CN103012598B - OGP-rhLeptin fusion protein for treating osteoporosis and obesity and nucleic acid encoding same - Google Patents
OGP-rhLeptin fusion protein for treating osteoporosis and obesity and nucleic acid encoding same Download PDFInfo
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- CN103012598B CN103012598B CN201210585618.XA CN201210585618A CN103012598B CN 103012598 B CN103012598 B CN 103012598B CN 201210585618 A CN201210585618 A CN 201210585618A CN 103012598 B CN103012598 B CN 103012598B
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Abstract
The invention provides an OGP-rhLeptin fusion protein for treating osteoporosis and obesity and a nucleic acid encoding the same, relating to a fusion protein and a nucleic acid encoding the same and overcoming the defect that drugs for effectively treating osteoporosis and obesity at the same time do not exist at present. The amino acid sequence of the OGP-rhLeptin fusion protein for treating osteoporosis and obesity is shown in SEQ ID NO:2. The sequence of the nucleic acid of the fusion protein is shown in SEQ ID NO:1. The fusion protein can be used in the field of drugs.
Description
Technical field
The present invention relates to the nucleic acid of a kind of fusion rotein and this fusion rotein of encoding.
Background technology
Senile osteoporosis sickness rate is higher, and there are 200,000,000 sufferers of osteoporosis face in the whole world, and women is more than the male sex.According to the standard of the World Health Organization (WHO), American National health and nutrition survey (NHANES III, 1988 ~ 1994 years) result shows, osteoporosis has a strong impact on life of elderly person quality, more than 50 years old in crowd, can there is in life osteoporotic fracture theirs in 1/2 women, 1/5 the male sex, once patient experience osteoporotic fracture for the first time, the danger of secondary fracture obviously strengthens.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 Asia at that time, and the overwhelming majority is in China.There is scholar, to the year of 1995 ~ 1996 years U.S.'s osteoporosis, myocardial infarction, palsy and mammary cancer, number occurs and investigate demonstration, annual osteoporotic fracture 1,500,000 times, wherein vertebral fracture 700,000 times, the Wrist fracture 200,000 times of occurring, Hip Fracture 300,000 times, other fracture 300,000 times.
Fat show with the clinical observation of bone magnitude relation, fatly improve relevant with bone density (BMD).Obesity is the protection factor of bone amount, and after the postmenopausal women of Overweight and obesity, non-overweight women's bone amount more of the same age is high and bone loss is slower.Weight loss is the Hazard Factor of perimenopause bone loss, improves weight index and can slow down bone loss after menopause.But obesity also usually causes hyperglycemia, hypertension, hyperlipidemia, high blood viscosity, high lithemia, hyperinsulinemia, coronary heart disease, cerebral apoplexy, Varicose veins of lower extremity, fatty liver, cholelithiasis, sleep-apnea, diabetes, gout and sacroiliitis, is one of inducement of a lot of diseases.
Summary of the invention
The present invention should reduce obese person's body weight, makes it away from hyperglycemia, hypertension, hyperlipidemia, high blood viscosity, high lithemia, hyperinsulinemia, coronary heart disease, cerebral apoplexy, Varicose veins of lower extremity, fatty liver, cholelithiasis, sleep-apnea, diabetes, gout and sacroiliitis equivalent risk; Simultaneously increase again its bone amount, avoid osteoporotic generation, and provide a kind of for controlling osteoporosis and fat OGP-rhLeptin fusion rotein and the nucleic acid of this fusion rotein of encoding.
The present invention for the aminoacid sequence of controlling osteoporosis and fat OGP-rhLeptin fusion rotein as shown in SEQ ID NO:2.
The present invention above-mentioned for the nucleotide sequence of controlling osteoporosis and fat OGP-rhLeptin fusion rotein as shown in SEQ ID NO:1.
OGP-rhLeptin fusion rotein of the present invention adopts the Leptin of artificial reforming and the translation of sOGP genes encoding to form.There is the user of reduction body weight, and prevent and treat osteoporotic effect.
The present invention, for controlling osteoporosis and fat OGP-rhLeptin fusion rotein microbial preparation, does not produce 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 for the aminoacid sequence of controlling osteoporosis and fat OGP-rhLeptin fusion rotein as shown in SEQ ID NO:2.
Present embodiment for the nucleotide sequence of controlling osteoporosis and fat OGP-rhLeptin fusion rotein as shown in SEQ ID NO:1, this nucleotide sequence is synthesized by bio-engineering corporation, makes the plasmid vector pUC(OGP/Obese ' of the nucleic acid that contains fusion rotein).By pUC(OGP/Obese ') out (endonuclease reaction system is as shown in table 1 with BamH I and EcoR I double digestion for carrier, endonuclease reaction reacts 10h under 37 ℃ of conditions, then 1.5% agarose gel electrophoresis, object fragment is purified recovery with DNA GEL EXTRACTION KIT), and as shown in table 2 with BamHI and EcoR I double digestion pGEX-6P-1(endonuclease reaction system, endonuclease reaction reacts 10h under 37 ℃ of conditions, then 0.6% agarose gel electrophoresis, object fragment is purified recovery with DNA GEL EXTRACTION KIT); Again by present embodiment for the DAN fragment (OGP/Obese ') of controlling osteoporosis and fat OGP-rhLeptin fusion rotein and being connected of vector pGEX-6P-1 (ligation system is as shown in table 3) of passing through double digestion.
Table 1
Table 2
Table 3
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-OGP/Obese '; Then vector pGEX-OGP/Obese ' is transformed in e. coli bl21 (DE3), selection positive recombinant called after e. coli bl21 (DE3-OGP/Obese ').
On vector pGEX-6P-1, do not contain Kpn I restriction enzyme site, the synthetic pGEX-OGP/Obese ' of present embodiment all can open for Kpn I single endonuclease digestion, illustrates that present embodiment is used for controlling osteoporosis and fat OGP-rhLeptin antigen-4 fusion protein gene successfully imports plasmid pGEX-6P-1.
Random picking e. coli bl21 (DE3-OGP/Obese ') 37 ℃ of incubated overnight of bacterium colony, extract matter DNA, carry out single endonuclease digestion by Kpn I, and with corresponding blank plasmid pGEX-6P-1 in contrast, the plasmid that contains Kpn I restriction enzyme site is carried out to gene sequencing.Examining order entrusts biotech firm to carry out, and e. coli bl21 (DE3-OGP/Obese ') contains DNA shown in SEQ ID NO:1.
E. coli bl21 (DE3-OGP/Obese ') is placed under 30 ℃ of envrionment conditionss of LB substratum and cultivates 18h, then adopt GST tag fusion protein method to carry out the separation and purification of albumen, present embodiment is 98% for the purity of controlling osteoporosis and fat OGP-rhLeptin fusion rotein, and the expression amount of fusion rotein is 38.7%.
The experiment of present embodiment fusion rotein (OGP-rhLeptin fusion rotein) treatment osteoporosis:
Get female sd inbred rats and extract bilateral ovaries under aseptic condition, after 12 weeks, get 40 of survival healthy rats, be divided at random 5 groups (positive controls 1, positive controls 2, fusion rotein experimental group and negative control group), 8 every group.Separately get female sd inbred rats excision bilateral one fritter fat, after 12 weeks, get at random 8 of survival healthy rats as Sham-operated control group.Amount to 6 groups, respectively oral following medicine:
Sham-operated control group: the CMC-Na solution that mass concentration is 0.5%, gavage dosage is 5ml/kg;
Negative control group: the CMC-Na solution that mass concentration is 0.5%, gavage dosage is 5ml/kg;
Fusion rotein experimental group: the OGP-rhLeptin fusion rotein solution that mass concentration is 0.5%, gavage dosage is 5ml/kg; (the OGP-rhLeptin fusion rotein sterilized water that is used for the treatment of osteoporosis and thrombus of acquisition dissolves)
Positive controls 1: alendronate sodium (Alen) 5mg/kg;
Positive controls 2: the OGP protein solution that mass concentration is 0.5%, gavage dosage is 5ml/kg.
Positive controls 3: the rhLeptin solution that mass concentration is 0.5%, gavage dosage is 5ml/kg.
Successive administration three months, gets rat femur head after execution, immerse in 4% glutaraldehyde fixing, femoral head sagittal plane is cut with dentistry diamond saw, 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 osteoporosis therapy contrast and experiment, experimental result is in table 4, and result shows that fusion rotein experimental group, positive controls 1 and positive controls 2 all have the osteoporotic effect for the treatment of, and the effect of fusion rotein experimental group is best.
The comparison of table 4 bone trabecula width and surface of bone amass (X ± SD)
| Group | n | Dosage | Bone trabecula width X ± SD | Bone trabecula area X ± SD |
| Sham-operated control group | 8 | 5ml/kg | 112.40±15.31 | 0.6909±0.0502 |
| Negative control group | 8 | 5ml/kg | 51.30±16.8 | 0.5747±0.0559 |
| Positive controls 1 | 8 | 5ml/kg | 112.34±14.8 | 0.6589±0.0430 |
| Positive controls 2 | 8 | 5ml/kg | 115.48±13.4 | 0.6774±0.0475 |
| Positive controls 3 | 8 | 5ml/kg | 54.64±17.8 | 0.5784±0.0424 |
| Fusion rotein experimental group | 8 | 5ml/kg | 121.42±15.1? | 0.7118±0.0462 |
It is better that present embodiment fusion rotein (OGP-rhLeptin) is treated osteoporosis effect by intravenous administration.
Present embodiment fusion rotein (OGP-rhLeptin fusion rotein) body weight regulates experiment:
By 40 of female ob/ob mouse, take 10 as one group, be divided into 4 groups.Respectively injection Vehicle (0.7% physiological saline), rhLeptin, sCT-rhLeptin fusion rotein and VBT, every day 1mg, continue 28 days, record the body weight of mouse every day, experimental result is as shown in table 5.Present embodiment fusion rotein body weight regulating effect is the most obvious.
Table 5
| 0.7% physiological saline | rhLeptin | SCT-rhLeptin fusion rotein | VBT | |
| Original body weight (g) | 59.9±0.6 | 59.5±0.8 | 59.4±0.7 | 58.8±0.8 |
| Body weight after 28d (g) | 58.9±0.5 | 51.0±0.8 | 50.7±0.8 | 51.8±0.7 |
| Body weight changes (g) | -0.1 | -8.5 | -8.7 | -7.0 |
| P<d | 0.01 | 0.01 | 0.01 |
Present embodiment fusion rotein (OGP-rhLeptin fusion rotein) toxicity test:
Harbin Medical University's Experimental Animal Center provides SPF level kunming mice.Get mouse that 60 6 week age, male and female half and half, weights are 18 ± 2g 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, urine excrement is normal, hair color along white and glossy, activity freely, between reactive good, mouse without mutually baiting phenomenon.Raise 14 days and 30 days continuously, mouse does not all occur dead.
The administration of OGP-rhLeptin fusion rotein is after 14 days, 30 days, put to death mouse, pluck eyeball and get blood, 3000r/min, centrifugal 5 min, draw serum, use Beckman automatic clinical chemistry analyzer to detect Main Biochemical: 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
The administration of OGP-rhLeptin 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 statistics sample, in every group, randomly draw 7 mouse, core, liver, spleen, lung, kidney HE dyeing does 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 | Number of days | The heart | Liver | Spleen | Lung | Kidney |
| Control group | 14 | 3.59±0.63 | 32.11±2.37 | 4.46±0.90 | 2.71±0.92 | 7.72±0.93 |
| Experimental group | 14 | 3.48±0.89 | 30.61±3.21 | 4.68±0.73 | 2.92±0.79 | 7.71±0.88 |
| Control group | 30 | 3.61±0.67 | 32.12±2.22 | 4.59±0.88 | 2.92±0.63 | 7.85±0.78 |
| Experimental group | 30 | 3.53±0.88 | 30.66±3.18 | 4.48±0.61 | 2.95±0.58 | 7.84±0.81 |
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: respectively organize the heart, liver, spleen, lung, kidney and the marrow of mouse all without abnormal changes such as oedema, sex change, necrosis.
Pathological section and the bone marrow smear of this experiment to the heart, spleen, lung, kidney observed, and is showed no obvious damaging change.The organ coefficient statistical analysis of 5 kinds of internal organs is shown to each group difference is without significance.All illustrate that OGP-rhLeptin fusion rotein and meta-bolites thereof do not produce organic lesion to internal organs.The each group difference of Biochemistry test is without significantly illustrating that OGP-rhLeptin fusion rotein is to liver, the infringement of kidney non-functional.Show the good biocompatibility of OGP-rhLeptin fusion rotein, to mouse without acute toxicity, long term toxicity.
SOGP (OGP) is a kind of little peptide being made up of 14 amino acid, has the function such as skeletonization and hemopoietic in the external osteoblastic proliferation of promotion, body, and its aminoacid sequence and histone H
4c-terminal in full accord, with mouse T cell receptor β chain V district portion homologous, OGP can increase the NTx mrna expression of cell, increases the synthetic and calcium deposition of alkaline phosphatase activities, collagen.For the healing of fracture, osteoporosis, the prevention of acute and chronic anemia and treatment and the effect in the hematopoiesis of tumour Radiotherapy chemotherapy process moderate stimulation medullary cell thereof.
Leptin is the expression product of obese genes encoding, the secretory protein being formed by 167 amino acid being produced by white adipose cell and placenta, this albumen can be used as the input signal that affects energy balance, and its function is mainly to realize the effect of attenuating body weight by affecting energy intake and expenditure.Present embodiment has been carried out design again to obese gene and has been obtained Obese ' gene.Obese ' gene is by 105 based compositions, 35 the amino acid whose rhLeptin(Recombinant Human Leptin that encode, people's leptin of recombinating).
Present embodiment fusion rotein inserts GlyThr between OGP and rhLeptin, has changed the secondary structure of polypeptide, but does not only make OGP and rhLeptin 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 OGP and rhLeptin expression amount.
Present embodiment fusion rotein treatment osteoporosis and fat aspect effect also exceed single OGP or Leptin.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 present embodiment fusion rotein has security; And the change of secondary structure do not affect chromatography and purifying, present embodiment fusion rotein has separation and purification and is easy to feature.
Adopt gene engineering method production fermentation preparation OGP-rhLeptin fusion rotein to have production cost low, biological activity is high, and immunogenicity is low, active high, the advantage of long half time.
Medicine, reagent, enzyme, competent cell and the plasmids etc. that use in present embodiment are all bought acquisition, if without particular requirement concentration be product annotation concentration.
Embodiment two: present embodiment for the nucleotide sequence of controlling osteoporosis and fat OGP-rhLeptin fusion rotein as shown in SEQ ID NO:1.
Present embodiment is synthesized by bio-engineering corporation for the nucleotide sequence of controlling osteoporosis and fat OGP-rhLeptin fusion rotein.
Present embodiment has changed the gene of natural leptin (Leptin) for controlling the nucleic acid order of osteoporosis and fat OGP-rhLeptin fusion rotein, and between OGP and Obese ', insert Kpn I restriction enzyme site, both can improve the stability of coded fusion rotein, also change the secondary structure of fusion rotein, its biological property is improved simultaneously.
Present embodiment adds respectively BamHI and EcoR I restriction enzyme site in antigen-4 fusion protein gene both sides, and according to the feature of intestinal bacteria preference codon, has redesigned fusion gene coding base sequence.
Claims (2)
1. for controlling osteoporosis and fat OGP-rhLeptin fusion rotein, it is characterized in that aminoacid sequence for controlling osteoporosis and fat OGP-rhLeptin fusion rotein is as shown in SEQ ID NO:2.
2. coding claim 1, for controlling the nucleic acid of osteoporosis and fat OGP-rhLeptin fusion rotein, is characterized in that the sequence of this nucleic acid is as shown in SEQ ID NO:1.
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