CN101817878B - Monomer quick-acting insulin as well as preparing method and application thereof - Google Patents
Monomer quick-acting insulin as well as preparing method and application thereof Download PDFInfo
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Abstract
The invention relates to a new insulin B chain, monomer quick-acting insulin and a medical composition which comprises the monomer quick-acting insulin. The invention also relates to a preparing method and application of the monomer quick-acting insulin.
Description
The application is application on 04 29th, 2006, and application number is dividing an application of CN200610026236.8.
Technical field
The present invention relates to medical science and field of pharmacology.More specifically, the present invention relates to new insulin B chain, new monomer quick-acting insulin and the pharmaceutical composition that contains said monomer quick-acting insulin.The invention still further relates to the method for making of described monomer quick-acting insulin.
Background technology
The onset diabetes rate rises year by year, and it is deadly to cause cardiovascular disorder, is one of principal disease that threatens now human health.Thereby showing according to the result of the United Kingdom Prospective Diabetes Study that the treatment of diabetes measure is orientated as implements meticulous glycemic control and avoids diabetic syndrome.Although Regular Insulin is generally used for type i diabetes, recent research shows that 1/3 type ii diabetes people needs supplementation with insulin with controlling blood sugar concentration.
Being to come regulating and controlling blood sugar concentration with excreting insulin in the normal human, generally is that 30-60 minute blood physiology insulin concentration reaches peak value after dining, returns to base concentration after 4-5 hour, can guarantee that like this blood sugar concentration does not have big fluctuation because of dining.Regular Insulin must with monomeric form could with its receptors bind, bring into play its biological effect then.And common natural insulin preparation is a polymer at neutral injection concentration, absorbs slowly, can not analogue body in phase during the normal biology of Regular Insulin.And monomer insulin is owing to can absorb play a role (Fig. 1) in neutral solution not polymerization of high density very soon.Therefore monomer quick-acting insulin becomes the medicine of new generation of treatment mellitus and other diseases.
Phase during meticulous insulinize (intensive insulin treatment) simulation normal human insulin secretion.Through inject the steady postprandial blood sugar concentration of Semilente Insulin before the meal; And in the injection effect or protamine zine insulin or insulin pump to keep between meal and the blood sugar concentration at night; The hyperglycemia at night appears in prevention, thereby can reduce the sickness rate and the lethality rate of the caused syndromes of mellitus.
Semilente Insulin mainly obtains through gene engineering method at present.The monomer insulin Lyspro of Eli Lilly company exploitation was used for clinical respectively in European and the U.S. in 1996, obtain very good effect.Onset in 15 minutes behind the subcutaneous injection Lyspro.The another kind of monomer insulin Aspart of Novo company research and development at present also goes on the market.(Howey，D.C.，et al.，Diabetes，1994，43：396-402；Torlone，E.et al.，Diabetologia，1994，37：713-720；Rami，B.et al.，Eur.J.Pediatr.1999，156：838-840；Vajo，Z.et al.Pharmacological reviews.2000，52：1-9)。
Used over half a century clinically from the pork insulin that Pancreas Sus domestica extracts, its production technique is very ripe, and cost is also lower.But over past ten years, because genetically engineered insulin human's appearance and application, the market of pork insulin receives great impact, surplus occurs, possibly replaced by the recombinant human insulin fully and withdraw from the market at last.How utilizing this resource is a problem of compeling solving.
In sum, this area presses for the various new monomer insulins of exploitation, especially need develop pork insulin (comprise genetically engineered preparation pork insulin) is carried out process for subsequent treatment, so that easy and improve the utility value of pork insulin at low cost.
Summary of the invention
The object of the invention just provides a kind of new monomer quick-acting insulin.
Another object of the present invention provides the pharmaceutical composition that contains this monomer insulin.
Still a further object of the present invention provides the method for making and the purposes of said monomer insulin.
In first aspect of the present invention, a kind of insulin B chain is provided, described insulin B chain is corresponding to being acidic amino acid on the 22nd on the natural insulin B chain, described acidic amino acid is selected from L-glutamic acid or aspartic acid.
In another preference, described insulin B chain has following aminoacid sequence:
F V N Q H L C G S H L V E A L Y L V C G E X G FFYTPK formula I
Wherein, X is Glu, Asp.
In second aspect of the present invention, a kind of Regular Insulin or its pharmacy acceptable salt are provided, described Regular Insulin is made up of INSULIN A chain and B chain, and described insulin B chain is an insulin B chain of the present invention.
In another preference, described Regular Insulin is monomer insulin, thereby is Semilente Insulin.
In another preference, the structure of said Regular Insulin is following:
Wherein, X is Glu, Asp.
In the third aspect of the invention, a kind of pharmaceutical composition is provided, it contains the Regular Insulin described in the second aspect present invention or its pharmacy acceptable salt, and pharmaceutically acceptable carrier.
In another preference, the formulation of described pharmaceutical composition is tablet, injection, granule, solution.
In fourth aspect present invention, provide a kind of monomer insulin to express precursor, contain the described insulin B chain in the 1st aspect, connection peptides and INSULIN A chain respectively from aminoterminal to carboxyl terminal.
In another preference, described connection peptides is Ala-Ala-Lys.
Aspect the of the present invention the 5th, a kind of isolating polynucleotide are provided, the described insulin B chain that described polynucleotide encoding the present invention is above-mentioned, or the above-mentioned described monomer insulin of code book invention is expressed precursor.
In another preference, described polynucleotide are DNA.
In another preference, the sequence that described coding monomer insulin is expressed the polynucleotide of precursor is ttcgtt aac caa cac ttg tgc ggt tcc cac ttg gtt gag gct ttg tac ttg
Gtt tgcggt gaa gaa ggt ttc tTc tac act cct aag gct gct aag ggt att gtc gaa caatgc tgt acc tcc atc tgc tcc ttg tac caa ttg gaa aac tac tgc aac.
Aspect the of the present invention the 6th, a kind of expression vector is provided, it contains the polynucleotide described in the 5th aspect.
Aspect the of the present invention the 7th, a kind of host cell is provided, it contains the expression vector described in the 6th aspect.
In eight aspect of the present invention, a kind of method for preparing Regular Insulin is provided, may further comprise the steps:
(a) under expression condition, cultivate host cell, said host cell contains the coding monomer insulin expresses the DNA of precursor, expresses precursor thereby give expression to described monomer insulin;
Wherein said monomer insulin is expressed precursor and is contained insulin B chain, connection peptides and INSULIN A chain respectively from aminoterminal to carboxyl terminal, and wherein insulin B chain has following aminoacid sequence:
F V N Q H L C G S H L V E A L Y L V C G EXGFFYTPK，
In the formula, X is Glu, Asp,
And connection peptides is Ala-Ala-Lys;
(b) isolate described monomer insulin and express precursor;
(c) cut described monomer insulin with tryptic digestion and express precursor, thereby downcut connection peptides, form the Regular Insulin that constitutes by INSULIN A chain and insulin B chain;
(d) isolate monomer insulin.
In another preference, described monomer insulin has structure shown in the formula II:
Wherein, X is Glu, Asp.
In nineth aspect present invention; A kind of monomer insulin or its pharmacy acceptable salt are provided; Described Regular Insulin is made up of INSULIN A chain and insulin B chain; Described insulin B chain is removed 8 amino acid and is being added amino acid GX ' corresponding on 23 and 24 of natural insulin B chain at the B chain carboxy-terminal, and wherein X ' is Phe or Phe-NH
2
In another preference, described Regular Insulin has following structure:
In the formula, X ' is Phe, Phe-NH
2Or Phe-OH.
Aspect the of the present invention the tenth, a kind of preparation method who produces the monomer insulin described in the nineth aspect present invention is provided, comprise step:
(1) under trypsinase catalysis, will go B chain carboxylic end octapeptide Regular Insulin (DOI) to contact, thereby between carboxyl terminal that removes B chain carboxylic end octapeptide Regular Insulin (DOI) and GX ', form peptide bond with GX ', form the monomer insulin described in the 9th aspect;
Perhaps,
Under trypsinase catalysis, Regular Insulin or its precursor are contacted with excessive GX ', thereby taking place, enzymatic changes peptide on 22 of insulin Bs, form the monomer insulin described in the 9th aspect;
(2) isolate the monomer insulin described in formed the 9th aspect.
In another preference, it is 1: 10 to 1: 100 with the mol ratio between Regular Insulin or its precursor and the excessive GX '.
In another preference, described Regular Insulin or its precursor or go B chain carboxylic end octapeptide Regular Insulin to derive from people or pig.
In the tenth one side of the present invention, a kind of pharmaceutical composition is provided, it contains the said monomer insulin described in the nineth aspect present invention or its pharmacy acceptable salt, and pharmaceutically acceptable carrier.
In another preference, the formulation of described pharmaceutical composition is tablet, injection, granule or solution.
Description of drawings
Fig. 1 is the time phasor of haemoconcentration behind physiology excreting insulin, monomer insulin, the natural medicament injection of insulin.X-coordinate is the time, and ordinate zou is three kinds of concentration of insulin in the blood.
Fig. 2 has shown that the HPLC of B22Glu-desB30 Regular Insulin prepares the result.C
18Post (Beckman, 4.6x250mm), Mobile phase B is 70% acetonitrile that contains 0.08%TFA, and mobile phase A is the water that contains 0.1%TFA, and (Mobile phase B: 30-50%/30min), flow velocity is 2ml/min to gradient.X-coordinate is the elution time, and ordinate zou is A
280nm
Fig. 3 has shown pH 8.3 polyacrylamide gel electrophoresis results.Gum concentration is 15%, Coomassie brilliant blue dyeing.From left to right be respectively: swimming lane 1: the single-chain insulin precursor (on), pork insulin (descending); Swimming lane 2:B22Glu-desB30 Regular Insulin.
Fig. 4 has shown that dezincify pork insulin concentration in neutral solution is to the influence of its gel chromatography behavior.Superdex75 post, moving phase are the phosphoric acid buffer of pH7.4, and flow velocity is 0.5ml/min; Peptide concentration is respectively from low to high: 38 μ M (0.22mg/mL), 75 μ M (0.43mg/mL), 150 μ M (0.86mg/mL), 300 μ M (1.73mg/mL), 600 (3.46mg/mL) μ M.X-coordinate is RT (unit: minute), and ordinate zou is A
230nm
Fig. 5 has shown that the B22Glu-desB30 concentration of insulin is to the influence of its gel chromatography behavior in neutral solution.Superdex75 post, moving phase are the phosphoric acid buffer of pH7.4, and flow velocity is 0.5ml/min; Peptide concentration is respectively from low to high: 2,5, and 10mg/mL.X-coordinate is that (unit: milliliter), ordinate zou is A to RV
280nm
Fig. 6 shown B22Glu-desB30 Regular Insulin extreme ultraviolet (on) and near ultraviolet (descending) circular dichroism spectrum analysis.Solid line is represented sample B22Glu-desB30 Regular Insulin, and dotted line representes that sample removes B chain carboxylic end L-Ala Regular Insulin.Sample concentration is 35 μ M, and damping fluid is the 20 milli pH7.4 potassiumphosphates that rub.
Fig. 7 has shown DHI-NH in neutral solution
2Concentration to its gel filtration chromatography behavioral implications.Superdex75 post, moving phase are the phosphoric acid buffer of pH7.4, and flow velocity is 0.5ml/min; Peptide concentration is respectively from low to high: 38 μ M (0.19mg/mL), 75 μ M (0.38mg/mL), 150 μ M (0.76mg/mL), 300 μ M (1.52mg/mL), 600 μ M (3.04mg/mL).X-coordinate is RT (unit: minute), and ordinate zou is A
230nm
Fig. 8 has shown dezincify Regular Insulin and DHI-NH
2The resulting partition ratio of Superdex75 column chromatography (Kd) value and peptide concentration relation curve under neutral solution.X-coordinate is a peptide concentration, and ordinate zou is the Kd value.
Embodiment
The inventor finds insulin B chain C end peptide section through extensive and deep research, and the self-polymerization of Regular Insulin is had material impact.More unexpectedly; To go 22 of B30 insulin Bs to be sported by basic aminoacids can obtain insulin analog B22E unexpectedly behind the acidic amino acid and remove B30 Regular Insulin; This Regular Insulin not only has the character of monomer insulin (not polymerization when physiological pH value, higher concentration), and whole biologos is 50% of a normal Regular Insulin.In addition, replace this basic aminoacids, also reduce the proteolytic enzyme restriction enzyme site, thereby reduce the risk in the production process, be convenient to recombinant production.
Know B chain carboxylic end octapeptide Regular Insulin (DOI) at present and gone B chain carboxylic end seven peptide Regular Insulin (DHpI) almost not have insulin activity.The inventor finds that also insulin B chain C end peptide section all plays important effect to the structure and the function of Regular Insulin.Insulin crystal structural analysis proof B21-B23 forms β-turnover, and B24-B26 forms intermolecular β-antiparallel structure.It is of crucial importance that these form dimer to Regular Insulin.The inventor is unexpected to be found; On B chain carboxylic end octapeptide Regular Insulin (DOI) basis; Carboxyl terminal at insulin B chain synthesizes or trypsinase enzymatic transpeptidation reaction through the trypsinase enzymatic, can extremely make things convenient for and add amino acid GX ' cheaply, thereby form the little insulin molecule (having about 40% natural radioactivity) that had both had greater activity; And can not form dimer, thereby be a kind of monomer insulin molecule with greater activity.
Particularly, in first part of the present invention new insulin B chain (B22 is an acidic amino acid) is provided, contain this B chain monomer insulin, contain the pharmaceutical composition of said monomer insulin.Insulin analog B of the present invention
22E goes B30 Regular Insulin (B22 E Des-B30 Regular Insulin) or B22D to remove B30 Regular Insulin (B22DDes-B30 Regular Insulin), expresses its precursor with methanol yeast, cuts through enzyme and obtains highly purified sample.And proving that it has the character of monomer insulin (not polymerization when physiological pH value, higher concentration), whole biologos is 50% of a normal Regular Insulin.
Second section provides six peptide Regular Insulin (DHI) and the verivate (DHI-NH thereof that go that new B chain carboxylic end shortens
2).They are the big fragments with (1) people or pork insulin, promptly go B chain C end octapeptide Regular Insulin (DOI) for raw material warp and glycocoll phenylalanine dipeptide or the dipeptide amide enzymatic is synthetic or (2) pork insulin, insulin human or single-chain insulin precursor are that enzymatic commentaries on classics peptide gets in the B22 position through trypsinase for raw material and dipeptides or dipeptide amide.This para-insulin analogue of experiment proof be a monomer insulin still during with higher concentration in neutral solution, and has the interior vigor of 40% Regular Insulin body.
Term
As used herein, term " B22Glu-desB30 Regular Insulin " refer to B chain 22 amino acids be L-glutamic acid remove B chain carboxylic end L-Ala Regular Insulin.Wherein the substruction of insulin B chain is following:
F V N Q H L C G S H L V E A L Y L V C G E X G FFYTPK formula I (SEQ ID NO:1)
Wherein, X is Glu, Asp.
More preferably, described insulin B chain is as follows:
F V N Q H L C G S H L V E A L Y L V C G E EGFFYTPK(SEQ ID NO：2)
As used herein, term " DHI " is meant formed Regular Insulin behind B chain carboxylic end six peptides.The substruction of a kind of preferred DHI is:
(formula III)
In the formula, X ' is Phe, Phe-NH
2, Phe-OH or other can make it become monomeric group.
In formula III, RGX ' corresponds respectively to the 22nd, 23 and 24 of natural insulin B chain.
As used herein, " polypeptide of the present invention " refer to B22Glu-desB30, contain Regular Insulin and DHI, the DHI-NH of B22Glu-desB30
2And its verivate (comprising pharmacy acceptable salt).
As used herein, term " DOI " is meant that insulin B chain C end clips eight formed insulin analogs of amino acid.
Polypeptide of the present invention and preparation method thereof
Polypeptide of the present invention can be the synthetic polypeptide of recombinant polypeptide or enzymatic, preferred recombinant polypeptide.Polypeptide of the present invention can be an enzymatic synthetic product, or uses recombinant technology from protokaryon or eucaryon host (for example, bacterium, yeast, higher plant, insect and mammalian cell), to produce.
With B22Glu-desB30 Regular Insulin is example, and B22Glu-desB30 Regular Insulin Nucleotide full length sequence of the present invention or its fragment can use the method for pcr amplification method, recombination method or synthetic to obtain usually.In case obtained relevant sequence, just can come to obtain in large quantity relevant sequence with recombination method.This normally is cloned into carrier with it, changes cell again over to, from the host cell after the propagation, separates obtaining relevant sequence then through ordinary method.
Among the present invention, B22Glu-desB30 Regular Insulin polynucleotide sequence can be inserted in the recombinant expression vector.Term " recombinant expression vector " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral like adenovirus, retrovirus or other carriers.In a word, as long as can in host, duplicate and stablize, any plasmid and carrier can be used.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.
Method well-known to those having ordinary skill in the art can be used to make up and contains B22Glu-desB30 Regular Insulin DNA sequences encoding and suitable transcribing/the translate expression vector of wave.These methods comprise extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technology of body etc.Described dna sequence dna can effectively be connected on the suitable promotor in the expression vector, and is synthetic to instruct mRNA.The representative example of these promotors has: colibacillary lac or trp promoter eucaryon promotor comprise CMV immediate early promoter, HSV thymidine kinase promoter, early stage and late period SV40 promotor and some other known may command gene expression promoter in protokaryon or eukaryotic cell or its virus.Expression vector also comprises ribosome bind site and the transcription terminator that translation initiation is used.
In addition; Expression vector preferably comprises one or more selected markers; To be provided for selecting the phenotypic character of transformed host cells; Cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness like eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can marking protein.
Host cell can be a prokaryotic cell prokaryocyte, like bacterial cell; Or eukaryotic cell such as low, like yeast cell; Or higher eucaryotic cells, like mammalian cell.Representative example has: intestinal bacteria, streptomyces; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; Zooblasts such as CHO, COS cell.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results
2Method is handled, and used step is well-known in this area.Another kind method is to use MgCl
2If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (like temperature transition or chemically induced), cell is cultivated for some time again.
The extracellular can expressed or be secreted into to recombinant polypeptide in the above methods in cell or on cytolemma.If desired, can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, ultraly handle, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) is technological with other various LCs and the combination of these methods.
In a preference, B22Glu-desB30 Regular Insulin of the present invention is earlier to express precursor forms with monomer insulin to express, and obtains B22Glu-desB30 Regular Insulin cutting through enzyme to handle then.To carboxyl terminal, monomer insulin is expressed precursor and is contained insulin B chain of the present invention, connection peptides and INSULIN A chain respectively from aminoterminal.
The not special restriction of connection peptides has needed only ligation, and is that Lys or Arg get final product at the amino-acid residue that links to each other with the A chain.One type of connection peptides example is (Ala)
2-5Lys, for example Ala-Ala-Lys.
Enzyme is cut the enzyme that processing can be used any cutting Lys and/or Arg, for example trypsinase.The enzyme tangent condition can change according to the enzyme of selecting for use.A kind of preferred enzyme tangent condition is: solvent: the 100mM ammonium bicarbonate buffers, and pH:7-8, concentration of substrate, about 10mg/ml, the trypsinase consumption is about substrate quality 1/100, and 4-25 ℃, 1-6 hour.
After B22Glu-desB30 insulin precurosor enzyme is cut, separate enzyme with methods such as reverse hydrophobic chromatographies and cut product, can obtain the B22Glu-desB30 Regular Insulin of first part of the present invention.
Monomer insulin shown in the formula III of the present invention can use insulin human, pork insulin or its precursor or go B chain carboxylic end octapeptide Regular Insulin to be raw material, the synthetic or trypsinase enzymatic transpeptidation reaction preparation with the conventional trypsinase enzymatic in this area.
A kind of preferred method method is under trypsinase catalysis; To go B chain carboxylic end octapeptide Regular Insulin (DOI) to contact with GX '; Thereby between carboxyl terminal that removes B chain carboxylic end octapeptide Regular Insulin (DOI) and GX ', form peptide bond, thereby form the monomer insulin shown in the formula III.Then, isolate described monomer insulin.In preference, described trypsinase enzymatic synthesis condition is generally pH6-8.5,30 ℃-37 ℃ of temperature, time 6-24 hour.
Another kind of preferred method is under trypsinase catalysis, Regular Insulin or its precursor contacts with excessive GX ', thereby on 22 of insulin Bs generation enzymatic commentaries on classics peptide, form the monomer insulin shown in the formula II.Then, isolate described monomer insulin.Preferably, the mol ratio of Regular Insulin or its precursor and excessive GX ' is 1: 10 to 1: 100.In preference, described trypsinase enzymatic changes the peptide condition and is generally pH6.5-8.5,30 ℃-37 ℃ of temperature, time 6-20 hour.
Pharmaceutical composition
The present invention also provides a kind of pharmaceutical composition, and it contains monomer insulin of the present invention or its pharmacy acceptable salt of safe and effective amount, and pharmaceutically acceptable carrier or vehicle.
Be applicable to that pharmaceutically acceptable carrier of the present invention comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical prepn should be complementary with administering mode.Pharmaceutical composition of the present invention can be made into the injection form, for example prepares through ordinary method with the saline water or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can prepare through ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity.In addition, polypeptide of the present invention also can use with the other treatment agent.
Pharmaceutical composition of the present invention can be used for treating mellitus and complication thereof.When making pharmaceutical composition; Be that monomer quick-acting insulin of the present invention or its pharmacy acceptable salt with safe and effective amount is applied to Mammals (like the people); Wherein this safe and effective amount is usually at least about 10 micrograms/kg body weight; And in most of the cases be no more than about 10 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage is factor such as considered route of administration, patient health situation also, and these all are within the skilled practitioners skill.
Monomer insulin of the present invention not only can use separately, can also use with the medicine (like other Regular Insulin) of other treatment mellitus.
Major advantage of the present invention is:
(1) Regular Insulin of the present invention has strong monomer character, promptly under neutral pH (like physiological pH), and still can polymerization during higher concentration.
(2) route of administration beyond the pharmaceutical composition that contains the Regular Insulin of this structure is used to inject has very high bioavailability.
(3) the pork insulin world market is abundant and that be on the verge of to be eliminated changes the higher monomer quick-acting insulin analogue of added value into through enzymatic means; Having improved the use natural insulin must be through separating the lag-effect that collecting process brings, and method for making is easy, cheap simultaneously.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; People such as Sambrook for example; Molecular cloning: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
Gene engineering expression B22Glu-desB30 Regular Insulin
In the present embodiment; Hold the formation that links to each other to express precursor (EPIP) with connection peptides (like Ala-Ala-Lys) with its A chain N at the B chain C end of B22Glu-desB30 Regular Insulin, after biosynthesizing, fold and form correct disulfide linkage because the structure of precursor helps Regular Insulin.Step is following:
Obtain precursor-gene with the DNA compound method, sequence is as follows;
ttc gtt aac caa cac ttg tgc ggt tcc cac ttg gtt gag gct ttg tac ttg
gtt tgc ggt gaa gaa ggt ttc ttc tac act cct aag gct gct aag ggt att gtcgaa caa tgc tgt acc tcc atc tgc tcc ttg tac caa ttg gaa aac tac tgc aac(SEQ ID NO：3)
Two ends utilize BamH1 and EcoR1 restriction enzyme site to be cloned in pPIC9K (available from the INVITROGEN company) plasmid and obtain pPIC9K/EPIP after adding BamH1 and EcoR1 restriction enzyme site.PPIC9K/EPIP is transformed into after the Bg1II linearization process among the conventional methanol yeast P.pastoris (available from INVITROGEN company), selects the bacterial strain that changes precursor-gene over to high density G418, obtains the methanol yeast engineering bacteria of a plurality of expression EPIP.
Engineering bacteria to selecting passes through high density fermentation, obtains tunning.Tunning obtains EPIP through hydrophobic chromatography.
The result shows that EPIP is after tryptic digestion (solvent: 100mM ammonium bicarbonate buffers, pH:7.8,4 ℃, 2 hours) is handled, and through sieve chromatography, HPLC (Fig. 2) purifying obtains highly purified B22Glu-desB30 Regular Insulin (Fig. 3).The mass spectroscopy molecular weight is 5677.8, and is consistent with theoretical value.
The mensuration of dezincify Regular Insulin auto-polymerization character
In the present embodiment, measure auto-polymerization character with ordinary method to dezincify Regular Insulin.
The Regular Insulin of higher concentration exists with the form of mixtures of monomer, disome, hexasomic in neutral solution.Use sieve chromatography: Superdex 75 (HR 10/30) post; Moving phase is phosphate buffered saline buffer, and pH7.4, flow velocity are 0.5ml/min; Applied sample amount is 40 microlitres; Room temperature, 230nm detects, and peptide concentration is respectively from low to high: 38 μ M (0.22mg/mL), 75 μ M (0.43mg/mL), 150 μ M (0.86mg/mL), 300 μ M (1.73mg/mL), 600 (3.46mg/mL) μ M.The molecular-weight average of mixture is described with partition ratio Kd.Kd=(Vr-V
0)/(Vc-V
0), wherein Vr is an efflux volume, V
0Be void volume, Vc is total column volume.
The result shows that along with the rising of dezincify insulin concentration, its RT on Superdex 75 posts shortens, asymmetry of peak strengthens; The Kd value constantly reduces (Fig. 4; Table 1, Fig. 8), the Regular Insulin that higher concentration is described is the inhomogenous multiple polymeric mixture that contains in neutral solution.The natural insulin drug concentration is 250 μ M (1.5mg/ml), is that form with polymkeric substance exists according to The above results Regular Insulin under injection concentration.
The Kd value of table 1 natural insulin on molecular sieve Superdex 75 (HR 10/30) column chromatography
The mensuration of B22Glu-des-B30 Regular Insulin auto-polymerization character on molecular sieve column chromatography
In the present embodiment, the monomer insulin of embodiment 1 preparation is measured its auto-polymerization character with ordinary method.
Except that peptide concentration, implementation condition is with embodiment 2.Peptide concentration is: 2mg/ml, 5mg/ml, 10mg/ml. be along with the rising of the concentration of polypeptide, the appearance time of B22Glu-desB30 Regular Insulin, and peak shape and Kd value do not change (Fig. 5, table 2) with peptide concentration.This explanation is to exist with monomeric form at injection concentration 500 μ M (3mg/ml) B22Glu-desB30 Regular Insulin.
Table 2B22Glu-desB30 Regular Insulin
Kd value on molecular sieve column Superdex 75 (HR 10/30) column chromatography
Embodiment 4
The mensuration of B22Glu-desB30 Regular Insulin auto-polymerization character on the circular dichroism spectrometer
One, method:
PH 7.4 potassium phosphate buffers, sample concentration: 35 μ M, room temperature, the Jasco-715 spectropolarimeter, far-ultraviolet spectrum scans 190nm from 250nm in the wide sample pool of 0.1cm,, near-ultraviolet spectrum scans 300nm from 245nm in the wide sample pool of 1.0nm.
Two, result:
B22Gludes-B30 Regular Insulin is compared with the B chain carboxylic end L-Ala Regular Insulin that goes with concentration; Absorption negative value at 208nm and 273nm obviously reduces (Fig. 6); Even be illustrated under the lower peptide concentration of neutral solution, the monomer content in the B22Glu-des-B30 insulin solutions also will be higher than and removes B chain carboxylic end L-Ala Regular Insulin with concentration.Go the aggregation property of B chain carboxylic end L-Ala Regular Insulin identical with natural insulin.
Biological activity determination in the B22Glu-desB30 Regular Insulin body
One, method:
Mice convulsion method (Chinese Pharmacopoeia, 1985)
Male mouse of kunming, body weight are 18-22g, divide four groups at random by body weight, and 10 every group, fasting is 2 hours before the experiment.It is 2.5 physiological saline solution that sample uses hydrochloric acid to transfer to the pH value, and when peptide concentration was 1mg/ml, the 280nm absorption value was 1.Sample estimates are tired and are 50% of standard substance, and high dosage is 0.27U/ml, and low dosage is 0.135U/ml, and solvent is that hydrochloric acid adjusting pH is 2.5 saline water.25 ℃, every mouse subcutaneous injection 0.2ml in 15 minutes places 37 ℃ to observe 90 minutes, and is convulsions, dead, it is lain on the back can stand up the person voluntarily in back 3 seconds and all think positive reaction.Data are calculated by the qualitative response assay method.
Two, result:
The mice convulsion method records the active 13.5U/mg that is, fiducial limit rate FL% is 23.5% (＜30%) (table 3); Used standard substance are the Regular Insulin of the 27U/mg that middle inspection provided in the experiment.The activity of B22Glu-desB30 Regular Insulin is 50% of a natural insulin.
Table 3 mice convulsion method is measured the biological activity (Chinese Pharmacopoeia, 1985 editions) of B22Glu-desB30 Regular Insulin
Take DOI as the synthetic DHI-NH of raw material enzymatic
2Or DHI
The H-Gly-Phe-NH of 60mg DOI (removing B chain carboxylic end octapeptide pork insulin) and 120mg
2Be dissolved in 2 milliliters and contain 0.2M Tris, among 70%1.4-butyleneglycol and the 10%DMSO, pH transfers to 7.6, adds the trypsinase that TPCK-handles, and solution reacted 20 hours down at 37 ℃, and enzyme is used 6mg altogether, divides three addings, promptly 0,2 and 6 hour.Reacted after enzyme and unnecessary little peptide are removed in Sephadex G50 gel-filtration, the main peak freeze-drying is after DEAE-Sepharose CL-6B column chromatography for separation obtains pure DHI-NH
2
DHI-NH
2Mass spectrum be accredited as 5067, meet theoretical molecular 5068.With DOI is that raw material can get DHI with GlyPhe and trypsinase enzymatic condensation, and the mass spectroscopy of DHI is 5069, meets theoretical molecular 5069.6.Condition by embodiment 5 records DHI and DHI-NH
2Whole biologos is 10.8IU/mg, is 40% of natural insulin.
The trypsinase enzymatic changes the peptide method and prepares DHI and DHI-NH
2
Pork insulin, insulin human or their precursor obtain DHI or DHI-NH through the transpeptidation reaction of trypsinase on the B22 position in the presence of excessive GX '
2Existing is example with the human insulin precursor:
The human insulin precursor lyophilized powder dissolves with DMSO, adds 10 times of excessive GX ' (mole ratio, wherein X '=Phe).Add 1, the 4-butyleneglycol is transferred pH to 6.5-7.0 with ammoniacal liquor, adds water to DMSO: water: 1, and 4-butyleneglycol=15: 15: 70 (volume ratio).The concentration of precursor is 20-30mg/ml, adds the trypsinase of precursor 1/5 weight then.Reaction is spent the night under 35 degrees centigrade.Reaction solution is used sour acetone precipitation, and is centrifugal, removes supernatant, and deposition is separated with IX or HPLC after removing acetone, gets the purpose product.
The result is identical with embodiment 6, and the mass spectroscopy of DHI is 5069, meets theoretical molecular 5069.6.Condition by embodiment 5 records DHI and DHI-NH
2Whole biologos is 10.9IU/mg, is 40% of natural insulin.
DHI-NH
2The mensuration of Regular Insulin auto-polymerization character
Implementation condition is fully with embodiment 2.
Along with the rising of peptide concentration, DHI-NH
2The appearance time of Regular Insulin, peak shape and Kd value hardly with the variation of peptide concentration change (Fig. 7, table 4, Fig. 8).This explanation DHIX series Regular Insulin is monomer insulin.
Table 4DHI-NH
2Kd value on molecular sieve Superdex 75 (HR 10/30) column chromatography
Pharmaceutical composition
Mix each component by following prescription, make the injection of insulin B 22Glu-Des30 (formula II) and DHI (formula III).
Prescription A
| B22Glu-Des30 (formula II) | 2g |
| Water for injection | 1L |
| NaCl | 9g |
| Human serum albumin | 5g |
Prescription B
| DHI (formula III) | 2g |
| Water for injection | 1L |
| NaCl | 9g |
| Human serum albumin | 5g |
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
< 110>Shanghai Bio-Tai Life Sciences Research Co., Ltd.
< 120>monomer quick-acting insulin and method for making thereof and purposes
<130>061725
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<221>misc_feature
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<400>1
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr
1 5 10 15
Leu Val Cys Gly Glu Xaa Gly Phe Phe Tyr Thr Pro Lys
20 25
<210>2
<211>29
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Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr
1 5 10 15
Leu Val Cys Gly Glu Glu Gly Phe Phe Tyr Thr Pro Lys
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<222>(1)..(159)
<223>
<400>3
ttc gtt aac caa cac ttg tgc ggt tcc cac ttg gtt gag gct ttg tac 48
Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr
1 5 10 15
ttg gtt tgc ggt gaa gaa ggt ttc ttc tac act cct aag gct gct aag 96
Leu Val Cys Gly Glu Glu Gly Phe Phe Tyr Thr Pro Lys Ala Ala Lys
20 25 30
ggt att gtc gaa caa tgc tgt acc tcc atc tgc tcc ttg tac caa ttg 144
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu
35 40 45
gaa aac tac tgc aac 159
Glu Asn Tyr Cys Asn
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Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr
1 5 10 15
Leu Val Cys Gly Glu Glu Gly Phe Phe Tyr Thr Pro Lys Ala Ala Lys
20 25 30
Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu
35 40 45
Glu Asn Tyr Cys Asn
50
Claims (4)
1. a monomer insulin or its pharmacy acceptable salt; Described Regular Insulin is made up of INSULIN A chain and insulin B chain; It is characterized in that; Described insulin B chain is removed 8 amino acid and is being added amino acid GX' corresponding on 23 and 24 of natural insulin B chain at people or pork insulin B chain carboxy-terminal, and wherein X' is Phe or Phe-NH
2
2. monomer insulin as claimed in claim 1 or its pharmacy acceptable salt is characterized in that, described Regular Insulin has following structure:
In the formula, X' is Phe, Phe-NH
2Or Phe-OH.
3. a preparation method who produces the described monomer insulin of claim 1 is characterized in that, comprises step:
(1) under trypsinase catalysis, will go B chain carboxylic end octapeptide Regular Insulin DOI to contact, thereby between carboxyl terminal that removes B chain carboxylic end octapeptide Regular Insulin DOI and GX', form peptide bond with GX', form the described monomer insulin of claim 1;
Perhaps,
Under trypsinase catalysis, Regular Insulin or its precursor are contacted with excessive GX', thereby taking place, enzymatic changes peptide on 22 of insulin Bs, form the described monomer insulin of claim 1;
(2) isolate the described monomer insulin of formed claim 1.
4. a pharmaceutical composition is characterized in that, it contains the said monomer insulin of claim 1 or its pharmacy acceptable salt, and pharmaceutically acceptable carrier.
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Non-Patent Citations (2)
| Title |
|---|
| Cui DF et al.Monomeric Destetrapeptide human insulin from a precursor expressed in Saccharomyces cerevisiae.《J Pept Res.》.2001,第57卷(第3期),188-192. * |
| 张弘等.一种重组抗胰蛋白酶单体人胰岛素突变体的设计、制备和鉴定.《生物化学与生物物理学报》.2000,第32卷(第6期), * |
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