CN1557954A

CN1557954A - Expression and application of transduction peptides - human insulinogen fusion protein

Info

Publication number: CN1557954A
Application number: CNA200410039129XA
Authority: CN
Inventors: 红刘; 刘红
Original assignee: BEIJING RICHUDONGFANG TECHNOLOGY DEVELOPMENT Co Ltd
Current assignee: BEIJING RICHUDONGFANG TECHNOLOGY DEVELOPMENT Co Ltd
Priority date: 2004-02-10
Filing date: 2004-02-10
Publication date: 2004-12-29
Anticipated expiration: 2024-02-10
Also published as: CN1255543C

Abstract

The present invention relates to cloning human proinsulin cDNA of furin enzyme incising site to terminal N or C of transduction peptide gene to synthesize the polynucleotides of transduction peptide-human proinsulin fusion protein; and to constituting fusion expressed prokaryotic expression vector with the polynucleotides to express the transduction peptide-human proinsulin fusion protein. Under the induction of the transduction peptide, the denaturated and purified fusion protein makes proinsulin protein enter effectively to cell, such as skin cell, muscle cell, alveolar cell, etc. Under the action of furin enzyme inside these non-endocritic cells, proinsulin is processed into insulin with natural physiologic activity into blood to lower blood sugar. The present invention may be taken by diabetics via different modes to treat diabetes effectively.

Description

The expression of Tranduced peptide-huamn proinsulin fusion protein and application

[technical field]

The invention belongs to the genetically engineered field.More specifically, the present invention relates to the proinsulin human cDNA that contains the furin restriction enzyme site is cloned into the N or the C-terminal sequence of transduction peptide gene, the polynucleotide of synthetic Tranduced peptide-huamn proinsulin fusion protein; Also relate to the prokaryotic expression carrier that utilizes these polynucleotide to make up amalgamation and expression simultaneously, the expression and purification fusion rotein.

[background technology]

Diabetes are one of the mortality ratio mankind's three big diseases of being only second to cardiovascular and tumor disease.Regular Insulin is the specific medicament of treatment diabetes, is used for clinical treatment existing 80 years, is the protein drug of consumption maximum clinically.

Along with development of biology, the recombinant human insulin has become the main source of present human Regular Insulin.The Eli Lilly company of the Novo company of Denmark and the U.S. is two major vendors that produce the recombinant human insulin at present, adopt two kinds of expression system productions of intestinal bacteria and yeast respectively, the former aftertreatment difficulty but expression amount height, latter's aftertreatment is easy relatively but expression amount is low.Coli expression system is with fusion protein form expression secretion proinsulin human, first abduction delivering proinsulin human, be converted into stable S-sulfonic acid type after, through sex change renaturation and disulfide linkage pairing, be folded into the proinsulin human of native conformation.The proinsulin human removes the C-peptide and obtains crystalline insulin (Owens DR.et al., Biotechnology of Insulin Therapy.Oxford:Blackwell, 1991:24-41 again through trypsinase and protaminase combined action; Kemmler, et al, J.Bio.Chem.1971,246:6786).But yeast expression system expression-secretion proinsulin human analogue, the insulin precurosor that secretion is come out has had natural disulfide linkage and correct N end, thereby expression product does not need again through the sex change renaturation, directly changeing peptide or trypsinase and protaminase by trypsinase handles, obtain crystalline insulin (MarkussenJ.Protein Engineering, 1987,1:205 and Thim L.Proc.Natl.Acad.Sci.USA, 1986,86:6766).Therefore protokaryon still is that the expression product of yeast expression system all needs just can be converted into the insulin human through complicated processing, is used for treatment of diabetes.

Regular Insulin all adopts the administration of subcutaneous injection mode at present, and the subcutaneous injection administration can be brought into play good drug action.Diabetes are diseases of a kind of administration throughout one's life, and more serious patient needs one day twice, even three, four subcutaneous injection administrations, and side effect is obvious, causes amyotrophy, so that can't inject at last, has brought great misery to patient.Injection medication inconvenience easily has an accident simultaneously.Come out soon at Regular Insulin, just constantly relevant for research oral, that lung is inhaled multiple medications such as people, nasal mucosa and anus bolt.Oral administration system is the drug delivery system that makes things convenient for the most, is easy to accept, but because Regular Insulin is a kind of unsettled protein drug, be easy to by protease hydrolysis at gi tract, enter into the first pass effect of the Regular Insulin of blood through gi tract, determined that the bioavailability of Regular Insulin is extremely low by liver.Although the employing of new technologies and methods in recent years makes oral administration system obtain suitable progress, its complicated process of preparation, the quality control difficulty, the organic solvent that uses in the process may cause the Regular Insulin inactivation.Mucoadhesive delivery system, how with in the nose or the form of oral spray occur, this class preparation is many to be carrier with liposome or micro-capsule, the gathering of liposome or micro-capsule and the heterogeneity of size distribution make the unusual difficulty of accurate quantification by the spray pattern administration.The internal rectum hydrolytic enzyme activities is low, so rectal administration is a possible route of administration, but rectum is very difficult to the absorption of Regular Insulin, and bioavailability is lower.Although can improve the osmotic absorption of medicine slightly by the method for using chemical infiltration accelerating agent, often bring misery to patient by rectum.These methods all have a tangible common issue with to fail satisfactory solution so far, and promptly bioavailability is too low, and cost is too high.Some method also has side effect or uses inconvenient in addition.

Percutaneous dosing is a kind of ideal administering mode of Regular Insulin, and the hydrolytic enzyme activities in the skin is very low, thereby can avoid the inactivation of Regular Insulin.The main difficulty and the problem of Regular Insulin percutaneous dosing are: (1) Regular Insulin relative molecular weight is bigger, and easily forms aggregate, and fine and close stratum corneum (S.C.) structure of skin surface makes it be difficult to infiltrate.(2) Regular Insulin, owing to the variation of inducing the meeting recurring structure of skin environment and is detained in skin through in the process of dermal osmosis.(3) the novel short technology of oozing of development at present, although reduced cuticular resistance to mass transfer to a great extent, but Regular Insulin be still through the skin transmission that the passive diffusion that relies on concentration difference to promote realizes, because medicine delay, concentration polarization, medicine are in the influence of profit phase partition ratio, the infiltration capacity of Regular Insulin is still lower.

The drug delivery system that this shows Regular Insulin is except that injection system, and other drug delivery system is all immature.Along with the development of medical industry, the curative properties for Regular Insulin has proposed increasing requirement clinically.So along with development of biology, having does not have possibility to produce the product that not only need not to inject but also can simulate the Regular Insulin under the islet secretion normal physiological conditions, and the present invention describes from above two angles.

Regular Insulin is to be synthesized in the β of pancreas islet cell and to secrete under normal physiological conditions, arrives target tissue with blood circulation, by membrane receptor associative list single-minded with it reveal physiological function (Bell GI.Et al., Nature, 1979,282:525).The biosynthetic process of Regular Insulin comprises that the mRNA of preproinsulin is translated into the preproinsulin of a peptide chain at the endoplasm face of endoplasmic reticulum, newborn polypeptide chain passes endoplasmic reticulum and enters endoplasmic under the guiding of signal peptide, removing signal peptide simultaneously becomes proinsulin.Proinsulin enters golgi body then, and process is folding, disulfide linkage matches and packing enters secretory granules, the last Regular Insulin that is processed into A, two chains of B by the PC2 and the single-minded proteolytic ferment of PC3 of trypsin-like and protaminase.

Know that at present expression of insulin gene in common non-internal secretion zooblast generally can only obtain proinsulin.Because the greatest problem of non-endocrine cell is to lack the regulation of secretion approach and proinsulin is converted to the enzyme (PC2 and PC3) of Regular Insulin, can not excise the C peptide in the proinsulin, make it to change into sophisticated Regular Insulin, therefore the product that gives expression to is a proinsulin.But a class intracellular protein precursor processive enzyme is arranged in the ordinary cells with composition Secretory Pathway, as Furin or PACE (Seidah NG, Chretien M.Trend EndocrinolMetabol, 1992,3:133), can discern and cut Arg/Lys-X-Lys-Arg-original mold formula sequence on composition Secretory Pathway excretory protein.Use site-directed mutagenesis technique to transform insulin gene, the Insulinogen C peptide two ends are introduced in Fmin identification and cleavage site, in common non-endocrine cell, express improved insulin gene, just can produce mature insulin (Groskrentz DJ with Furin recognition site, Sliwkowski XM, Gorman CM.J Bio Chem, 1994,269:6241).

Nexin transduction domain (Protein Transduction Domain, PTD) (SchwarzeSR, et al., Science, 1999,285:1569-1572.) be automatically permeates cell membranes of a class, and can other biomacromolecule be carried a section of entering in the cytolemma as carrier and be rich in arginic polypeptide.Generally speaking, eukaryotic cytolemma all is impermeables to exhausted big number protein and length above 6 amino acid whose polypeptide.(Cell.1988 55:1179-1188) reports that the trans-activator TAT from HIV (human immunodeficiency virus) 1 (HIV-1) that joins in the cell culture fluid can automatically enter in the culturing cell trans-activation LTR promotor to Green in 1988 etc.Studies confirm that subsequently, it is owing to be positioned at the effect of one section polypeptide of 47-57 (YGRKKRRQRRR) position on the TAT albumen that TAT can stride across cytolemma automatically.The PTD aminoacid sequence (47-57) of the TAT of synthetic (is called for short tat peptide, together following) and utilize the method for chemosynthesis that N-end or the C-that other macro-molecular protein is connected it held, or the fusion rotein of being made up of other macro-molecular protein and tat peptide that the method for utilizing genetic recombination makes up all can permeates cell membranes enter in the cell, and find this penetrating (or transduction) effect and cultured cells kind and culture temperature irrelevant (all can transduce for 4 ℃ and 37 ℃), and enter intracellular amount only be added in nutrient solution in the amount of fused protein relevant, and metabolic enzyme inhibitor has no effect to this transduction process.That is to say, the caused this transduction of tat peptide is that a kind of and present known biomacromolecule enters intracellular mode, as depends on acceptor, translocator or endocytosis fully different (Nagahara H, et al, NatureMedicine.1998,4 (12): 1449-1452; Xia H, Nature biotechnology.2001,19:640-644).Except that tat peptide, transcribe peptide sequence (Derossi D, et al.The Journal of Biologicalchemistry.1996,27 (30): 18188-18193.) of the 43-58 position of factors A NTP from fruit bat feeler foot homology abnormal shape; 267-300 sequence (Elliott C from the conjugated protein VP22 of the single viral DNA of sore rash, Ohare P.Cell.1997,88:223-233) all have and on all four transduction function of tat peptide and transduction mechanism, therefore they also be collectively referred to as PTD (Ford K G, et al.Gene Therapy.2001,8:1-4).Schwarze in 1999 etc. have confirmed the transduction function of tat peptide the first time under the situation of live body.Molecular weight is injected in the mouse peritoneal under the live body situation greater than the fusion rotein that 120KD is made up of beta-galactosidase enzymes and tat peptide sequence, after 4 hours, mouse the institute in a organized way, comprise that brain (can penetrate hemato encephalic barrier) has all detected the activity of beta-galactosidase enzymes, do not have the contrast fusion rotein of TAT sequence then to detect activity less than enzyme.Result of study confirms the peculiar biological function of tat peptide, and it is not destroying under the bioactive situation of cytolemma, high molecular weight protein can be carried to enter in the cell, and make high molecular weight protein such as enzyme etc. keep its original biological activity.Infer that at present this process is relevant with the existence of protein transduction domain neutral and alkali amino acid (arginine and Methionin), these amino acid have strong positive charge, may interact to mediate by direct and cell surface Suleparoid protein-polysaccharide and wear membrane process (Tyagi M.et al.J Bio Chem, 2001,276 (5): 3254-3261).Transduction peptide system is considered to a kind of up-and-coming launch vehicle, in the prospect that all has a very wide range of applications aspect basic medical research and the clinical treatment.

Use at present this technology with the tens of kinds of fusion rotein transfered cells of the about 15～120kD of molecular weight.Express the fusion rotein of N end by prokaryotic expression system for the transduction peptide, protein purification can carry out under the sex change condition, this not only makes the purge process of those fusion roteins that exist with the inclusion body form simplify greatly, the more important thing is albumen through sex change, become chain-like structure relatively flexibly by original native conformation, broken away from the restriction of space conformation, possesses higher energy, thereby make transduction efficiency improve (Nagahara H.et al. greatly, Nat Med, 1998,4:1449-1452).After fusion rotein changes cell over to, folding again under the mate molecule effect, recover native conformation, and make target protein bring into play its biologic activity, the protein transduction domain covalently bound with it can not influence the normal configuration or the function of cell.The speed that imports of the peptide-mediated exogenous molecules of endocellular transduction is exceedingly fast in addition, promptly can finish in the general 15min, and each intracellular protein concentration is close to identical.Thus, both can control intracellular protein concentration, control action kou time accurately again, the final concentration of common 20～200umol/L can produce the biology phenotype (Dowdy SF et al.Methods, 2001,24:247-256).Therefore, compare with the traditional method of albumen being introduced cell such as microinjection, perforin and liposome method etc., the peptide-mediated protein transduction of transduceing has significant superiority.The research report of peptide and the insulin human's fusion aspect of also will not transduceing at present.

[summary of the invention]

[technical problem that will solve]

The present invention overcomes the recombinant human insulin's of present use shortcoming, a kind of transduce peptide technology and non-internal secretion type cell processing insulin human's technology bonded technology is provided, a kind of polynucleotide and corresponding fusion rotein thereof of Tranduced peptide-huamn proinsulin fusion protein specifically are provided, make diabetic can pass through sublingual administration, lung suction people, nasal mucosa and multiple mode medications such as anus bolt or skin absorption, and need not to inject the purpose that can reach effective treatment disease.

[technical scheme]

One of purpose of the present invention provides a kind of polynucleotide of Tranduced peptide-huamn proinsulin fusion protein, and it contains and is selected from following group sequence:

(a) nucleotide sequence shown in the SEQ ID NO.1;

(b) nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO.2;

(c) (a) or the variant sequence of sequence (b), wherein contain one or more insertions, disappearance, interpolation or substitute, and basic (a) or (b) activity of sequence of keeping;

(d) with (a) or sequence (b) sequence at least about 75% identity is arranged;

(e) with (a) or sequence (b) sequence at least about 90% identity is arranged;

(f) (a) or the degeneracy variant of sequence (b); With

(g) complementary sequence of above sequence.

The sequence of these polynucleotide is to have the human proinsulin gene sequence clone of furin restriction enzyme site to the N of transduction peptide or the sequence of C-terminal.

Wherein the human proinsulin gene sequence is the sequence according to design of intestinal bacteria preference codon and chemosynthesis.The transduction peptide sequence is transcribed factors A NTP and is wrapped the PTD of exanthema virus I type VP22 transcription factor merely for PTD sequence, the fruit bat homology abnormal shape of the trans-activator TAT of human immunodeficiency virus's genes encoding, is used for transduction human Regular Insulin and expands to all analogue sequences of using PTD.

Another object of the present invention provides a kind of Tranduced peptide-huamn proinsulin fusion protein, and it contains and is selected from following group aminoacid sequence:

(a) aminoacid sequence shown in the SEQ ID NO.2;

(b) aminoacid sequence of the polynucleotide encoding of SEQ ID NO.1;

(c) with the sequence of the polynucleotide encoding of SEQ ID NO.1 sequence at least about 70% identity is arranged; With

(d) with the sequence of the polynucleotide encoding of SEQ ID NO.1 sequence at least about 90% identity is arranged.

Wherein said fusion rotein, the proinsulin human who it is characterized in that containing the transduction peptide and have the furin restriction enzyme site.

The present invention also provides a kind of Tranduced peptide-huamn proinsulin fusion protein expression vector, it is characterized in that containing the polynucleotide that comprise the SEQ ID NO.1 that operationally is connected with expression control sequenc.

The present invention also provides a kind of Tranduced peptide-huamn proinsulin fusion protein to express and the excretory method, it is characterized in that following steps realize:

(a) design of Tranduced peptide-huamn proinsulin gene is with synthetic;

(b) contain the structure of the expression plasmid of Tranduced peptide-huamn proinsulin dna fragmentation;

(c) contain the Expression of Fusion Protein of Tranduced peptide-huamn proinsulin dna fragmentation.

Described expression method, it adopts the mode of gst fusion protein, His fusion rotein or temperature-induced type fusion rotein to express.

The host cell of described expression vector conversion or transfection can be Bacillus coli cells or yeast cell or insect cell or mammalian cell.

The present invention also provides a kind of Tranduced peptide-huamn proinsulin fusion protein to be used for hypoglycemic application, and it can pass through sublingual administration, lung is inhaled people, nasal mucosa and multiple mode medications such as anus bolt or skin absorption.

Described application comprises the disconnected or composition of the present invention to polypeptide or derivatives thereof of the present invention, polymkeric substance, concatermer or the cyclized structure of patient's administering therapeutic significant quantity, antibody of the present invention or its antigen binding fragment.

Use and can be undertaken by any suitable method, comprise by in intravenously, intraperitoneal, intramuscular, subcutaneous, the nose, using of carrying out of intracutaneous, eye drip, spray nose, suction, anus, vagina, part and oral route.

[beneficial effect]

The Tranduced peptide-huamn proinsulin fusion protein that the present invention expresses by prokaryotic expression system can efficiently pass through various cells and tissue, under the molecular chaperones in born of the same parents and the effect of folding enzymes, through folding, disulfide linkage pairing, recovers native conformation.And contain the proinsulin human of furin recognition site, can in non-endocrine cell, be processed into Regular Insulin, so route of administration can comprise sublingual administration, lung suction, nasal mucosa and multiple modes such as anus bolt or skin absorption with natural insulin physiologically active.

Simultaneously, the present invention is by prokaryotic expression Tranduced peptide-huamn proinsulin fusion protein, through protein purification, not needing that expression product is carried out protein renaturation or trypsinase and protaminase handles, can obtain being used for sublingual administration, lung is inhaled the pharmaceutical grade protein of the treatment diabetes of people, nasal mucosa and mode administrations such as anus bolt or skin absorption, therefore corresponding have a very big advantage at production cost and on the time.

[description of drawings]

This specification sheets comprises four width of cloth accompanying drawings, wherein:

Accompanying drawing 1 is the synoptic diagram of Tranduced peptide-huamn proinsulin gene of the present invention; Wherein 1 mark B of accompanying drawing represents the B chain, and A represents the A chain, and C represents the C chain, and arrow is the restriction enzyme site of furin;

Accompanying drawing 2 is SDS PAGE electrophorograms of pGEX-Pins expression product of the present invention; Wherein: 1, the bacterium before inducing, 2,3,4,5, IPTG inductive transformed bacteria, 6, protein molecular Marker;

Accompanying drawing 3 is SDS PAGE electrophorograms of pBV-Pins expression product of the present invention; Wherein 1,2, IPTG inductive transformed bacteria, 3, the bacterium before inducing, 4, protein molecular Marker;

Accompanying drawing 4 is Westernblotting of pGEX-Pins of the present invention and pBV-Pins expression product; Wherein: 1, pGEX-Pins; 2, pBV-Pins.

[embodiment]

The design of embodiment 1 tat peptide-human proinsulin gene is with synthetic

According to the sub-designer's proinsulin gene of the amino acid code sequence of A, the B of known Regular Insulin, C chain amino acid sequence and intestinal bacteria preference, and carry out transgenation, add one section tat peptide at the N of Regular Insulin end according to the aminoacid sequence of Furin identification.Tat peptide-human proinsulin gene sequence is synthetic by Shanghai Bo Ya company, total length 369bp, SEQ ID NO.1, gene constructed in the pDM-18 cloning vector, the gene two ends are respectively the restriction enzyme site of 5 ' BamHI and 3 ' EcoRI or 5 ' EcoRI and 3 ' BamHI, and synoptic diagram is seen accompanying drawing 1.

Embodiment 2 contains the structure of the expression plasmid of tat peptide-proinsulin human's dna fragmentation

Methods such as the recovery of the extraction of plasmid, PCR, restriction enzyme reaction, agarose electrophoresis, endonuclease bamhi, the connection of DNA and conversion are all carried out (Sambrook J with reference to the method slightly modified of Maniatis laboratory manual introduction, Fritsch EF, Maniatis F. Molecular Cloning:Alaboratory Manual, 2nded, NewYork:Cold Spring Harbor LaboratoryPress, 1989).

With EcoRI and BamHI complete degestion expression vector pGEX-4T-1 (PharmaciaBiotech company) and pBV220 (Beijing preventive medicine academy of sciences structure), reclaim carrier segments; Obtain tat peptide-proinsulin human with EcoRI and BamHI double digestion pDM-Pins plasmid, reclaim the gene fragment of 369bp; PGEX-4T-1 is connected with tat peptide-proinsulin human's fragment with pBV220 carrier endonuclease bamhi, makes up recombinant expression vector pGEX-Pins and pBV-Pins.Through BamH-EcoRI double digestion pGEX-Pins and pBV-Pins, produced the 369bp fragment of expection, gene to insertion checks order simultaneously, and enzyme is cut evaluation and shown with the dna sequence analysis result, and the sequence dna fragment of being cloned in each plasmid is correct with coding reading frame.

Embodiment 3 contains the expression of the gst fusion protein of tat peptide-proinsulin human's dna fragmentation

With recombinant expression plasmid pGEX-Pins transformed into escherichia coli TG1, JM109, DH5 α and BL21 (DE3) (is Time Inc. available from sky, Beijing).Inoculate single bacterium colony in LB/ penbritin (100mg/L) nutrient solution, 37 ℃ of overnight incubation.Get the above-mentioned bacterium 2% that spends the night and be inoculated in fresh LB/ penbritin nutrient solution, 37 ℃ are cultivated 2～3h to A600 is 0.4～0.6, uses 0.1mmo/L IPTG37 ℃ of inducing culture 6h then.A600 is surveyed in sampling, and centrifugal collection thalline adds the ratio suspended sample of 10 μ l water and equal-volume 2 * SDSPAGE sample-loading buffer in the thalline of every part of nutrient solution (A600=0.1), boil 5min after, get 15 μ l and identify expression product by SDSPAGE.The SDS PAGE result of cellular lysate liquid shows, is that the 40kD place presents tangible abduction delivering band (accompanying drawing 2) at molecular weight, and size conforms to the theoretical value of corresponding fusion expressed product respectively.

Collect and express bacterium, the ratio suspension back carrying out ultrasonic bacteria breaking that adds 10ul PBS in the thalline of every part of nutrient solution (A600=0.1), the centrifugal 15min of 15000r/min (4 ℃), precipitation suspends with the PBS of supernatant with equating volume, get an amount of supernatant liquor then respectively and precipitate suspension and add isopyknic 2 * SDS PAGE sample-loading buffer, boil 5min, get on the 15 μ l sample and carry out SDSPAGE and analyze, to determine the solvability of expression product.Solvability analytical results to expression product shows that fusion rotein has the soluble fractions that is present in cellular lysate liquid more than 80%.

Embodiment 4 contains the temperature-induced type Expression of Fusion Protein of tat peptide-proinsulin human's dna fragmentation

With recombinant expression plasmid pBV-Pins transformed into escherichia coli TG1, JM109, DH5 α and BL21 (DE3).Inoculate single bacterium colony in LB/ penbritin (100mg/L) nutrient solution, 37 ℃ of overnight incubation.Get the above-mentioned bacterium 2% that spends the night and be inoculated in fresh LB/ penbritin nutrient solution, 30 ℃ are cultivated 3～4h to A600 is 0.4～0.6, then elevated temperature to 42 ℃ inducing culture 6h.A600 is surveyed in sampling, and centrifugal collection thalline adds the ratio suspended sample of 10 μ l water and equal-volume 2X SDS PAGE sample-loading buffer by the thalline of every part of nutrient solution (A600=0.1), boil 5min after, get 15 μ l by SDS PAGE evaluation expression product.The SDS PAGE result of cellular lysate liquid shows, is that the 13kD place presents tangible abduction delivering band (accompanying drawing 3) at molecular weight, and size conforms to the theoretical value of corresponding fusion expressed product respectively, and density scan discloses and accounts for 24% of total bacterial protein.

Under the inducing of 42 ℃ of temperature, the expression product of pBV-Pins transformed bacteria is the fusion rotein of PTD, 147 amino acid of this albumen total length, infer that molecular weight is about 13kD, collect and express bacterium, the ratio suspension back carrying out ultrasonic bacteria breaking that adds 10 μ l PBS in the thalline of every part of nutrient solution (A600=0.1), the centrifugal 15min of 15000r/min (4 ℃), precipitation suspends with the PBS of supernatant with equating volume, get an amount of supernatant liquor then respectively and precipitate suspension and add isopyknic 2 * SDS PAGE sample-loading buffer, boil 5min, get on the 15 μ l sample and carry out SDS PAGE and analyze, to determine the solvability of expression product.Solvability analytical results to expression product shows that expression product is present in the bacterium with the inclusion body form.

The immunoblotting of embodiment 5 expression products is identified

The expression product of pGEX-Pins and pBV-Pins is behind 12% SDS PAGE electrophoresis, with the half dry type electroporation protein transduction is moved on the nitrocellulose filter, again by with the reaction of the anti-insulin antibody of HRP mark, diaminobenzidine (DAB) colour developing can be analyzed the antigenicity of expression product.

Immunoblotting assay shows that they can produce specific reaction (accompanying drawing 4) with anti-insulin antibody.

The purifying of embodiment 6 pGEX-Pins expression products

Contain glutathione S-transferase (GST) in the fusion protein molecule that pGEX-Pins expresses, it is centrifugal to contain the expression bacteria culture fluid of plasmid pGEX-Pins by 100ml, the thalline that obtains is suspended in 5ml and contains among the PBS (pH7.4) of 10mg/L PMSF and 1%Triton X-100, carrying out ultrasonic bacteria breaking, the centrifugal 20min of 15000r/min (4 ℃), use PBS equilibrated glutathione S eparose 4B post (1ml) on the supernatant in advance, behind PBS flush away foreign protein, cut processing with the zymoplasm enzyme, with 5ml elutriant (50mmol/L Tris HCl, pH8.0) tat peptide-proinsulin human of wash-out cutting-out.

The purifying of embodiment 7 pBV-Pins expression products

Getting 100ml, to contain the expression bacteria culture fluid of plasmid pBV-Pins centrifugal, the thalline that obtains is suspended in 5ml and contains among the PBS (pH7.4) of 10mg/L PMSF and 1%Triton X-100, carrying out ultrasonic bacteria breaking, the centrifugal 20min of 15000r/min (4 ℃) expresses bacterium and obtains inclusion body through ultrasonic degradation, with PBSU solution (20mmol/L phosphate buffered saline buffer, pH7.4,0.5mol/LNaCl, 8mol/L urea) dissolving inclusion body after, carry out Separose G-50 gel chromatography.

The antigenic activity of embodiment 8 expression products is measured

According to measuring the formula that the insulin standard product are set up simultaneously:

Y＝15369.5-2854.5×1nX(r＝-0.97)

Wherein Y represents CPM (countper minute), X representative sample concentration (μ IU/ml), and r is a relation conefficient.CPM value when pressing sample dilution in 1: 1 is calculated the antigenic activity of Regular Insulin.

Radioimmunoassay shows that the fusion rotein of purifying presents the Regular Insulin antigenic activity.

Sequence table

＜110〉The sun comes in the east in Beijing development in science and technology responsibility company limited

＜120〉expression of Tranduced peptide-huamn proinsulin fusion protein and application

<160>2

<210>1

<211>369

<212>DNA

＜213〉artificial sequence

<400>1

ATGTACGGTC?GTAAAAAGCG?TCGCCAGCGT?CGTCGCATGG?CTCTGTGGAT?GCGCCTCCTG?60

CCGCTGCTGG?CGCTTCTGGC?TCTCTGGGGC?CCAGACCCAG?CTGCAGCCTT?CGTTAACCAG?120

CACCTGTGCG?GCTCTCACCT?GGTTGAAGCT?CTCTACCTGG?TTTGCGGTGA?ACGTGGCTTC?180

TTCTACACTC?CGAAAACCCG?CCGTAAACGT?GAGGACCTGC?AGGTGGGTCA?GGTTGAACTG?240

GGTGGCGGTC?CGGGTGCTGG?CAGCCTGCAG?CCGCTGGCTC?TGGAAGGCTC?CCGTCGTAAA?300

CGTGGTATCG?TTGAACAGTG?CTGTACCTCT?ATCTGCTCCC?TGTACCAGCT?GGAGAACTAC?360

TGCAACTGA?369

<210>2

<211>127

<212>PRT

＜213〉artificial sequence

<400>2

M?Y?G?R?K?K?R?R?Q?R?R?R?M?A?L

5 10 15

W?M?R?L?L?P?L?L?A?L?L?A?L?W?G

20 25 30

P?D?P?A?A?A?F?V?N?Q?H?L?C?G?S

35 40 45

H?L?V?E?A?L?Y?L?V?C?G?E?R?G?F

50 55 60

F?Y?T?P?K?T?R?R?K?R?E?D?L?Q?V

65 70 75

G?Q?V?E?L?G?G?G?P?G?A?G?S?L?Q

80 85 90

P?L?A?L?E?G?S?R?R?K?R?G?I?V?E

95 100 105

Q?C?C?T?S?I?C?S?L?Y?Q?L?E?N?Y

110 120 125

C?N

127

Claims

1. the polynucleotide of a Tranduced peptide-huamn proinsulin fusion protein, it contains and is selected from following group sequence:

(a) nucleotide sequence shown in the SEQ ID NO.1;

(b) nucleotide sequence of aminoacid sequence shown in the coding SEQ ID NO.2;

(d) with (a) or sequence (b) sequence at least about 75% identity is arranged;

(e) with (a) or sequence (b) sequence at least about 90% identity is arranged;

(f) (a) or the degeneracy variant of sequence (b); With

(g) complementary sequence of above sequence.

2. polynucleotide according to claim 1, wherein, described sequence is to have the human proinsulin gene sequence clone of furin restriction enzyme site to the N of transduction peptide or the sequence of C-terminal.

3. polynucleotide according to claim 1 and 2, wherein said human insulin gene sequence are the sequences according to design of intestinal bacteria preference codon and chemosynthesis.

4. polynucleotide according to claim 1 and 2, wherein said transduction peptide sequence is transcribed factors A NTP and is wrapped the PTD of exanthema virus I type VP22 transcription factor merely for PTD (ProteinTransduction Domain) sequence, the fruit bat homology abnormal shape of the trans-activator TAT of human immunodeficiency virus's genes encoding, is used for transduction human Regular Insulin and expands to all analogue sequences of using PTD.

5. Tranduced peptide-huamn proinsulin fusion protein, it contains and is selected from following group aminoacid sequence:

(a) aminoacid sequence shown in the SEQ ID NO.2;

(b) aminoacid sequence of the polynucleotide encoding of claim 1;

(c) with the aminoacid sequence of the polynucleotide encoding of claim 1 sequence at least about 70% identity is arranged; With

(d) with the sequence of the polynucleotide encoding of claim 1 sequence at least about 90% identity is arranged.

6. albumen according to claim 5, the proinsulin human who it is characterized in that containing the transduction peptide and have the furin restriction enzyme site.

7. a Tranduced peptide-huamn proinsulin fusion protein expression vector is characterized in that containing the polynucleotide that comprise the claim 1 that operationally is connected with expression control sequenc.

8. a Tranduced peptide-huamn proinsulin fusion protein is expressed and the excretory method, it is characterized in that following steps realize:

(a) design of Tranduced peptide-huamn proinsulin gene is with synthetic;

9. method according to claim 8, it adopts the mode of gst fusion protein, His fusion rotein or temperature-induced type fusion rotein to express.

10. the described expression vector of claim 7 transforms or the host cell of transfection, and it is Bacillus coli cells or yeast cell or insect cell or mammalian cell.

11. a Tranduced peptide-huamn proinsulin fusion protein is used for hypoglycemic application, wherein the albumen with claim 5 or 6 can pass through sublingual administration, lung is inhaled people, nasal mucosa and multiple mode medications such as anus bolt or skin absorption.

12. application according to claim 11 comprises to the proteic antibody of proteic its derivatives of the claim 5 of patient's administering therapeutic significant quantity or 6 or polymkeric substance or concatermer or cyclized structure, claim 5 or 6 or its antigen binding fragment is disconnected or claim 5 or 6 proteic compositions.