CN102965327B - msCT-rhLeptin fusion protein transgenic engineering strain - Google Patents
msCT-rhLeptin fusion protein transgenic engineering strain Download PDFInfo
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- CN102965327B CN102965327B CN201210534879.9A CN201210534879A CN102965327B CN 102965327 B CN102965327 B CN 102965327B CN 201210534879 A CN201210534879 A CN 201210534879A CN 102965327 B CN102965327 B CN 102965327B
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Abstract
The invention discloses an msCT-rhLeptin fusion protein transgenic engineering strain and relates to a fusion protein transgenic engineering strain. The defect that no medicaments for simultaneously effectively treating osteoporosis and obesity exist in the prior art can be overcome. Bacillus coli BL21 (DE3-msCT/Obese') of the msCT-rhLeptin fusion protein transgenic engineering strain is prepared according to the following steps of: 1) obtaining a fusion protein DNA (deoxyribonucleic acid) fragment; 2) performing double digestion on a plasmid; 3) performing enzyme linkage; and 4) transforming the bacillus coli BL21. The msCT-rhLeptin fusion protein transgenic engineering strain disclosed by the invention can be used for the field of pharmaceutical preparation.
Description
Technical field
The present invention relates to a kind of fusion rotein transgenic engineering bacterial strain.
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 the year two thousand fifty 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 will solve and there is no at present simultaneously the effectively defect for the treatment of osteoporosis and fat medicine, and a kind of msCT-rhLeptin fusion rotein transgenic engineering bacterial strain providing.
MsCT-rhLeptin fusion rotein transgenic engineering bacterial strain is called e. coli bl21 (DE3-msCT/Obese '), e. coli bl21 (DE3-msCT/Obese ') preparation according to the following steps:
One, by the plasmid vector pUC(msCT/Obese ' that contains msCT-rhLeptin antigen-4 fusion protein gene) use BamH I and EcoR I double digestion, obtain the DNA fragmentation of msCT-rhLeptin fusion rotein;
Two, with BamHI and EcoR I double digestion plasmid pGEX-6P-1;
Three, the DNA fragmentation of msCT-rhLeptin fusion rotein with carry out enzyme through the vector pGEX-6P-1 of double digestion and be connected, then place to connect and spend the night for 16 DEG C, obtain vector pGEX-msCT/Obese ';
Four, transform e. coli bl21 (DE3) with vector pGEX-msCT/Obese ', select positive recombinant, obtain msCT-rhLeptin fusion rotein transgenic engineered bacteria e. coli bl21 (DE3-msCT/Obese ').
In the present invention, the gene order of msCT-rhLeptin fusion rotein is as shown in SEQ ID NO:1.
In the present invention, the aminoacid sequence of msCT-rhLeptin fusion rotein is as shown in SEQ ID NO:2.
MsCT-rhLeptin fusion rotein of the present invention adopts the Leptin of artificial reforming and the translation of salmon's calcitonin gene coding to form.There is the user of reduction body weight, and prevent and treat osteoporotic effect.
The present invention, for controlling osteoporosis and fat msCT-rhLeptin fusion rotein microbial preparation, does not produce antagonism reaction, uses safety.
MsCT-rhLeptin fusion rotein totally 70 amino acid that msCT-rhLeptin fusion rotein transgenic engineered bacteria e. coli bl21 of the present invention (DE3-msCT/Obese ') fermentation produces.The present invention adopts biological gene engineering means to obtain e. coli bl21 (DE3-msCT/Obese ').Adopt genetic engineering bacterium e. coli bl21 of the present invention (DE3-msCT/Obese ') to produce msCT-rhLeptin fusion rotein by large scale fermentation, there is wide application prospect.The present invention has established basic substance for treating osteoporosis and obesity.
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 msCT-rhLeptin fusion rotein transgenic engineering bacterial strain is called e. coli bl21 (DE3-msCT/Obese '), e. coli bl21 (DE3-msCT/Obese ') preparation according to the following steps:
One, by the plasmid vector pUC(msCT/Obese ' that contains msCT-rhLeptin antigen-4 fusion protein gene) with BamH I and EcoR I double digestion, obtain the DNA fragmentation (msCT/Obese ') of msCT-rhLeptin fusion rotein;
Two, with BamHI and EcoR I double digestion plasmid pGEX-6P-1;
Three, the DNA fragmentation of msCT-rhLeptin fusion rotein with carry out enzyme through the vector pGEX-6P-1 of double digestion and be connected, then place to connect and spend the night for 16 DEG C, obtain vector pGEX-msCT/Obese ';
Four, transform e. coli bl21 (DE3) with vector pGEX-msCT/Obese ', select positive recombinant, obtain msCT-rhLeptin fusion rotein transgenic engineered bacteria e. coli bl21 (DE3-msCT/Obese ').
Present embodiment step 4 adopts electric shock conversion method to transform e. coli bl21 (DE3).
Embodiment two: the difference of present embodiment and embodiment one is: plasmid vector pUC(msCT/Obese ' in step 1) endonuclease reaction system is
Other steps and parameter are identical with embodiment one.
Embodiment three: the difference of present embodiment and embodiment one or two is: in step 3, plasmid pGEX-6P-1 endonuclease reaction system is
Other steps and parameter are identical with embodiment one or two.
Embodiment four: the difference of present embodiment and embodiment one, two or three is: in step 2, enzyme ligation system is
Other steps and parameter are identical with embodiment one, two or three.
Embodiment five: the difference of one of present embodiment and embodiment one to four is: in step 1, the gene order of msCT-rhLeptin fusion rotein is as shown in SEQ ID NO:1.Other steps and parameter are identical with one of embodiment one to four.
The nucleotide sequence of present embodiment msCT-rhLeptin fusion rotein is synthesized by bio-engineering corporation, and is made up the plasmid vector pUC(msCT/Obese ' of the nucleic acid that contains fusion rotein of bio-engineering corporation).
Embodiment six: the difference of one of present embodiment and embodiment one to five is: in step 1, the aminoacid sequence of msCT-rhLeptin fusion rotein is as shown in SEQ ID NO:2.Other steps and parameter are identical with one of embodiment one to five.
Embodiment seven: present embodiment msCT-rhLeptin fusion rotein transgenic engineering bacterial strain is called e. coli bl21 (DE3-msCT/Obese '), e. coli bl21 (DE3-msCT/Obese ') preparation according to the following steps:
One, by the plasmid vector pUC(msCT/Obese ' that contains msCT-rhLeptin antigen-4 fusion protein gene) with BamH I and EcoR I double digestion, obtain the DNA fragmentation (msCT/Obese ') of msCT-rhLeptin fusion rotein;
Two, with BamHI and EcoR I double digestion plasmid pGEX-6P-1;
Three, the DNA fragmentation of msCT-rhLeptin fusion rotein with carry out enzyme through the vector pGEX-6P-1 of double digestion and be connected, then place to connect and spend the night for 16 DEG C, obtain vector pGEX-msCT/Obese ';
Four, transform e. coli bl21 (DE3) with vector pGEX-msCT/Obese ', select positive recombinant, obtain msCT-rhLeptin fusion rotein transgenic engineered bacteria e. coli bl21 (DE3-msCT/Obese ');
Wherein plasmid vector pUC(msCT/Obese ' in step 1) endonuclease reaction system is:
Wherein in step 3, plasmid pGEX-6P-1 endonuclease reaction system is:
Wherein in step 2, enzyme ligation system is:
Wherein in step 1 the gene order of msCT-rhLeptin fusion rotein as shown in SEQ ID NO:1; In step 1, the aminoacid sequence of msCT-rhLeptin fusion rotein is as shown in SEQ ID NO:2.
In present embodiment, the nucleotide sequence of msCT-rhLeptin fusion rotein is synthesized by bio-engineering corporation, and is made up the plasmid vector pUC(msCT/Obese ' of the nucleic acid that contains fusion rotein of bio-engineering corporation).Present embodiment has changed natural salmon calcitonin see calcimar (salmon calcitonin in the time building msCT-rhLeptin fusion rotein transgenic engineered bacteria e. coli bl21 (DE3-msCT/Obese '), the gene of gene sCT) and leptin (Leptin), and between msCT 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.And add 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, redesign fusion gene coding base sequence.
On vector pGEX-6P-1, do not contain Kpn I restriction enzyme site, the synthetic pGEX-msCT/Obese ' of present embodiment all can open for Kpn I single endonuclease digestion, illustrates that present embodiment msCT-rhLeptin fusion rotein successfully imports in plasmid pGEX-6P-1.Then plasmid pGEX-msCT/Obese ' is imported in e. coli bl21 (DE3), choose positive colony.
Random picking positive colony e. coli bl21 (DE3-msCT/Obese ') 37 DEG C 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-msCT/Obese ') contains DNA shown in SEQ ID NO:1.
E. coli bl21 (DE3-msCT/Obese ') is placed under 28 DEG C of envrionment conditionss of LB substratum and cultivates 15h, then adopt GST tag fusion protein purification process to carry out the separation and purification of albumen, the purity that present embodiment is used for the treatment of osteoporosis and fat fusion rotein is 98%, and the expression amount of fusion rotein is 39%.
The msCT-rhLeptin fusion rotein that utilizes present embodiment e. coli bl21 (DE3-msCT/Obese ') fermentation to obtain is tested:
The experiment of msCT-rhLeptin fusion rotein treatment osteoporosis:
Get female sd inbred rats, under aseptic condition, extract bilateral ovaries, wherein negative control group excision bilateral one fritter fat.After 5 days, get 48 of survival healthy rats, be divided at random 6 groups, 8 every group, oral following medicine respectively:
Model 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;
Experimental group: the msCT-rhLeptin fusion rotein solution that mass concentration is 0.5%, gavage dosage is 5ml/kg; (acquisition for controlling osteoporosis and fat msCT-rhLeptin fusion rotein aseptic aqueous solution dissolves)
Positive controls 1: alendronate sodium (Alen) 5mg/kg.
Positive controls 2: the msCT 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, respectively 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 1, and result shows that positive controls 1, positive controls 2, positive controls 3 and experimental group all have the osteoporotic effect for the treatment of, and the effect of experimental group is best.
The comparison of table 1 bone trabecula width and surface of bone amass (X ± SD)
| Group | n | Dosage | Bone trabecula width X ± SD | Bone trabecula area X ± SD |
| Model group | 8 | 5ml/kg | 50.40±13.7 | 0.3557±0.071 |
| Negative control group | 8 | 5ml/kg | 114.35±13.5 | 0.6382±0.0344 |
| Positive controls 1 | 8 | 5mg/kg | 111.35±14.7 | 0.6590±0.0422 |
| Positive controls 2 | 8 | 5ml/kg | 118.46±13.0 | 0.6882±0.0478 |
| Positive controls 3 | 8 | 5ml/kg | 54.34±17.27 | 0.5782±0.0413 |
| Experimental group | 8 | 5ml/kg | 126.69±15.8 | 0.7335±0.0417 |
MsCT-rhLeptin fusion rotein body weight regulates experiment:
By 40 of female ob/ob mouse, taking 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 2.Present embodiment fusion rotein body weight regulating effect is the most obvious.
Table 2
| ? | 0.7% physiological saline | rhLeptin | SCT-rhLeptin fusion rotein | VBT |
| Original body weight (g) | 57.6±0.6 | 59.8±0.7 | 60.2±0.7 | 57.9±0.8 |
| Body weight after 28d (g) | 56.6±0.5 | 51.3±0.7 | 51.1±0.6 | 50.9±0.8 |
| Body weight changes (g) | -0.1 | -8.5 | -9.1 | -7.0 |
| P<d | ? | 0.01 | 0.01 | 0.01 |
MsCT-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 msCT-rhLeptin fusion rotein is after 14 days, 30 days, put to death mouse, pluck eyeball and get blood, 3000r/min, centrifugal 5min, 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 3.
Table 3
The administration of msCT-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 4.
Table 4
| Group | Number of days | The heart | Liver | Spleen | Lung | Kidney |
| Control group | 14 | 3.59±0.60 | 32.02±2.28 | 4.33±0.90 | 2.72±0.93 | 7.63±0.90 |
| Experimental group | 14 | 3.51±0.86 | 30.52±3.27 | 4.49±0.74 | 2.88±0.73 | 7.62±0.65 |
| Control group | 30 | 3.63±0.63 | 32.01±2.15 | 4.36±0.75 | 2.89±0.61 | 7.75±0.80 |
| Experimental group | 30 | 3.55±0.70 | 30.55±3.27 | 4.29±0.62 | 2.92±0.79 | 7.78±0.65 |
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 msCT-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 msCT-rhLeptin fusion rotein is to liver, the infringement of kidney non-functional.Table msCT-rhLeptin fusion rotein msCT-rhLeptin fusion rotein good biocompatibility, to mouse without obviously acute toxicity, long term toxicity.
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 leucine of the 16th is replaced with L-Ala, deletes the tyrosine of the 22nd), the biological activity of improved msCT can reach 8600IU/mg.
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).
The msCT-rhLeptin fusion rotein that present embodiment msCT-rhLeptin fusion rotein transgenic engineered bacteria e. coli bl21 (DE3-msCT/Obese ') fermentation produces inserts GlyThr between msCT and rhLeptin, change the secondary structure of polypeptide, but not only do not make msCT and rhLeptin loss of biological activity, improved on the contrary its biological activity; And add Arg at fusion gene C-end, can remove the group of C-terminal amide.The expression amount of the interior msCT-rhLeptin fusion rotein of present embodiment e. coli bl21 (DE3-msCT/Obese ') is also high than single CTx or the expression amount of msCT in intestinal bacteria.
The fusion rotein that present embodiment e. coli bl21 (DE3-msCT/Obese ') produces treatment osteoporosis and fat aspect effect also exceed single msCT or CTx, and this fusion rotein can carry out administration by injection or oral mode, does not have first pass effect.
The change of present embodiment e. coli bl21 (DE3-msCT/Obese ') the fusion rotein secondary structure of producing does not produce toxicity in vivo, has security; And the change of secondary structure do not affect chromatography and purifying, the fusion rotein that produces has separation and purification and is easy to feature.
Adopt present embodiment e. coli bl21 (DE3-msCT/Obese ') fermentation preparation msCT-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.
Claims (5)
1.msCT-rhLeptin fusion rotein transgenic engineering bacterial strain, it is characterized in that msCT-rhLeptin fusion rotein transgenic engineering bacterial strain is called e. coli bl21 (DE3-msCT/Obese '), e. coli bl21 (DE3-msCT/Obese ') preparation according to the following steps:
One, by the plasmid vector pUC(msCT/Obese ' that contains msCT-rhLeptin antigen-4 fusion protein gene) use BamH I and EcoR I double digestion, obtain the DNA fragmentation of msCT-rhLeptin fusion rotein;
Two, with BamHI and EcoR I double digestion plasmid pGEX-6P-1;
Three, the DNA fragmentation of msCT-rhLeptin fusion rotein with carry out enzyme through the vector pGEX-6P-1 of double digestion and be connected, then place to connect and spend the night for 16 DEG C, obtain vector pGEX-msCT/Obese ';
Four, transform e. coli bl21 (DE3) with vector pGEX-msCT/Obese ', select positive recombinant, obtain msCT-rhLeptin fusion rotein transgenic engineered bacteria e. coli bl21 (DE3-msCT/Obese ');
In step 1, the gene order of msCT-rhLeptin fusion rotein is as shown in SEQ ID NO:1.
2. msCT-rhLeptin fusion rotein transgenic engineering bacterial strain according to claim 1, is characterized in that plasmid vector pUC(msCT/Obese ' in step 1) endonuclease reaction system is
3. msCT-rhLeptin fusion rotein transgenic engineering bacterial strain according to claim 1, is characterized in that in step 2, plasmid pGEX-6P-1 endonuclease reaction system is
4. msCT-rhLeptin fusion rotein transgenic engineering bacterial strain according to claim 1, is characterized in that in step 3, enzyme ligation system is
5. msCT-rhLeptin fusion rotein transgenic engineering bacterial strain according to claim 1, is characterized in that the aminoacid sequence of msCT-rhLeptin fusion rotein in step 1 is as shown in SEQ ID NO:2.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101319012A (en) * | 2008-07-21 | 2008-12-10 | 黑龙江大学 | Calcitonin-gene-related peptide and trout calcitonin amalgamation polypeptide |
| CN102292346A (en) * | 2009-01-22 | 2011-12-21 | 优尼金实验室公司 | Treatment for obesity |
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| CN101319012A (en) * | 2008-07-21 | 2008-12-10 | 黑龙江大学 | Calcitonin-gene-related peptide and trout calcitonin amalgamation polypeptide |
| CN102292346A (en) * | 2009-01-22 | 2011-12-21 | 优尼金实验室公司 | Treatment for obesity |
Non-Patent Citations (1)
| Title |
|---|
| 文辉才 等.基于人脂肪组织获取基因构建pET28a-Lepin（瘦素）重组子.《中国组织工程研究与临床康复》.2009,第13卷(第7期),1317-1319. * |
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