CN1380304A - N-terminal deletion lipocyte complement related protein and its preparation method - Google Patents
N-terminal deletion lipocyte complement related protein and its preparation method Download PDFInfo
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- CN1380304A CN1380304A CN 01126950 CN01126950A CN1380304A CN 1380304 A CN1380304 A CN 1380304A CN 01126950 CN01126950 CN 01126950 CN 01126950 A CN01126950 A CN 01126950A CN 1380304 A CN1380304 A CN 1380304A
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Abstract
The present invention belongs to the field of biological technology, and relates to a protein structure of new-type recombinant lipocyte complement related protein, and its preparation and application. According to the structure characteristics and action made of apMI said invention can obtain new-type N-terminal deletion lipocyte complement related protein structure. Said invention uses RT-PCR to obtain site-directed mutant N-terminal delation gene, then recombines it with prokaryotic expression vector pLY4 and transforms colibacillus JF 1125, so that said ivnention adopts three-step method to purify N-terminal delation lipocyte complement related protein. After having deen freeze-dried said obtained product can reduce blood sugar, blood-lipid and weight of body, so that it can be used for curing diabetes and adiposis.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of novel reorganization lipocyte complement related protein.More specifically, the present invention relates to N-terminal deletion lipocyte complement related protein and its production and application.
Background technology
Total length people's lipocyte complement related protein (adipose most abundanttranscript 1 is called for short apM1) can be by extracting in the human plasma, and its structure is divided into four parts: N-end signal peptide, the complementary district of no homology, the similar district of collagen and head-like structure territory district.Be a kind of newfound in recent years adipocyte specifically expressing excretory albumen that is, this gene is expressed in the adipocyte atomization can improve 100 times.Expression product and complement c1q have 40% homology, so be called complement related protein.There are some researches show that itself and ob gene product leptin have 35.7% and 48% similarity respectively on aminoacid sequence and protein sequence.Expression product is secreted in the blood plasma, and secretion process is subjected to the regulation and control of Regular Insulin.Norther Blot detects this gene of confirmation and only expresses in white adipose.In recent years, the researchist finds that this albumen significantly reduces in adiposis patient and obesity mice, give the intravenous injection of high fat diet obesity mice this albumen, can be under the situation that does not influence Regular Insulin and leptin level, and remarkable lowering blood glucose and blood lipid level.Other has research to be reported in the blood plasma and to detect the protein product that is about apM1 total length 70% with monoclonal antibody.
Summary of the invention
The purpose of this invention is to provide a kind of recombinant protein N-terminal deletion lipocyte complement related protein that has the lowering blood glucose and the two kinds of functions that lose weight concurrently, compare with the total length lipocyte complement related protein, this molecular weight of albumen is less, acts on more rapid.
The present invention is according to the constructional feature and the mode of action of apM1, prepare novel N-terminal deletion lipocyte complement related protein, it only is 50% sequence of total length that lipocyte complement related protein is lost in N-breakthrough of the present invention, and has blood fat reducing and blood sugar simultaneously, slimming effect.Preparation technology's handy and safe of the present invention utilizes genetic engineering means production, and products obtained therefrom can also reduce blood triglyceride effectively, rapidly except having the full length fragment function.
Total length apM1 contains 244 amino acid whose protein, and molecular weight is about 37KD, and the site that mainly plays a role is at globular domain.The present invention has designed the deletant that contains globular domain, and Phe176 is replaced with Asp 176, has increased polarity, the performance that helps acting on.Table 1 is sequence table SEQ ID NO:1, shows the aminoacid sequence of N-terminal deletion lipocyte complement related protein of the present invention.
Table 2 is sequence table SEQ ID NO:2, shows the nucleotide sequence of N-terminal deletion lipocyte complement related protein of the present invention.
Another object of the present invention provides a kind of production method of the present invention, the cDNA fragment that comprises clones coding N-terminal deletion lipocyte complement related protein sequence part, with carrier reorganization back transformed host cell, carry out large scale culturing under suitable condition, from host cell, reclaim and purifying N-terminal deletion lipocyte complement related protein.
The preparation of N-terminal deletion lipocyte complement related protein comprises RT-PCR clone and rite-directed mutagenesis gene fragment among the present invention, recombinate with plasmid pLY4, DNA digestion with restriction enzyme evaluation and screening positive colony, whether nucleotide sequence analysis checking fragment sequence and connection be correct.
With cDNA fragment of the present invention and expression vector reorganization, form expression plasmid.The invention is not restricted to specific expression vector,, form suitable plasmid of expressing as long as can recombinate with described cDNA fragment.As pLY4 and pET32 (+), in a preferred embodiment, the present invention uses prokaryotic expression carrier pLY4.
Above-mentioned recombinant expression vector imports suitable host cell according to a conventional method.The present invention is not limited to any specific host cell, as long as it can express described recombinant expression vector.As intestinal bacteria JF1125 and e. coli bl21, in an embodiment preferred, the present invention uses intestinal bacteria JF1125.
Expression product of the present invention only need carry out ultrasonic broken bacterium in inclusion body, sex change renaturation, promptly separable and purifying desired product.
All basic molecular biology operations are all with reference to " molecular cloning experiment guide " in the above technical scheme.
Comparison shows that with total length lipocyte complement related protein character N-end deletant lowering blood glucose of the present invention and blood fat ability are eager to excel in whatever one does than total length.
Embodiment
The design of embodiment 1.N-terminal deletion lipocyte complement related protein, preparation and character are identified:
(1) structure of the clone of N-terminal deletion lipocyte complement related protein gene and prokaryotic expression plasmid gapM1-pLY4
According to the design amino-acid sequence of N-terminal deletion lipocyte complement related protein, according to colibacillary preference codon design nucleotide sequence.The design primer clones gene and carries out rite-directed mutagenesis with RT-PCR.With the pUC19 reorganization, change e. coli jm109 over to earlier, enzymolysis screening positive clone, nucleotide sequence analysis confirm that gene order is identical with design.The purpose fragment is cut out again, with the pLY4 reorganization, constitute prokaryotic expression carrier gapM1-pLY4, change intestinal bacteria JF1125 over to, enzymolysis screening positive clone, nucleotide sequence analysis confirm that gene order is identical with design.Confirm to obtain positive colony.
Restriction enzyme is available from BRL company, and e. coli jm109, JF1125, plasmid pLY4, pUC19 are that preserve this chamber.
(2) gapM1 Prokaryotic Expression
Several positive colony bacterium colonies of picking, 30 ℃ of shaking culture are spent the night in LB solution, and then, with M9CA substratum 1: 50 dilution, 30 ℃ of shaking culture are to be warming up to 42 ℃, abduction delivering 3 hours at 0.6 o'clock to A600.After inducing end, centrifugal collection bacterium with PBS (PH=7.4) washing 2 times, is analyzed expression product with SDS-PAGE, and after the Xylene Brilliant Cyanine G R-250 dyeing, purity, molecular weight are decided in Pharmacia Imagenaster VDS scanning.As seen induce the back supernatant liquor band that concentrates to be arranged at the about 16KD of molecular weight place.Through scanning, target protein accounts for 30% of bacterial protein.Screen high gapM1 cloning by expression.SDS-PAGE is undertaken by the Laemmli method.
(3) the abduction delivering engineering bacteria is chosen the high expression level bacterial strain, carries out the low density fermentation with 5 liters of fermentor tanks, collects thalline behind the stability abduction delivering, the PBS washing, and-70 ℃ of preservations are stand-by.Fermentation parameter: 30 ℃, PH6.8, dissolved oxygen 60%, stirring velocity is with the oxygen level interlock, is to be warming up to 42 ℃ of abduction deliverings at 2 o'clock to A600.
(4) separation of inclusion body, dissolving and renaturation
Wet bacterium suspends with the PB damping fluid, presses with the high-pressure homogenization pump pressure.After centrifugal, the proteic existence of SDS-PAGE analysis purposes.The result shows that target protein is present in the inclusion body precipitation.
Squeeze centrifugal gained precipitation through washings (0.05M PB, PH7.4,0.1M NaCl) washing three times.After centrifugal, with lysate [0.05MPB (PH7.4), 8M urea, 2.5% sucrose, 0.5% beta-mercaptoethanol] dissolving, room temperature effect 2 hours is to clear.Abandon precipitation behind the ultracentrifugation, get the supernatant dilution refolding.Renaturation solution is 0.05M PB, PH7.4,0.1M NaCl, 2.5% sucrose, 0.5% beta-mercaptoethanol.
(5) gel-filtration
Sephadex G-50 post (Phamacis company) with the direct upper prop of dilution refolding liquid, is used the PB wash-out after using 20mmol/L PB (Ph7.4) balance then, and flow velocity 10ml/min collects active peak.
(6) S-Sepharose Fast Flow column chromatography
With 50mmol/LPB (Ph6.0) the balance S-Sepharose Fast Flow post of 10 times of volumes, with the significant part upper prop of collecting after the gel-filtration, Waters chromatographic instrument control flow velocity and detection protein peak.Behind the end of the sample, be washed till baseline with 50mmol/L PB (pH6.0) damping fluid, 0-1M NaCl gradient elution is collected elution fraction, and SDS-PAGE analysis purposes albumen distributes, with target protein packing lyophilize, and-40 ℃ of preservations.
(7) purity is identified and molecular weight determination:
Sample carries out the 15%SDS-PAGE electrophoresis, and after the Xylene Brilliant Cyanine G R-250 dyeing, purity, molecular weight are decided in Pharmacia Imagenaster VDS scanning.Products obtained therefrom purity is more than 93%, the about 16KD of molecular weight.
(8) measure short Fatty Acid Oxidation activity
With the C2C12 cell in differentiation liquid (DMEM, 2.5% horse serum) cultivated seven days in, add target protein and changed preincubation liquid (MEM in preceding 1 hour into, 3mM/L glucose, the 4mM glutamine synthetase, 25mM Hepes, the BSA of 1% no free fatty acid, 0.25mM oleate, 5ug/ml Gentamycin).Experiment begins to add C
-14The target protein of the oleic acid of mark and 5ml purifying and full-length proteins were hatched 90 minutes.Measure C in the solution
-14The CO of mark
2Content.The result shows that N-end deletant is short Fatty Acid Oxidation specific activity full-length proteins high about 30% in the time of 90 minutes.The application of embodiment 2 N-terminal deletion lipocyte complement related proteins:
Use that the prepared N-terminal deletion lipocyte complement related protein of the present invention is lost weight and the experiment of lowering blood glucose, confirm that it has important effect in the control of fat and type ii diabetes.
(1) reduces body weight
Form the obesity mice model June for 3 monthly age C57BL/6J mouse high fat diets.Control group gives physiological saline and continues high fat diet, and gapM1 treatment group vein gives 25ugN-terminal deletion lipocyte complement related protein, and once a day, total length treatment group vein gives full-length proteins 25ug, all continues high fat diet.After 16 days, treatment group body weight begins obvious reduction.Table 3 is fed the influence of the body weight of raising the back mouse to high fat diet for gapM1.
Table 3
| Group | The mouse number | Original body weight (g) | Body weight after 16 days (g) | Decline per-cent | The P value |
| Control group | ????8 | ????51.2±4.5 | ????51.0±4.2 | ????0.001％ | ＞0.05 |
| GapM1 treatment group | ????8 | ????54.3±3.2 | ????50.23±2.8 | ????7.5％ | 0.001 |
| Total length treatment group | ????8 | ????51.9±4.1 | ????50.04±3.8 | ????3.2％ | 0.025 |
(2) lowering blood glucose, blood free fatty acid and blood triglyceride effect
Give the high fat diet of C57BL/6J mouse, the quiet notes physiological saline of while control group (n=8), the administration group (n=8) give gapM1 25ug, surveys mouse blood sugar, blood free fatty acid and blood triglyceride levels respectively at 45min and 105min.The result shows, gapM1 can significantly reduce blood sugar after the meals, blood free fatty acid and blood triglyceride levels (p＜0.05) and see Table 2.Blood sugar, blood free fatty acid and blood triglyceride detect uses Sigma company blood glucose, blood free fatty acid and the detection of blood triglyceride detection kit.Table 4 be gapM1 to meals after the effect of blood sugar, blood free fatty acid and blood triglyceride
Table 4
| Group | ?45min | ??105min | |
| Blood sugar (mg/dl) | Control group | ?200±7.5 | ??240±2.5 |
| The medication group | ?2004±7.5 | ??175±6.5 | |
| Blood free fatty acid (Mm) | Control group | ?0.94±0.033 | ??1.1±0.03 |
| The medication group | ?0.84±0.025 | ??0.9±0.024 | |
| Blood triglyceride (mg/dl) | Control group | ?130±5 | ??140±16 |
| The medication group | ?140±3 | ??100±4 |
Need not further to elaborate, believe and adopt the disclosed content in front, those skilled in the art can use the present invention to greatest extent.Therefore, the preferred specific embodiments of front should be understood that only to illustrate, but not limits the scope of the invention by any way.
From the above description, those skilled in the art can understand essential characteristic of the present invention at an easy rate, and under the situation that does not depart from its purport and scope, can carry out various changes and improvement to the present invention.
Table 1 SEQ ID NO:1
1?????????????????????????????????????????30
AYVYRSAFSVGLETYVTIPNMPIRFTKIFY
31????????????????????????????????????????60
NQQNHYDGSTGKFHCNIPGLYYFAYHITVY
61????????????????????????????????????????90
MKDVKVSLDKKDKAMLFTYDQYQENNVDQ
91????????????????????????????????????????120
ASGSVLLHLEVGDQVWLQVYGEGERNGLYA
121
DNDNDSTFTGFLLYHDTN
Table 2 SEQ ID NO:2GCC TAT GTA TAC CGC TCA GCA TTC AGT GTG GGA TTG GAG ACT TAC GTTACT ATC CCC AAC ATG CCC ATT CGC TTT ACC AAG ATC TTC TAC AAT CAGCAA AAC CAC TAT GAT GGC TCC ACT GGT AAA TTC CAC TGC AAC ATT CCTGGG CTG TAC TAC TTT GCC TAC CAC ATC ACA GTC TAT ATG AAG GAT GTGAAG GTC AGC CTC GAC AAG AAG GAC AAG GCT ATG CTC TTC ACC TAT GATCAG TAC CAG GAA AAT AAT GTG GAC CAG GCC TCC GGC TCT GTG CTC CTGCAT CTG GAG GTG GGC GAC CAA GTC TGG CTC CAG GTG TAT GGG GAA GGAGAG CGT AAT GGA CTC TAT GCT GAT AAT GAC AAT GAC TCC ACC TTC ACAGGC TTT CTT CTC TAC CAT GAC ACC AAC
Claims (11)
1. novel recombinant human N-terminal deletion lipocyte complement related protein is characterized in that changing the structure of people's total length lipocyte complement related protein, has the aminoacid sequence of SEQ ID NO:1 and the nucleotide sequence of SEQ ID NO:2.
2. recombinant human N-terminal deletion lipocyte complement related protein according to claim 1, it is characterized in that its molecular weight ratio full-length proteins of described reorganization associated protein is little, its structural modification is that Phe176 is replaced with Asp176, has changed 176 amino acids polarity.
3. a method for preparing Novel Human N-terminal deletion lipocyte complement related protein is characterized in that adopting the following step: (1) design Novel Human N-terminal deletion lipocyte complement related protein molecular structure; (2) expressing human N-terminal deletion lipocyte complement related protein gene in the carrier that is suitable for expressing; (3) host cell that is suitable for expressing with the recombinant vectors transfection; (4) cultivate host cell being suitable for expressing under the segmental condition of this cDNA, therefrom reclaim and the required product of purifying.
4. method according to claim 3, wherein employed carrier is not limited to specific expression vector, as long as it can be recombinated with described cDNA fragment, forms suitable plasmid of expressing.
5. method according to claim 4, wherein employed carrier is a prokaryotic expression carrier.
6. method according to claim 5, wherein employed carrier is pLY4.
7. method according to claim 3, wherein employed host cell is not limited to specific host cell, as long as it can express described recombinant expression vector.
8. method according to claim 7, use therein host cell are intestinal bacteria JF1125.
9. method according to claim 3, the method for wherein cultivating the host bacterium is a fermentation process, with 30 ℃ of amplifications of low nutritional medium engineering bacterias, reaches 42 ℃ of abduction deliverings behind certain cell concentration.Fermentation parameter is: 42 ℃ of temperature, and oxygen capacity is controlled between 33-40%, and PH is 5, stirring velocity and OD interlock.
10. method according to claim 3, wherein the final product of recombinant human N-terminal deletion lipocyte complement related protein is by homogenate behind the engineering bacterium fermentation, obtains through sex change renaturation, gel-filtration, ion exchange method purifying.
11. according to the application of the fatty complement related protein of claim 1-3 described people N-end disappearance in preparation control obesity and diabetes disease medicament.
SEQ?ID?NO：?1
1???????????????????????????????????????30
AYVYRSAFSVGLETYVTIPNMPIRFTKIFY
31??????????????????????????????????????60
NQQNHYDGSTGKFHCNIPGLYYFAYHITVY
61??????????????????????????????????????90
MKDVKVSLDKKDKAMLFTYDQYQENNVDQ
91??????????????????????????????????????120
ASGSVLLHLEVGDQVWLQVYGEGERNGLYA
121
DNDNDSTFTGFLLYHDTN
SEQ?ID?NO：2GCC?TAT?GTA?TAC?CGC?TCA?GCA?TTC?AGT?GTG?GGA?TTG?GAG?ACT?TAC?GTTACT?ATC?CCC?AAC?ATG?CCC?ATT?CGC?TTT?ACC?AAG?ATC?TTC?TAC?AAT?CAGCAA?AAC?CAC?TAT?GAT?GGC?TCC?ACT?GGT?AAA?TTC?CAC?TGC?AAC?ATT?CCTGGG?CTG?TAC?TAC?TTT?GCC?TAC?CAC?ATC?ACA?GTC?TAT?ATG?AAG?GAT?GTGAAG?GTC?AGC?CTC?GAC?AAG?AAG?GAC?AAG?GCT?ATG?CTC?TTC?ACC?TAT?GATCAG?TAC?CAG?GAA?AAT?AAT?GTG?GAC?CAG?GCC?TCC?GGC?TCT?GTG?CTC?CTGCAT?CTG?GAG?GTG?GGC?GAC?CAA?GTC?TGG?CTC?CAG?GTG?TAT?GGG?GAA?GGAGAG?CGT?AAT?GGA?CTC?TAT?GCT?GAT?AAT?GAC?AAT?GAC?TCC?ACC?TTC?ACAGGC?TTT?CTT?CTC?TAC?CAT?GAC?ACC?AAC
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1740195B (en) * | 2005-04-15 | 2012-01-04 | 复旦大学 | Polypeptide gapM1 and its prepn process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1740195B (en) * | 2005-04-15 | 2012-01-04 | 复旦大学 | Polypeptide gapM1 and its prepn process |
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