CN1737150A - Adiponectin-Glucagon-like peptide-1-like peptide recombinant protein expression vector and construction - Google Patents
Adiponectin-Glucagon-like peptide-1-like peptide recombinant protein expression vector and construction Download PDFInfo
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Abstract
Disclosed is an adiponectin-Glucagon-like peptide-1-like peptide recombinant protein expression vector and construction, wherein adiponectin and GLP-1-A encoded gene are obtained by utilizing PCR technology, the method comprises obtaining adiponectin through amplification of adiponectin spherical domain gene, then obtaining GLP-1-A encoded gene by mutating codons encoding No. 8 amino acid alanine in GLP-1-(7-37) encoded gene, then cloning GLP-1-A and adiponectin encoded genes directionally to expression vector by utilizing enzyme digestion technology, obtaining adiponectin-glucagon-like peptide-1 analogue peptide fusion protein expression vector, finally expressing adiponectin-GLP-1-A fusion protein through transforming protein expression bacteria.
Description
Technical field
The present invention relates to biological field, relate in particular to similar peptide fusion protein expression vector of adiponectin-Glucagon-like peptide-1 and construction process.
Background technology
(Diabetes Mellitus DM) is the disease of serious harm human health to diabetes B, and number of patients is just along with growth in the living standard, aging population, and improving of diagnostic techniques and increasing sharply.In recent years studies show that insulin resistant and hypoinsulinism are the distinctive pathophysiological changes of diabetes B, and the two-way interaction finally causes the generation of diabetes B.Moreover, insulin resistant has also participated in generation, the development of chronic complicating diseases of diabetes such as cardiovascular complication, diabetic nephropathy etc., is an important pivot of contact diabetes, hypertension, hyperlipidaemia, coronary heart disease.Therefore at the pathogenesis of diabetes B, adopt effective medicine to alleviate insulin resistant, increase insulin secretion and have great importance for the treatment of diabetes B and complication thereof.Still do not have at present effective medicine can be simultaneously at both insulin resistant and hypoinsulinism were treated aspect pathogenetic two of the diabetes B.Although the orally-taken blood sugar reducing medicine of euglycemic agent such as thiazolidinediones medicine and numerous species has been arranged to be used clinically, if but these drug mains are treated at diabetes B pathogenetic in a certain respect (merely at insulin resistant or hypoinsulinism or act on sugar absorb or metabolic a certain link), and they improve insulin resistant respectively, promote insulin secretion, the ability of lowering blood glucose is also not fully up to expectations, prolonged application effect meeting reduces gradually, the side effect that these medicines had has simultaneously also greatly limited their application clinically.Even Regular Insulin, also because the blood sugar reducing function that exists of insulin resistant reduces greatly, heavy dose of application Regular Insulin also may increase the weight of the development of chronic complicating diseases of diabetes.Given this, seeking the new medicine that improves insulin resistant, promotion insulin secretion in the world energetically, in the hope for the treatment of diabetes B more effective, safely.
Adiponectin (Adiponectin) is the adipocyte excretory specific proteins of finding in recent years, its diacrisis plays an important role in the generation of insulin resistant and diabetes B, development, and Serum adiponectin concentration can be predicted the risk of the individual insulin resistant that takes place in the future.Diabetes B patient blood plasma adiponectin level obviously descends, perspective longitudinal research shows, basis blood plasma adiponectin concentration is the independent risk factor of diabetes B morbidity, the blood plasma adiponectin prompting of high density has less diabetes B onset risk, to develop into the individuality of diabetes B afterwards, its blood plasma adiponectin concentration significantly is lower than control group, still continues after the diabetes B morbidity to descend, and parallels with the susceptibility decline of Regular Insulin.Apm 1 gene may be the tumor susceptibility gene of diabetes B, and the homozygote mouse that apm 1 gene lacks (Adipo-/-mouse) shows the insulin resistant and the impaired glucose tolerance of moderate.These studies show that unusual generation, the development that has participated in insulin resistant and diabetes B that adiponectin produces, and can significantly improve insulin resistant, lowering blood glucose with the adiponectin of recombinating after to diabetes and insulin resistant treatment of animals.Adiponectin does not stimulate insulin secretion, but strengthens Regular Insulin to the glycogenetic inhibition of liver cell grape, makes the Regular Insulin of inferior physiological concentration can suppress glycogen output equally.Adiponectin also can reduce mouse circulation free fatty acids and the triglyceride levels that high fat high-sucrose is fed, alleviate the mouse body weight, increase picked-up, oxidation and the energy expenditure of Skeletal Muscle Cell to free fatty acids, the skeletal muscle content of triglyceride is reduced, thereby the conduction of enhances skeletal myocyte's insulin signaling increases insulin sensitivity.Adiponectin participates in regulating outside sugar, the lipid metabolism except improving insulin resistant, goes back the atherosclerotic formation of antagonism.Low-level blood plasma adiponectin is the independent risk factor that cardiovascular disorder takes place the diabetic subject.In addition, the diabetes B patient's of companion's diabetic retinopathy blood plasma adiponectin concentration significantly is lower than the diabetes B patient who does not accompany retinopathy, and the minimizing that the prompting adiponectin produces also may participate in generation, the development of diabetic microvascular complication.
In a word, adiponectin has hypoglycemic, lipopenicillinase, increase the effect of insulin sensitivity, but the atherosclerotic formation of antagonism can not make weight increase simultaneously, does not find the existence of adiponectin opposing in human body, and adiponectin is the albumen that body self produces, human body almost is free from side effects, thus adiponectin might become the treatment diabetes a kind new medicine, energetically it is researched and developed in the world.Human adiponectin is totally 244 amino acid, holds the C-end to be followed successively by signal sequence (1-18aa), unspiralized region (19-41aa) from N-, 22 collagen tumor-necrosis factor glycoproteinss (42-107aa), C-holds spherical territory, and (be the spherical territory of adiponectin, 108-244aa), the spherical territory of adiponectin is its functional domain.Have the investigator to obtain containing the segment in the spherical territory of adiponectin with the adiponectin of trypsinase cracking total length, its activity is much larger than the adiponectin of total length.Studies show that the spherical territory of the adiponectin of bacterial expression has good biological activity, this provides advantageous conditions for further researching and developing adiponectin.
Glucagon-like peptide-1 (Glucagons-like Peptide-1, GLP-1) be a kind of hormone of alpha Cell of islet and intestinal mucosa L emiocytosis, alpha Cell of islet and intestinal mucosa L cell produce pancreas earlier and rise sugared plain former, further become the GLP-1 (7-37) of biologically active through hydrolysis, an amino acid of wherein a part of molecule C-end is decomposed, and surplus end is turned to GLP-1 (7-36) acid amides of biologically active by acid amides.80% GLP-1 exists with the form of GLP-1 (7-36) acid amides in vivo, and the form with GLP-1 (7-37) exists on a small quantity, but both physiologically actives are identical.The active territory of GLP-1 is positioned at the N-end, can with the GLP-1 receptors bind on the beta Cell of islet, increase the activity of the sweet cyclase of acid of islet cells inner gland, promote the release of Regular Insulin, its effect is to rely on glucose, when blood sugar increasing, venoclysis GLP-1 can obviously increase insulin secretion, along with the decline of glucose level, and the disappearance that glucose stimulates, GLP-1 promotes the effect of insulin secretion to diminish, and continues infusion GLP-1 blood sugar and can not continue to descend.The diabetes B patient, the GLP-1 of diet induced secretion is impaired, causes the deterioration of beta Cell of islet excreting insulin, thereby causes the generation of diabetes B.GLP-1 also has increases the synthetic of liver starch and muscle glycogen, promotes lipid acid synthetic, suppresses pancreas and rises the plain secretion of sugar, suppresses digestive tract power, delay the absorption of food, suppress the islet cells apoptosis, promote islet cell hyperplasia, depress appetite loses weight, and delays the effects such as generation of diabetes.
Because GLP-1 has multiple pharmacological effect, is trying out the treatment in diabetes B.Studies show that the exogenous GLP-1 of input can obviously improve diabetes B patient empty stomach and postprandial blood sugar, thereby reduce the consumption of Regular Insulin, even inactive Regular Insulin, and hypoglycemia can not take place, long-term infusion GLP-1 can significantly increase by 1 secretion mutually of diabetes B patient Regular Insulin.But GLP-1 is the triglyceride reducing level also, increases the low-density lipoprotein particle volume, improves the endothelial function disorder of diabetes B companion patients with coronary heart disease, delays generation, the development of diabetes B cardiovascular complication.Although GLP-1 has lot of advantages aspect the treatment diabetes B, but remain the shortcoming that is being difficult to overcome, the transformation period is very short in the body of GLP-1, the transformation period of venoclysis is about 5 minutes, thereby make that GLP-1 is difficult to use clinically, therefore, actively seeking the transformation period that effective means prolongs GLP-1 has very important significance.GLP-1 mainly removes with two kinds of forms in vivo: enzyme is removed with organ and is removed.GLP-1 loses N-end under the effect of dipeptide peptidase i V two amino-acid residues become GLP-1 (9～36) acid amides of non-activity.Because dipeptide peptidase i V has the substrate specificity of height, the n terminal amino acid composition that changes GLP-1 can increase the resistibility of GLP-1 to dipeptide peptidase i V.Deacon etc. replace the similar peptide of GLP-1 (the Glucagons-like Peptide-1 Analogues that obtains behind GLP-1 (7～37) the 8th amino acids L-Ala with glycine, GLP-1-A), resistibility to dipeptide peptidase i V significantly increases, its vitro half-lives extends to 159 minutes, and and GLP-1 (7～37) have close biological activity.But the participation that dipeptide peptidase i V is only arranged can not be explained GLP-1 all purge mechanisms in vivo, and the transformation period of GLP-1 in external blood plasma is 20 minutes, draws the transformation period in 3～11 minutes the body considerably beyond many institutes.Deacon etc. replace the vitro half-lives significant prolongation of the GLP-1-A that obtains behind GLP-1 (7～37) the 8th amino acids L-Ala with glycine, but in the body transformation period do not improved accordingly, prompting still has other mechanism to participate in removing in the body of GLP-1.Kidney plays an important role in this course, and GLP-1 is a small-molecular peptides, easily filters from kidney, in the renal cells brush border DPP-IV is arranged, and GLP-1 can be absorbed by renal cells and decompose after being filtered by kidney.The clearance rate of rat kidney excision back GLP-1 descends, and vitro study shows that kidney once can filter the GLP-1 that surpasses 80% in the perfusion liquid and remove.Therefore, seeking effective means delays GLP-1 and has great importance for the transformation period in the body that prolongs GLP-1 from the removing of kidney.
At occurring in nature, the protein of many Multidomains (or multi-functional territory) is formed by several polypeptide chain fragment assemblies together, and being assembled into bigger multifunctional protein by relative small construction territory is the important factor that nature is evolved.The development of gene recombination technology makes the mankind to merge different genes or gene fragment mutually, the be stitched together artificial fusion rotein of the novel Multidomain that forms of the albumen that obtains difference in functionality by protein expression, present this method has been applied in the research in many fields, and demonstrates higher value.In the process that albumen merges, in order to keep each proteic space conformation and biological activity on the fusion rotein to greatest extent, often different albumen is linked to each other by the bigger joint of spatial activity, the folding small peptide that is rich in glycine of simple in structure, difficult formation is commonly used to as joint.Because the functional domain of adiponectin is positioned at the C-end, the functional domain of GLP-1 is positioned at the N-end, thereby but the applying gene recombinant technology links to each other the N-end of adiponectin and the C-end of GLP-1 by a bigger joint of spatial activity, produce a kind of new albumen, this albumen has the C-end of adiponectin and the N-end of GLP-1 simultaneously, so can have two kinds of functions of adiponectin and GLP-1, new albumen is because the molecular volume increase, be difficult for filtering and removing from kidney, can overcome single GLP-1 polypeptide easily removes from kidney, the shortcoming that transformation period is short, replace with glycine as the 8th amino acids L-Ala simultaneously GLP-1, then new proteic N-end also has the ability of opposing dipeptide peptidase i V, can eliminate two kinds of factors that GLP-1 easily is eliminated in vivo simultaneously, the transformation period in the body of significant prolongation GLP-1.Because the activity in the spherical territory of adiponectin is greater than the adiponectin of total length, and the spherical territory of the adiponectin of bacterial expression has activity, in this fusion rotein as further with the adiponectin of the spherical territory replacement of adiponectin total length, then can make the adiponectin in this fusion rotein have bigger activity.
The present invention at home, use gene recombination technology outward first, structure contains the prokaryotic expression carrier PET28a-Adiponectin-GLP-1-A (this carrier is the similar peptide fusion protein expression vector of adiponectin-Glucagon-like peptide-1) of the similar peptide fusion gene of human adiponectin-Glucagon-like peptide-1 (Adiponectin-GLP-1-A fusion gene), wherein the adiponectin encoding gene obtains by the spherical territory of amplification adiponectin encoding gene, the GLP-1-A encoding gene is by being that codon glycine obtains with GLP-1 (7-37) encoding gene the 8th amino acids L-Ala codon mutation, the Adiponectin-GLP-1-A fusion gene is formed by connecting adiponectin encoding gene and GLP-1-A encoding gene by joint sequence, and this joint sequence coded product is the small peptide that is rich in glycine.The present invention is for further expressing bacterium with prokaryotic expression carrier PET28a-Adiponectin-GLP-1-A transforming protein, obtain the similar peptide fusion protein of human adiponectin-Glucagon-like peptide-1 (Adiponectin-GLP-1-A fusion rotein) through protein purification, the activity of research Adiponectin-GLP-1-A fusion rotein, measure the transformation period of GLP-1-A on the fusion rotein, acquisition can promote the beta Cell of islet excreting insulin, can improve insulin resistant again, can significantly correct the disorder of diabetes B internal metabolism, the new albumen that side effect is little, set up the method for the Adiponectin-GLP-1-A fusion rotein of expressing biologically active, early-stage Study is provided.The further new albumen that obtains of this research might replace the orally-taken blood sugar reducing medicine of considerable part, and blood sugar reducing function can be better, safer, more meet the physiological requirements of human body, have great social effect and economic implications.
Summary of the invention
One of purpose of the present invention provides the cDNA sequence of people Adiponectin-GLP-1-A fusion gene, total length is totally 567 bp, 1-15bp coding enteropeptidase recognition sequence, 16-108bp coding people GLP-1-A, the long 93bp of sequence (wherein 19-21bp sports codon glycine GGT with the codon GCT of coding the 8th amino acids L-Ala in people GLP-1 (7-37) encoding gene), 109-153bp coding joint peptide dna sequence dna, the long 45bp of sequence, the 154-564bp adiponectin of encoding, the long 411bp of sequence, 565-567bp is a terminator codon, and this fusion gene cDNA sequence is seen SEQ ID NO 1.
Two of purpose of the present invention provides the construction process of people Adiponectin-GLP-1-A fusion gene, realize by following steps: the adiponectin encoding gene obtains by the spherical territory of amplification adiponectin encoding gene, sport codon glycine GGT by codon GCT and obtain people GLP-1-A gene coding the 8th amino acids L-Ala in people GLP-1 (7-37) encoding gene, when designer GLP-1-A gene primer, on the upstream primer P1 of people GLP-1-A, design the nucleotide sequence that adds a restriction enzyme site of coding respectively, the complementary sequence of the nucleotide sequence of enteropeptidase recognition sequence and people GLP-1-A gene, downstream primer P2 design respectively and add people GLP-1-A gene complementation sequence, the nucleotide sequence of the joint peptide-coding sequence and the restriction enzyme site of encoding; During design apm 1 gene primer, joint peptide-coding sequence, the nucleotide sequence of a restriction enzyme site of coding and the complementary sequence of the spherical domain gene of adiponectin are added in design respectively on upstream primer P3, wherein P2 is identical with the restriction enzyme site of P3, and downstream primer P4 designs the complementary sequence of the spherical domain gene of adiponectin respectively and the nucleotide sequence of the restriction enzyme site of encoding.By PCR two genes that increase respectively, the utilization enzyme incision technology, successively GLP-1-A and apm 1 gene are recombinated in the prokaryotic expression carrier, utilize restriction enzyme site that two gene fusion are obtained people Adiponectin-GLP-1-A fusion gene, in this fusion gene, the encoding gene of GLP-1-A is connected with the nucleotide sequence of adiponectin encoding gene by the flexible joint peptide of encoding.GLP-1-A encoding gene primer P1, P2 sequence are seen SEQ.ID NO2, SEQ.ID NO3, and adiponectin encoding gene primer P3, P4 sequence are seen SEQ.ID NO4, SEQ.ID NO5; The adiponectin coding gene sequence is seen SEQ.ID NO6, and the GLP-1-A coding gene sequence is seen SEQ.ID NO7, and the nucleotide sequence of flexible joint peptide is seen SEQ.IDNO8.
Three of purpose of the present invention provides the expression vector (this carrier is the similar peptide fusion protein expression vector of adiponectin-Glucagon-like peptide-1) that contains the Adiponectin-GLP-1-A fusion gene.
Four of purpose of the present invention provides the construction process of the expression vector that contains the Adiponectin-GLP-1-A fusion gene, realizes by following steps:
(1) extract the total RNA of human fat tissue, RT-PCR obtains the adiponectin encoding gene, and sequence is seen SEQ.ID NO6.
(2) extract the human genome DNA, PCR obtains the GLP-1-A encoding gene, and sequence is seen SEQ.IDNO7.
(3) utilization enzyme incision technology obtains to contain the prokaryotic expression carrier PET28a-Adiponectin-GLP-1-A (this carrier is the similar peptide fusion protein expression vector of adiponectin-Glucagon-like peptide-1) of Adiponectin-GLP-1-A fusion gene among the prokaryotic expression carrier PET28a that successively GLP-1-A and adiponectin encoding gene recombinated.
(4) express bacterium with the PET28a-Adiponectin-GLP-1-A transforming protein, confirm that this carrier can express the Adiponectin-GLP-1-A fusion rotein that contains the enteropeptidase recognition sequence well, the sequence that contains the Adiponectin-GLP-1-A fusion rotein of enteropeptidase recognition sequence is seen SEQ.ID NO9.
The invention has the advantages that:
1. first the encoding gene of adiponectin and GLP-1-A is constructed in the world and obtain the Adiponectin-GLP-1-A fusion gene.
2. use enzyme incision technology in the world first, make up the prokaryotic expression carrier PET28a-Adiponectin-GLP-1-A that contains the Adiponectin-GLP-1-A fusion gene.
3.Adiponectin-GLP-1-A fusion gene is formed by connecting by a joint sequence, make to be connected by a joint peptide with GLP-1-A, help keeping to greatest extent each proteic space conformation and biological activity on the fusion rotein by the adiponectin in the Adiponectin-GLP-1-A fusion rotein of this fusion gene expression.
4.Adiponectin-GLP-1-A the adiponectin encoding gene in the fusion gene obtains by the spherical territory of amplification adiponectin encoding gene, makes that expressing the adiponectin that obtains in the Adiponectin-GLP-1-A fusion by this fusion gene has bigger biological activity.
5. in the Adiponectin-GLP-1-A fusion gene, also introduced the enteropeptidase recognition sequence, what make that this fusion gene at first expresses acquisition is the Adiponectin-GLP-1-A fusion rotein that contains the enteropeptidase recognition sequence, promptly comprise an enteropeptidase recognition sequence Asp-Asp-Asp-Asp-Lys at the N-of Adiponectin-GLP-1-A fusion rotein end, can by the enteropeptidase effect its cutting be removed in the research afterwards, the N-end dissociative of GLP-1-A is come out, obtain the Adiponectin-GLP-1-A fusion rotein.
6. the present invention has the transformation period in the long body by the GLP-1-A on the Adiponectin-GLP-1-A fusion rotein of two kinds of mechanism assurance acquisitions in the future.On the one hand the GLP-1-A gene in the Adiponectin-GLP-1-A fusion gene is to be that codon glycine obtains with GLP-1 (7-37) encoding gene the 8th amino acids L-Ala codon mutation, and this sudden change can make its expression product have the ability of significant opposing dipeptide peptidase i V degraded; The molecular weight of fusion rotein own increases on the other hand, is difficult for filtering and removing from kidney, can overcome single GLP-1 polypeptide easily from kidney removing, short shortcoming of transformation period.Method by construction of fusion protein prolongs that the transformation period belongs to initiative in the world in the body of GLP-1.
7. express bacterium with PET28a-Adiponectin-GLP-1-A prokaryotic expression carrier transforming protein, find that this carrier can express the Adiponectin-GLP-1-A fusion rotein that contains the enteropeptidase recognition sequence well, this fusion rotein is for obtaining first in the world, for further obtaining the Adiponectin-GLP-1-A fusion rotein through protein purification, enteropeptidase cutting, the activity of research Adiponectin-GLP-1-A fusion rotein, the transformation period of measuring GLP-1-A on the fusion rotein provides good early-stage Study.
8. follow-up study of the present invention if can be succeedd, obtain the Adiponectin-GLP-1-A fusion rotein of biologically active, the method of the Adiponectin-GLP-1-A fusion rotein of expressing biologically active will be set up at home, outward first, acquisition can promote the beta Cell of islet excreting insulin, can improve insulin resistant again, can significantly correct the disorder of diabetes B internal metabolism, the new albumen that side effect is little has great economic implications and social effect.
Description of drawings
Fig. 1 GLP-1-A, adiponectin encoding gene agarose gel electrophoresis.
Fig. 2 PET28a-Adiponectin-GLP-1-A enzyme is cut agarose gel electrophoresis as a result.
The people Adiponectin-GLP-1-A expressing fusion protein that Fig. 3 contains the enteropeptidase recognition sequence is the 12%SDS-polyacrylamide gel electrophoresis as a result.
The people Adiponectin-GLP-1-A fusion rotein Western Blot that Fig. 4 contains the enteropeptidase recognition sequence analyzes.
Embodiment
Following examples only are used to the present invention is described and are not used in the restriction scope of the invention.
Embodiment one
1. material and instrument
1.1 material
Coli strain E.coli DH5 α, BL21 (DE3) is provided by the attached second hospital institute of oncology of Zhejiang University, prokaryotic expression plasmid PET28a is available from German novagen company, and RNA extract (Trizol) is available from U.S. Gibcol BRL company, diethylpyrocarbonate, Oligo (dT)
15, UNIQ-10 pillar genome DNA extraction test kit all gives birth to worker bio-engineering corporation, M-MLV Reverse Transcriptase, RNasin, dNTPs, Taq DNA polymerase, agarose, T available from Shanghai
4Dna ligase, restriction enzyme NheI, BamHI, HindIII, XbaI are all available from U.S. Promega company, and the lymphocyte extracting solution is available from Shanghai permanent letter chemical reagent company limited, GeneRuler
TMDNA Ladder Mix is available from German Fermentas company, DNA Marker DL2000, isopropylthio-(Isopropyl-beta-D-thiogalactopyranoside, IPTG) available from Japanese TaKaRa company, SDS polyacrylamide gel electrophoresis lower molecular weight standard protein is available from Shanghai Bo Ya Bioisystech Co., Ltd, 6 * His Monoclonal Antibody (anti-Histidine monoclonal antibody) is available from U.S. BD Biosciences company, the goat anti-mouse IgG of horseradish enzyme labelling is available from Beijing Zhong Shan Bioisystech Co., Ltd, plasmid extraction kit is available from Shen, Shanghai energy lottery industry Bioisystech Co., Ltd, Western Blotting LuminolReagent is available from U.S. Santa Cruz company, PVDF membrane is available from U.S. Bio-Rad company, and QIA quick Gel Extraction kit is available from German QIAGEN company.
1.2 key instrument
High speed low temperature centrifugal machine (Biofuge 28RS) is a German Heraeus company product, bacterium constant temperature shaking table (Orbital Shaker) is a U.S. Forma Scientific company product, uv-spectrophotometric instrument (8453 type) is available from U.S. Hewlett Packard company, GeneAmp PCR system 2400 is available from U.S. PerkinElmer company, vertical electrophoresis instrument (Mini Protean 3 cell) and electrotransfer instrument are U.S. Bio-Rad company product, vacuum lyophilization instrument (UVS400A) is available from U.S. Savant Instruments company, ultrasonic cell disruptor (JY88-2 type) is available from Ningbo Xin Zhike device institute, and the ABI377DNA sequenator is available from U.S. PIERCE company.
2. primer design
According to the gene order of the people GLP-1 (7-37) that announces among prokaryotic expression carrier PET28a and the GenBank with adiponectin, the primer of design GLP-1-A and adiponectin encoding gene (obtaining) and the position of inserting carrier by the spherical territory of amplification adiponectin.GLP-1-A encoding gene primer: P1:5 '-cgcgctagc
GacgatgacgataagCatggtgaagggaccttt-3 ', P2:5 '-cgcggatccagaaccagaaccagaaccagaaccagaaccagaaccagatcctcggc ctttcacca-3 '; Adiponectin encoding gene primer: P3:5 '-cccggatccgcctatgtataccgctca-3 ', P4:5 '-cccaagctttcagttggtgtcatggta-3.P1 sports codon glycine GGT (adding bold Italic represents) with GLP-1 (7-37) encoding gene the 8th amino acids L-Ala codon GCT, comprise that also N end enteropeptidase recognition sequence (represent by underscore, PET28a expressed proteins N end contains histidine-tagged, so designed the enteropeptidase recognition sequence on the primer, with removing the histidine-tagged of fusion rotein N end), NheI restriction enzyme site (GCTAGC, overstriking is represented), P2, P3 contains joint sequence (italic is represented) and BamHI restriction enzyme site (GGATCC, overstriking is represented), P4 contains HindIII restriction enzyme site (AAGCTT, overstriking is represented).Joint sequence is the nucleotide sequence 5 '-tctggttctggttctggttctggttctggttctggttctggatcc-3 ' of 45 bases, connects GLP-1-A and two genes of adiponectin, 15 amino acid of encoding, and sequence is: N-(Serine-glycine)
7-Serine-C.Primer is given birth to worker bio-engineering corporation by Shanghai and is synthesized.GLP-1-A encoding gene primer sequence is seen SEQ.ID NO2, SEQ.ID NO3, and adiponectin encoding gene primer sequence is seen SEQ.ID NO4, SEQ.ID NO5, the nucleotide sequence of coding flexible joint peptide is seen SEQ.ID NO8.
3. the RT-PCR of adiponectin encoding gene amplification
3.1 the extracting of the total RNA of fatty tissue
Get the about 100mg of human fat tissue of-80 ℃ of preservations, grinding powder in liquid nitrogen is with reference to total RNA of Trizol reagent specification sheets extracting fatty tissue, with uv-spectrophotometric instrument determination and analysis RNA purity, all RNAA
260/ A
280Ratio is more than 1.80.
3.2 reverse transcription
With mRNA is template, Oligo (dT)
15Be primer, through the synthetic cDNA of reverse transcription, reaction conditions is: get the total RNA of 2 μ g, 1.0 μ l 10pmol/ μ l ligo (dT)
15, add 0.1%DEPC water 9 μ l, cumulative volume 10 μ l, mixing, 65 ℃, sex change 10min, place immediately on ice, the cooling back adds 4 μ l, 5 * M-MLV Buffer, 1 μ l 25mM dNTPs, 0.5 μ l RNasin, 0.5 μ l M-MLV Reverse Transcriptase, add 0.1%DEPC water to 20 μ l, 37 ℃ of reaction 1h, gained cDNA is used for pcr amplification.
3.3 amplification
Reaction system: 10 * PCR Buffer, 5 μ l, 25mM MgCl
23 μ l, 10mM dNTPs 1 μ l, 10 μ M P
31 μ l, 10 μ M P
41 μ l, cDNA 2 μ l, Taq DNA polymerase (5u/ μ l) 0.5 μ l, ddH
2O 36.5 μ l, cumulative volume 50 μ l.The pcr amplification condition is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s, 60 ℃ of annealing 1min, 68 ℃ are extended 2min, and 40 circulations are extended 7min for back 68 ℃.Amplified production is identified (voltage 80V, electrophoresis 30min) through 1.5% agarose gel electrophoresis.Reclaim goal gene with QIA quick GelExtraction kit.Amplification is seen Fig. 1, wherein 1.DNA mark; 2.GLP-1-A; 3. adiponectin, the adiponectin coding gene sequence is seen SEQ.ID NO6.
4.GLP-1-A the pcr amplification of encoding gene
4.1 human genome DNA's extraction
Get people's peripheral vein 5ml anticoagulation, separation of human peripheral blood karyocyte extracts the human genome DNA with UNIQ-10 pillar genome extraction agent box.
4.2 pcr amplification
Reaction system is: 10 * PCR Buffer, 5 μ l, 25mM MgCl
23 μ l, 10mM dNTPs 1 μ l, 10 μ M P
11 μ l, 10 μ M P
21 μ l, cDNA 2 μ l, Taq DNA polymerase (5u/ μ l) 0.5 μ l, ddH
2O 36.5 μ l, reaction cumulative volume 50 μ l.The pcr amplification condition is: 94 ℃ of pre-sex change 5min, and 94 ℃ of sex change 30s, 62 ℃ of annealing 30s, 72 ℃ are extended 30s, and 40 circulations are extended 5min for back 72 ℃.Amplified production is identified (voltage 80V, electrophoresis 30min) through 1.5% agarose gel electrophoresis.Reclaim goal gene with QIA quickGel Extraction kit.Amplification is seen Fig. 1, and the GLP-1-A coding gene sequence is seen SEQ.ID NO7.
5.Adiponectin-GLP-1-A fusion gene and contain the structure of the prokaryotic expression carrier PET28a-Adiponectin-GLP-1-A of this fusion gene
5.1 PET28a-GLP-1-A construction of recombinant plasmid
1. enzyme is cut and is connected: GLP-1-A encoding gene and the PET28a plasmid that reclaims of will tapping rubber respectively carries out endonuclease reaction with NheI and BamHI under 37 ℃ condition, the enzyme system of cutting is: ddH
2O 10 μ l, 10 * buffer MC, 2 μ l, BSA (10 μ g/ μ l) 0.5 μ l, GLP-1-A encoding gene or PET28a plasmid 6.5 μ l, NheI 0.5 μ l, BamHI 0.5 μ l, reaction cumulative volume 20 μ l, reaction 3h.Then enzyme is cut product and carried out 1.5% agarose gel electrophoresis (voltage 80V respectively, electrophoresis 30min) after, reclaim purpose fragment after enzyme is cut with QIA quick Gel Extraction kit rubber tapping, will reclaim product and spend the night with 4 ℃ of connections of T4 dna ligase, linked system is: ddH
2O 4.5 μ l, 10 * buffer, 1 μ l, PET28a enzyme cut back to close product 3 μ l, and GLP-1-A encoding gene enzyme cuts back to close product 1 μ l, T4 dna ligase 0.5 μ l, cumulative volume 10 μ l.To connect product transformed competence colibacillus bacterium DH5 α next day, connect plasmid with amplification.
2. identify: the single bacterium colony that has grown on the several flat boards of picking, in containing the 7ml LB liquid nutrient medium amplification pipe of (containing kantlex 80 μ g/ml) 37 ℃ respectively, the 300rpm jolting is spent the night, extract plasmid with plasmid extraction kit, the double digestion plasmid under 37 ℃ condition with XbaI and BamHI, the enzyme system of cutting is: ddH
2O 10 μ l, 10 * buffer MC, 2 μ l, BSA (10 μ g/ μ l) 0.5 μ l, connect plasmid 6.5 μ l, XbaI 0.5 μ l, BamHI 0.5 μ l, cumulative volume 20 μ l, reaction 3h, enzyme is cut product behind 1.5% agarose gel electrophoresis (voltage 80V, electrophoresis 30min), observes enzyme and cuts the result, leave and take the bacterium liquid and the plasmid of positive colony, obtain the PET28a-GLP-1-A recombinant plasmid.
5.2 Adiponectin-GLP-1-A fusion gene and contain the structure of the prokaryotic expression carrier PET28a-Adiponectin-GLP-1-A of this fusion gene
1. enzyme is cut and is connected: respectively the PET28a-GLP-1-A plasmid that builds and the adiponectin goal gene that reclaims of tapping rubber are carried out endonuclease reaction with BamHI, HindIII under 37 ℃ condition, the enzyme system of cutting is: ddH
2O 10 μ l, 10 * buffer E, 2 μ l, BSA (10 μ g/ μ l) 0.5 μ l, PET28a-GLP-1-A or adiponectin goal gene 6.5 μ l, BamHI 0.5 μ l, HindIII 0.5 μ l, cumulative volume 20 μ l, reaction 3h.Then enzyme is cut product and carried out 1.5% agarose gel electrophoresis (voltage 80V, electrophoresis 30min) respectively, rubber tapping is reclaimed enzyme and is cut back purpose fragment, reclaims product and spends the night with 4 ℃ of connections of T4 dna ligase, and linked system is: ddH
2O 14.5 μ l, 10 * buffer, 2 μ l, PET28a-GLP-1-A enzyme cut back to close product 2 μ l, and adiponectin goal gene enzyme cuts back to close product 1 μ l, T4 dna ligase 0.5 μ l, cumulative volume 20 μ l.To connect product transformed competence colibacillus bacterium DH5 α next day, connect plasmid with amplification.
2. identify: the single bacterium colony that has grown on the several flat boards of picking, in the 7ml LB liquid nutrient medium amplification pipe of (containing kantlex 80 μ g/ml) 37 ℃, the 300rpm jolting is spent the night, and extracts plasmid with plasmid extraction kit, and (the enzyme system of cutting is: ddH to use BamHI, HindIII respectively
2O 10 μ l, 10 * buffer E, 2 μ l, BSA (10 μ g/ μ l) 0.5 μ l connects plasmid 6.5 μ l, BamHI 0.5 μ l, HindIII 0.5 μ l, cumulative volume 20 μ l, 37 ℃ of reaction 3h) and XbaI, HindIII (the enzyme system of cutting is: ddH
2O 10 μ l, 10 * bufferB, 2 μ l, BSA (10 μ g/ μ l) 0.5 μ l, connect plasmid 6.5 μ l, XbaI 0.5 μ l, HindIII 0.5 μ l, cumulative volume 20 μ l, 37 ℃ of reaction 3h) double digestion plasmid, enzyme is cut product through 1.5% agarose gel electrophoresis (voltage 80V, electrophoresis 30min) after, the small pieces that 426bp and 683bp occur, enzyme is cut product and is met desired value, illustrates that the Adiponectin-GLP-1-A fusion gene successfully inserts the expection restriction enzyme site of PET28a, has obtained the Adiponectin-GLP-1-A fusion gene and has contained the prokaryotic expression carrier PET28a-Adiponectin-GLP-1-A (this carrier is the similar peptide fusion protein expression vector of adiponectin-Glucagon-like peptide-1) of this fusion gene.See Fig. 2, wherein M.DNAmarker; 1.PET28a-Adiponectin-GLP-1-A enzyme is not cut; 2.BamHI/HindIII enzyme is cut the result; 3.XbaI/HindIII enzyme is cut the result.Through the PET universal primer by the ABI377DNA sequenator to the PET28a-Adiponectin-GLP-1-A evaluation of checking order, show that the direction of insertion of each gene and order are entirely true, institute checks order and is listed as and expects in full accord.Adiponectin-GLP-1-A fusion gene sequence is seen SEQ.ID NO1, total length is totally 567 bp, 1-15bp coding enteropeptidase recognition sequence, 16-108bp coding people GLP-1-A, the long 93bp of sequence, 109-153bp coding joint peptide dna sequence dna, the long 45bp of sequence, the 154-564bp adiponectin of encoding, the long 411bp of sequence, 565-567bp is a terminator codon, sees SEQ.ID NO1.
6. prokaryotic expression carrier PET28a-Adiponectin-GLP-1-A expresses the preliminary study of the Adiponectin-GLP-1-A fusion rotein that contains the enteropeptidase recognition sequence
Get the correct PET28a-Adiponectin-GLP-1-A transformed competence colibacillus of order-checking and express bacterium BL21 (DE3), next day, picking list bacterium colony was in 7ml LB liquid nutrient medium (containing kantlex 80 μ g/ml), 37 ℃ of joltings are spent the night, draw 70 μ l in 7ml LB liquid nutrient medium (containing kantlex 80 μ g/ml), 37 ℃ of jolting 2h make bacterium liquid at the absorbancy (A of 600nm place
600) be 0.4～0.6, leave and take and induce preceding bacterium liquid 1ml in contrast, add isopropylthio-(IPTG) in the remaining bacterium liquid and induce, 37 ℃ of difference jolting 1～6h collect 1～6h day part bacterium respectively, every pipe amount of bacteria are adjusted, until A
600Basically identical, with 5, the centrifugal 10min of 000rpm abandons supernatant with the bacterium liquid collected, after precipitation is washed 3 times with PBS, resuspended with an amount of PBS, bacterium liquid is got 2 * sds gel sample loading buffer that 10 μ l add equivalent, 100 ℃ are boiled 5min, 13,000rpm goes up sample behind the centrifugal 1min, determine whether target protein expresses and the expression amount maximum time period behind the row 12%SDS-polyacrylamide gel electrophoresis.The result shows, with induce before bacterium liquid phase ratio, IPTG induces the back at 25KD place denseer protein band to be arranged, and is consistent with the fusion rotein size of expection, and IPTG induces the protein band of 5～6h the denseest, induces preceding equal showed increased.Get and induce ultrasonication bacterium on the 5h bacterium liquid 1ml ice bath of back, stay supernatant and precipitation respectively, to precipitate and use and the isopyknic PBS mixing of supernatant, get 10 μ l respectively, 2 * sds gel the sample loading buffer that adds equivalent, 100 ℃ are boiled 5min, 13,000rpm goes up sample behind the centrifugal 1min, with the expression of 12%SDS-polyacrylamide gel electrophoresis analysis purposes albumen in last cleer and peaceful precipitation, found that, at the 25KD place denseer protein band being arranged, is dense with sedimentary electrophoretic band, consistent with the fusion rotein size of expection.See Fig. 3, wherein M: albumen marker, 1.IPTG induce back 5h protein precipitation, and 2.IPTG induces back 5h to go up white protein, and 3.IPTG induces preceding bacterium liquid, and 4-9.IPTG induces back 1h～6h total protein; Capable Western Blot analysis shows in supernatant and the precipitation that specific protein expression is all arranged to product with anti-Histidine monoclonal antibody.See 4, wherein 1.IPTG induces back 5h protein precipitation, and 2.IPTG induces back 5h to go up white protein.Expressing what at first obtain by the PET28a-Adiponectin-GLP-1-A prokaryotic expression carrier is the people Adiponectin-GLP-1-A fusion rotein that contains the enteropeptidase recognition sequence, totally 188 amino acid, comprise 1-5aa coding enteropeptidase recognition sequence, 6-36aa coding GLP-1-A (wherein the 7th amino acids is the amino acid glycine of sudden change), the 37-51aa connection peptides of encoding, the 52-188aa adiponectin of encoding.Sequence is seen SEQ.IDNO9.Can further cut acquisition Adiponectin-GLP-1-A fusion rotein to this fusion rotein in the research afterwards with enteropeptidase.
The sequence that the present invention relates to
SEQ.ID NO1 (Adiponectin-GLP-1-A fusion gene cDNA sequence):
The information of SEQ.ID NO1:
(i) sequence signature
(A) length: 567 bases
(B) type: nucleic acid
(C) chain: two strands
(D) topology: linearity
(ii) molecule-type: cDNA
(iii) sequence description:
5’-gacgatgacgataagcatggtgaagggacctttaccagtgatgtaagttcttatttggaag
gccaagctgccaaggaattcattgcttggctggtgaaaggccgaggatctggttctggttctgg
ttctggttctggttctggttctggatccgcctatgtataccgctcagcattcagtgtgggattg
gagacttacgttactatccccaacatgcccattcgctttaccaagatcttctacaatcagcaaa
accactatgatggctccactggtaaattccactgcaacattcctgggctgtactactttgccta
ccacatcacagtctatatgaaggatgtgaaggtcagcctcttcaagaaggacaaggctatgctc
ttcacctatgatcagtaccaggaaaataatgtggaccaggcctccggctctgtgctcctgcatc
tggaggtgggcgaccaagtctggctccaggtgtatggggaaggagagcgtaatggactctatgc
tgataatgacaatgactccaccttcacaggctttcttctctaccatgacaccaactga-3’
SEQ.ID NO2 (GLP-1-A encoding gene primer P1 sequence):
The information of SEQ.ID NO2:
(i) sequence signature
(A) length: 42 bases
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ii) molecule-type: DNA
(iii) sequence description:
5’-cgcgctagc
gacgatgacgataagcatggtgaagggaccttt-3’
SEQ.ID NO3 (GLP-1-A encoding gene primer P2 sequence):
The information of SEQ.ID NO3:
(i) sequence signature
(A) length: 65 bases
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ii) molecule-type: DNA
(iii) sequence description:
5’-cgcggatccagaaccagaaccagaaccagaaccagaaccagaaccagatcctcggccttt
cacca-3’
SEQ.ID NO4 (adiponectin encoding gene primer P3 sequence):
The information of SEQ.ID NO4:
(i) sequence signature
(A) length: 27 bases
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ii) molecule-type: DNA
(iii) sequence description:
5’-cccggatccgcctatgtataccgctca-3’
SEQ.ID NO5 (adiponectin encoding gene primer P4 sequence):
The information of SEQ.ID NO5:
(i) sequence signature
(A) length: 27 bases
(B) type: nucleic acid
(C) chain: strand
(D) topology: linearity
(ii) molecule-type: DNA
(iii) sequence description:
5’-cccaagctttcagttggtgtcatggta-3
SEQ.ID NO6 (adiponectin coding gene sequence):
The information of SEQ.ID NO6:
(i) sequence signature
(A) length: 426 bases
(B) type: nucleic acid
(C) chain: two strands
(D) topology: linearity
(ii) molecule-type: cDNA
(iii) sequence description: 5 ' end is introduced BamH I restriction enzyme site (6bp, box indicating), and 3 ' end is introduced HindIII restriction enzyme site (6bp, box indicating).
5’-ggatccgcctatgtataccgctcagcattcagtgtgggattggagacttacgttactatcccc
aacatgcccattcgctttaccaagatcttctacaatcagcaaaaccactatgatggctccactggta
aattccactgcaacattcctgggctgtactactttgcctaccacatcacagtctatatgaaggatgt
gaaggtcagcctcttcaagaaggacaaggctatgctcttcacctatgatcagtaccaggaaaataat
gtggaccaggcctccggctctgtgctcctgcatctggaggtgggcgaccaagtctggctccaggtgt
atggggaaggagagcgtaatggactctatgctgataatgacaatgactccaccttcacaggctttct
tctctaccatgacaccaactga
-3’
SEQ.ID NO7 (GLP-1-A coding gene sequence):
The information of SEQ.ID NO7:
(i) sequence signature
(E) length: 159 bases
(F) type: nucleic acid
(G) chain: two strands
(H) topology: linearity
(ii) molecule-type: cDNA
(iii) sequence description: 5 ' end is introduced Nhe I restriction enzyme site (6bp, box indicating), enteropeptidase recognition sequence (15bp, overstriking is represented), 3 ' end is introduced joint sequence (45bp, overstriking is represented), BamH I restriction enzyme site (6bp, box indicating, be positioned at joint sequence), total length 159bp, wherein 25-27bp sports codon glycine GGT (adding bold Italic represents) with the codon GCT of coding the 8th amino acids L-Ala in people GLP-1 (7-37) encoding gene.
5’-
gacgatgacgataagcatggtgaagggacctttaccagtgatgtaagttcttatttg
gaaggccaagctgccaaggaattcattgcttggctggtgaaaggccgaggatctggttctggttctg
gttctggttctggttctggttct
-3’
SEQ.ID NO8 (Adiponectin-GLP-1-A fusion gene flexible joint nucleotide sequence):
The information of SEQ.ID NO8:
(i) sequence signature
(I) length: 45 bases
(J) type: nucleic acid
(K) chain: two strands
(L) topology: linearity
(ii) molecule-type: DNA
(iii) sequence description:
5’-tctggttctggttctggttctggttctggttctggttctggatcc-3’
SEQ.ID NO9 (the Adiponectin-GLP-1-A fusion rotein sequence that contains the enteropeptidase recognition sequence):
The information of SEQ.ID NO9:
(i) sequence signature
(A) length: 188 amino acid
(B) type: amino acid
(C) chain: strand
(ii) molecule-type: polypeptide
(iii) sequence description:
Asp?Asp?Asp?Asp?Lys?His?Gly?Glu?Gly?Thr?Phe?Thr?Ser?Asp?Val?Ser?Ser?Tyr?Leu
Glu?Gly?Gln?Ala?Ala?Lys?Glu?Phe?Ile?Ala?Trp?Leu?Val?Lys?Gly?Arg?Gly?Ser?Gly?Ser
Gly?Ser?Gly?Ser?Gly?Ser?Gly?Ser?Gly?Ser?Gly?Ser?Ala?Tyr?Val?Tyr?Arg?Ser?Ala?Phe
Ser?Val?Gly?Leu?Glu?Thr?Tyr?Val?Thr?Ile?Pro?Asn?Met?Pro?Ile?Arg?Phe?Thr?Lys?Ile
Phe?Tyr?Asn?Gln?Gln?Asn?His?Tyr?Asp?Gly?Ser?Thr?Gly?Lys?Phe?His?Cys?Asn?Ile
Pro?Gly?Leu?Tyr?Tyr?Phe?Ala?Tyr?His?Ile?Thr?Val?Tyr?Met?Lys?Asp?Val?Lys?Val?Ser
Leu?Phe?Lys?Lys?Asp?Lys?Ala?Met?Leu?Phe?Thr?Tyr?Asp?Gln?Tyr?Gln?Glu?Asn?Asn
Val?Asp?Gln?Ala?Ser?Gly?Ser?Val?Leu?Leu?His?Leu?Glu?Val?Gly?Asp?Gln?Val?Trp
Leu?Gln?Val?Tyr?Gly?Glu?Gly?Glu?Arg?Asn?Gly?Leu?Tyr?Ala?Asp?Asn?Asp?Asn?Asp
Ser?Thr?Phe?Thr?Gly?Phe?Leu?Leu?Tyr?His?Asp?Thr?Asn
Claims (5)
1. the cDNA sequence of the similar peptide fusion gene of human adiponectin-Glucagon-like peptide-1, it is characterized in that: total length is totally 567 bp, 1-15bp coding enteropeptidase recognition sequence, 16-108bp coding people GLP-1-A, the long 93bp of sequence, wherein 19-21bp sports codon glycine GGT with the codon GCT of coding the 8th amino acids L-Ala in people GLP-1 (7-37) encoding gene, 109-153bp coding joint peptide dna sequence dna, the long 45bp of sequence, the 154-564bp adiponectin of encoding, the long 411bp of sequence, 565-567bp are terminator codon, and this fusion gene cDNA sequence is seen SEQ ID NO 1.
2. the similar peptide fusion gene of human adiponectin-Glucagon-like peptide-1 construction process, it is characterized in that realizing by following steps: the adiponectin encoding gene obtains by the spherical territory of amplification adiponectin encoding gene, sport codon glycine GGT by codon GCT and obtain people GLP-1-A gene coding the 8th amino acids L-Ala in people GLP-1 (7-37) encoding gene, when designer GLP-1-A gene primer, on the upstream primer P1 of people GLP-1-A, design the nucleotide sequence that adds a restriction enzyme site of coding respectively, the complementary sequence of the nucleotide sequence of enteropeptidase recognition sequence and people GLP-1-A gene, downstream primer P2 design respectively and add people GLP-1-A gene complementation sequence, the nucleotide sequence of the joint peptide-coding sequence and the restriction enzyme site of encoding; During design apm 1 gene primer, the joint peptide-coding sequence is added in design respectively on upstream primer P3, the nucleotide sequence of a restriction enzyme site of coding and the complementary sequence of the spherical domain gene of adiponectin, wherein P2 is identical with the restriction enzyme site of P3, downstream primer P4 designs the complementary sequence of the spherical domain gene of adiponectin respectively and the nucleotide sequence of the restriction enzyme site of encoding, by PCR two genes that increase respectively, the utilization enzyme incision technology, successively GLP-1-A and apm 1 gene are recombinated in the prokaryotic expression carrier, utilize restriction enzyme site that two gene fusion are obtained the similar peptide fusion gene of human adiponectin-Glucagon-like peptide-1, in this fusion gene, the encoding gene of GLP-1-A is connected with the nucleotide sequence of adiponectin encoding gene by the flexible joint peptide of encoding.
3. the similar peptide fusion gene of human adiponectin-Glucagon-like peptide-1 according to claim 2 construction process, it is characterized in that: GLP-1-A encoding gene primer P1, P2 sequence are SEQ.ID NO 2, SEQ.IDNO 3, adiponectin encoding gene primer P3, P4 sequence are SEQ.ID NO 4, SEQ.ID NO 5, the adiponectin coding gene sequence is SEQ.ID NO 6, and the GLP-1-A coding gene sequence is SEQ.ID NO 7, and the nucleotides sequence of flexible joint peptide is classified SEQ.ID NO 8 as.
4. the similar peptide of adiponectin-Glucagon-like peptide-1 according to claim 1 merges fusion gene, it is characterized in that: the expression vector that contains the similar peptide fusion gene of human adiponectin-Glucagon-like peptide-1.
5. the construction process that contains the expression vector of the similar peptide fusion gene of human adiponectin-Glucagon-like peptide-1 according to claim 4 is characterized in that realizing by following steps:
(1) extract the total RNA of human fat tissue, RT-PCR obtains the adiponectin encoding gene, and sequence is seen SEQ.ID NO 6;
(2) extract the human genome DNA, PCR obtains the GLP-1-A encoding gene, and sequence is seen SEQ.IDNO 7;
(3) utilization enzyme incision technology obtains to contain the prokaryotic expression carrier PET28a-Adiponectin-GLP-1-A of the similar peptide fusion gene of adiponectin-Glucagon-like peptide-1 among the prokaryotic expression carrier PET28a that successively GLP-1-A and adiponectin encoding gene recombinated.
(4) express bacterium with the PET28a-Adiponectin-GLP-1-A transforming protein, confirm that this carrier can express the similar peptide fusion protein of adiponectin-Glucagon-like peptide-1 that contains the enteropeptidase recognition sequence well, the sequence that contains the similar peptide fusion protein of adiponectin-Glucagon-like peptide-1 of enteropeptidase recognition sequence is seen SEQ.ID NO 9.
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