Fracture 300,000 times, other is fractured 300,000 times.
When fracture occured, the initial impression of patient was pain.In fracture early treatment process, pain from start to finish all exists.It not only affects patient's result for the treatment of, also directly affects the functional rehabilitation of limbs simultaneously.All there be pain, dysfunction, limb swelling in various degree in dissimilar fracture, and pain is at first to consider the problem that solves.Because pain can cause sympathetic activation, reflectivity suppresses gastrointestinal function then, causes appetite stimulator; Pain also can affect sleep quality, causes the release of multiple hormone in the body, produces corresponding pathophysiological change, and Abwehrkraft des Koepers is descended, and directly affects wound healing and functional rehabilitation.Therefore the pain of removing fracture patient is the important component part for the treatment of.
If the patient takes multi-medicament treatment osteoporosis and pain relieving, produce easily the drug antagonism reaction; Will by considering the transformation period of medicine, also will consider medicine effective concentration on the other hand, and bring inconvenience to the patient medicine time on the one hand if stagger.
Lack at present a kind of medicine that can effectively treat osteoporosis and pain relieving simultaneously.
Summary of the invention
The present invention will solve and there is no at present simultaneously the effectively defective for the treatment of osteoporosis and pain relieving medicine, and a kind of msCT-CTx fusion rotein of osteoporosis and pain relieving and the nucleic acid of this fusion rotein of encoding of being used for the treatment of that provides.
The present invention is used for the treatment of the aminoacid sequence of msCT-CTx fusion rotein of osteoporosis and pain relieving shown in SEQ ID NO:2.
The present invention is above-mentioned to be used for the treatment of the nucleotide sequence of msCT-CTx fusion rotein of osteoporosis and pain relieving shown in SEQ ID NO:1.
Because fracture patient is suddenly and causes injury, without mental preparation, to add relevant knowledge and lack, therapeutic process is long; The patient often is passive position because of fear of pain, and particularly the senile fracture patient tend to cause in the future dysfunction, and decubation is longer.The present invention is used for the treatment of the msCT-CTx fusion rotein of osteoporosis and pain relieving can treat osteoporotic while pain relieving, patient treatment effect for fracture that osteoporosis causes is remarkable, and owing to can obviously alleviate or eliminate the affected part pain, the patients ' psychological burden is little, rehabilitation is fast, and the rear functional rehabilitation phase is short.
The present invention is used for the treatment of the msCT-CTx fusion rotein microbial preparation of osteoporosis and pain relieving, does not produce the antagonism reaction, uses safety.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment is used for the treatment of the aminoacid sequence of msCT-CTx fusion rotein of osteoporosis and pain relieving shown in SEQ ID NO:2.
Present embodiment is used for the treatment of the nucleotide sequence of msCT-CTx fusion rotein of osteoporosis and pain relieving shown in SEQ ID NO:1, and this nucleotide sequence is synthetic by bio-engineering corporation, makes the plasmid vector pUC(msCT/CTx of the nucleic acid that contains fusion rotein).With pUC(msCT/CTx) carrier BamH I and EcoR I double digestion out (the endonuclease reaction system is as shown in table 1), and as shown in table 2 with BamHI and EcoR I double digestion pGEX-6P-1(endonuclease reaction system); Again present embodiment is used for the treatment of osteoporosis and is connected the DAN fragment (msCT/CTx) of msCT-CTx fusion rotein and through be connected (the ligation system is as shown in table 3) of the vector pGEX-6P-1 of double digestion with pain relieving.
Table 1
Figure BDA0000257181321
Table 2
Figure BDA0000257181322
Table 3
Figure BDA0000257181323
The DAN fragment of fusion rotein is placed to be connected with 16 ℃ of vector pGEX-6P-1 and is spent the night, and obtains vector pGEX-msCT/CTx; Then vector pGEX-msCT/CTx is transformed in the e. coli bl21 (DE3), selects positive recombinant called after e. coli bl21 (DE3-msCT/CTx).
Do not contain Kpn I restriction enzyme site on vector pGEX-6P-1, the synthetic pGEX-msCT/CTx of present embodiment all can open for Kpn I single endonuclease digestion, illustrates that the msCT-CTx antigen-4 fusion protein gene that present embodiment is used for the treatment of osteoporosis and pain relieving successfully imports among the plasmid pGEX-6P-1.
37 ℃ of incubated overnight of picking e. coli bl21 (DE3-msCT/CTx) bacterium colony are extracted matter DNA at random, carry out single endonuclease digestion with the Kpn I, and with corresponding blank plasmid pGEX-6P-1 in contrast, the plasmid that will contain Kpn I restriction enzyme site carry out gene sequencing.Examining order entrusts biotech firm to carry out, and e. coli bl21 (DE3-msCT/CTx) contains DNA shown in the SEQ ID NO:1.
E. coli bl21 (DE3-msCT/CTx) placed under 30 ℃ of envrionment conditionss of LB substratum cultivate 18h, then adopt GST tag fusion protein method to carry out the separation and purification of albumen, the purity that present embodiment is used for the treatment of the msCT-CTx fusion rotein of osteoporosis and pain relieving is 98%, and the expression amount of fusion rotein is 38.7%.
The experiment of present embodiment fusion rotein (msCT-CTx fusion rotein) treatment osteoporosis:
Get female sd inbred rats, extract bilateral ovaries under the aseptic condition, negative control group excision bilateral one fritter fat.Get 40 of survival healthy rats after 5 days, be divided at random 5 groups, 8 every group, the oral following medicine of difference:
Negative control group (control): mass concentration is 0.5% CMC-Na solution, and gavage dosage is 5ml/kg;
Model group: mass concentration is 0.5% CMC-Na solution, and gavage dosage is 5ml/kg;
Experimental group: mass concentration is 0.5% msCT-CTx fusion rotein solution, and gavage dosage is 5ml/kg; (the msCT-CTx fusion rotein that is used for the treatment of osteoporosis and pain relieving of acquisition dissolves with aseptic aqueous solution)
Positive controls 1: alendronate sodium (Alen) 5mg/kg.
Positive controls 2: mass concentration is 0.5% msCT protein solution, and gavage dosage is 5ml/kg.
Successive administration three months is respectively got the rat femur head after the execution, immerse in 4% glutaraldehyde fixing, with the dentistry diamond saw femoral head sagittal plane is cut, get its a slice, through cleaning, 10% clorox soaks 6h, ultrasonic cleaning 15min, Gradient elution using ethanol, ether soaks, dry, ion sputtering film coating, SX-40 scanning electron microscopic observation, acceleration voltage 20kV.
Observe the osteoporosis therapy contrast and experiment, experimental result sees Table 4, and the result shows that experimental group, positive controls 1 and positive controls 2 all have the osteoporotic effect for the treatment of, and the effect of experimental group is best.
Long-pending (the X ± SD) of the comparison of table 4 bone trabecula width and surface of bone
Group n Dosage Bone trabecula width X ± SD Bone trabecula area X ± SD
Model group 8 5ml/kg 50.01±19.9 0.3848±0.0490?
Negative control group 8 5ml/kg 112.50±15.41 0.6911±0.0512
Positive controls 1 8 5mg/kg 113.46±17.8 0.6019±0.0483
Positive controls 2 8 5ml/kg 118.47±13.2 0.6884±0.0479
Experimental group 8 5ml/kg 125.68±15.9? 0.7169±0.0425
The experiment of present embodiment fusion rotein (msCT-CTx fusion rotein) hot-plate analgesia:
70 of the big or small close female sd inbred rats of picking are divided into arbitrarily 7 groups, 10 every group.Regulating hot plate temperature is 55 ± 0.5 ℃, puts rat on hot plate, measures the normal pain reaction of each rat.Lick metapedes or lift metapedes also later as standard take rat, the pain fault time is that 5 ~ 30s is normal, and 60s is maximum value.
The blank group gives equivalent physiological saline, positive controls 1 gives the morphine (25 μ g/kg) of low dosage, positive controls 2 gives the morphine (125 μ g/kg) of high dosage, positive controls 3 gives CTx(0.75 μ g/kg), the fusion rotein low dose group gives present embodiment msCT-CTx fusion rotein 0.25 μ g/kg, the dosage group gives present embodiment msCT-CTx fusion rotein 0.75 μ g/kg in the fusion rotein, and the fusion rotein high dose group gives present embodiment msCT-CTx fusion rotein 1.5 μ g/kg.0.5h, lh, 2h, 3h repeat said determination behind the medicine, the record pain sensation reaction times.The pain fault of different time is obtained each cell mean after the record administration, and experimental result is as shown in table 5.Experimental result explanation present embodiment msCT-CTx fusion rotein has excellent analgesic effect.
Table 5
Figure BDA0000257181324
Present embodiment fusion rotein (msCT-CTx fusion rotein) toxicity test:
Harbin Medical University's Experimental Animal Center provides SPF level kunming mice.Get 60 6 week age, male and female half and half, weights be that the mouse of 18 ± 2g is as experimental subjects.Adopt maximum tolerance administration (according to clinical Interferon, rabbit consumption 50ug/kg).Experimental group injection total dose 1mg/kg, once daily 0.02mL.Control group mice injection Isodose physiological saline.
Administration process small mouse is acted normally, feed, drinking-water is normal, the urine excrement is normal, hair color along white and glossy, activity freely, between reactive good, the mouse without mutually baiting phenomenon.Raised 14 days and 30 days continuously, mouse does not all occur dead.
The administration of msCT-CTx fusion rotein is after 14 days, 30 days, put to death mouse, pluck eyeball and get blood, 3000r/min, centrifugal 5 min, draw serum, detect Main Biochemical with the Beckman automatic clinical chemistry analyzer: aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), blood urea nitrogen (BUN), uric acid (URCA), total bilirubin (TBIL), TOTAL BILE ACID (TBA).Blood biochemistry index is as shown in table 6.
Table 6
Figure BDA0000257181325
The administration of msCT-CTx fusion rotein is put to death mouse after 14 days, 30 days, cores, liver, spleen, lung, kidney, observes internal organs color, form, calculates each organ coefficient.Estimate according to the statistics sample, in every group, randomly draw 7 mouse, core, liver, spleen, lung, kidney HE dyeing do histopathologic examination; Get myeloid tissue Wright's staining observation of cell and have or not oedema, sex change, necrosis etc.The pathological observation result of main organs is as shown in table 7.
Table 7
Group Fate The heart Liver Spleen Lung Kidney
Control group 14 3.60±0.58 32.11±2.25 4.40±0.78 2.68±0.88 7.75±0.84
Experimental group 14 3.51±0.80 30.71±3.24 4.64±0.69 2.92±0.84 7.74±0.79
Control group 30 3.64±0.56 32.22±2.35 4.53±0.54 2.91±0.72 7.88±0.69
Experimental group 30 3.56±0.86 30.76±3.08 4.44±0.64 2.92±0.60 7.90±0.70
The HE dyeing of experimental group and the control group main organs heart, liver, spleen, lung, kidney and marrow Wright dyeing paired observation are found: each organizes the heart, liver, spleen, lung, kidney and the marrow of mouse all without abnormal changes such as oedema, sex change, necrosis.
This experiment is observed pathological section and the bone marrow smear of the heart, spleen, lung, kidney, is showed no obvious damaging change.Organ coefficient statistical analysis to 5 kinds of internal organs draws each group difference without significance.Illustrate that all msCT-CTx fusion rotein and meta-bolites thereof do not produce organic lesion to internal organs.Each group difference of Biochemistry test is without illustrating that significantly the msCT-CTx fusion rotein is to liver, the infringement of kidney non-functional.Show preferably biocompatibility of msCT-CTx fusion rotein, to mouse without obviously acute toxicity, long term toxicity.
Conotoxin (conotoxin, CTx) is the active polypeptide that a class derives from the cone shell venom.Normally formed by 10 ~ 46 amino-acid residues; Molecular weight is little, and structure is various, is rich in halfcystine, disulfide linkage skeleton with high conservative, Main Function is in different kinds of ions passage and neurotransmitter receptors such as sodium, potassium, calcium, and the transmission of blocking-up or enhancing nervous excitation signal is a class polypeptide toxin of finding up to now the molecular weight minimum.The conotoxin that designs in the present embodiment is comprised of 25 amino-acid residues.
Present embodiment is transformed natural salmon calcitonin see calcimar (salmon calcitonin, sCT), improved msCT ［ Gly 8, Ala 16, del-Tyr 22(α-amino-isovaleric acid that sCT is the 8th becomes glycine, and the 16th leucine is replaced with L-Ala, deletes the 22nd tyrosine), the biological activity of improved msCT can reach 8600IU/mg.
The present embodiment fusion rotein inserts GlyThr between CTx and msCT, changed the secondary structure of polypeptide, but does not only make msCT and CTx loss of biological activity, has improved on the contrary its biological activity.Present embodiment is added Arg at fusion gene C-end, can remove the group of C-terminal amide.The expression amount of present embodiment fusion rotein in intestinal bacteria is also high than single msCT and CTx expression amount.
The effect of present embodiment fusion rotein aspect treatment osteoporosis and pain relieving also surpasses single msCT or CTx.The present embodiment fusion rotein can carry out administration by injection or oral mode, does not have first pass effect.
The change of secondary structure does not produce toxicity in vivo, and the present embodiment fusion rotein has security; And the change of secondary structure do not affect chromatography and purifying, and the present embodiment fusion rotein has separation and purification and is easy to characteristics.
Adopting gene engineering method to produce fermentation preparation msCT-CTx fusion rotein, to have a production cost low, and biological activity is high, and immunogenicity is low, and is active high, the advantage of long half time.
The medicine that uses in the present embodiment, reagent, enzyme, competent cell and plasmid etc. are all bought acquisition, if without particular requirement then concentration be product annotation concentration.
Present embodiment msCT-CTx fusion rotein also can be used for the treatment of the illnesss such as anxiety disorder, Parkinson's disease, muscular tone and hypertension except analgesic effect is arranged; The tool double effect is on the one hand blocked pain sensation transmission and the tool analgesic effect, suppresses on the other hand the exitotoxicity neurotransmitter and discharges, and stops stream in the neuronal cell pathologic calcium and neuroprotective is arranged.
Embodiment two: present embodiment is used for the treatment of the nucleotide sequence of msCT-CTx fusion rotein of osteoporosis and pain relieving shown in SEQ ID NO:1.
The nucleotide sequence that present embodiment is used for the treatment of the msCT-CTx fusion rotein of osteoporosis and pain relieving is synthesized by bio-engineering corporation.
Present embodiment is used for the treatment of in the nucleic acid order of msCT-CTx fusion rotein of osteoporosis and pain relieving and has changed natural salmon calcitonin see calcimar (salmon calcitonin, sCT) gene, and between msCT and CTx, insert Kpn I restriction enzyme site, both can improve the stability of coded fusion rotein, also change simultaneously the secondary structure of fusion rotein, its biological property is improved.
Present embodiment adds respectively BamHI and EcoR I restriction enzyme site in the antigen-4 fusion protein gene both sides, and according to the characteristics of intestinal bacteria preference codon, has redesigned fusion gene coding base sequence.
Figure IDA00002571813900021

Claims (2)

1. the msCT-CTx fusion rotein that is used for the treatment of osteoporosis and pain relieving is characterized in that being used for the treatment of the aminoacid sequence of msCT-CTx fusion rotein of osteoporosis and pain relieving shown in SEQ ID NO:2.
2. coding claim 1 is used for the treatment of the nucleic acid of the msCT-CTx fusion rotein of osteoporosis and pain relieving, it is characterized in that the sequence of this nucleic acid is shown in SEQ ID NO:1.
CN201210533953.5A 2012-12-12 2012-12-12 msCT-CTx fusion protein for treating osteoporosis and reliving pain and nucleic acid encoding fusion protein Expired - Fee Related CN102964451B (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1740193A (en) * 2005-09-15 2006-03-01 浙江大学 Non-amidated omega-conotoxin VIIA and its prepn process and application
CN101319012A (en) * 2008-07-21 2008-12-10 黑龙江大学 Calcitonin-gene-related peptide and trout calcitonin amalgamation polypeptide

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1740193A (en) * 2005-09-15 2006-03-01 浙江大学 Non-amidated omega-conotoxin VIIA and its prepn process and application
CN101319012A (en) * 2008-07-21 2008-12-10 黑龙江大学 Calcitonin-gene-related peptide and trout calcitonin amalgamation polypeptide

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