CN1162600A - Mutant human growth hormones and their uses - Google Patents

Abstract

In accordance with the present invention, there are provided mutant human growth hormone proteins which exhibit enhanced affinity for growth hormone but lowered hormone activity, base sequences encoding the same and their production processes as well as uses of said proteins. The proteins according to the present invention, with their enhanced affinities for the growth hormone receptor, can inhibit the binding of growth hormone to its receptor, while they retain lowered growth hormone activities, thus finding application as a medicament for the treatment of acromegaly and gigantism.

Description

Mutant human growth hormones and their application

The present invention relates to the mutant human growth hormones albumen of aminoacid sequence that a kind of Fig. 1 of having gives, a kind of deoxynucleotide with base sequence of encode such amino acid sequences, the mutant human growth hormones albumen that all has certain aminoacid sequence, wherein its amino-acid residue part is partly replaced, insert or lack extremely to a certain degree, but do not cause it that growth hormone receptor is shown the enhanced affinity and keep its active characteristic feature forfeiture of tethelin that has reduced, and relate to their application in the medicine of preparation treatment gigantosoma and acromegaly.

The active human growth hormone of lifeless matter according to the present invention works as the antagonist of normal growth hormone receptor, thereby disorder and hypertrophy that inhibition causes owing to excessive secretion, and can be used as the medicine that the treatment of gigantosoma and acromegaly is had the security of improvement.

Because by the gene unconventionality that tethelin brings, gigantosoma and the acromegaly having known growth retardation that the deficiency owing to tethelin causes already and caused owing to overexpression.For growth hormone deficiency, be extensive use of the therapy of supplementation with growth hormones, but also untappedly so far gone out for gigantosoma and the effective medicine of acromegaly.

In 1978, reported first such as Kowarski were found the active tethelin of a kind of lifeless matter (a kind of mutant tethelin) (Kowarski .J.Clin.Endocrinol.Metab. such as A.A., 47:461,1978).Yet, on molecular level, mutant tethelin is not described yet so far, though openly be reported in the unusual polymer (Valena that identifies a kind of tethelin in the blood from the child who suffers from nanism, L.J. etc., N.Engl.J.Med., 312:214,1985).In the blood of the recycle system, be found the child of containing the active tethelin of lifeless matter and demonstrate the high blood levels of mutant tethelin, and the low haemoconcentration of rhIGF-1 (IGF-1), thereby slow g and D caused.But this kind growth retardation is feature (Hayek A. etc., Pediatr.Res., 12:413,1973 the normal growth hormone of using is had good reaction; Rudman, D. etc., N.Engl.J.Med., 305:123,1981; Plotnick, L.P. etc., Pediatrics 71:324,1983; Bright, C.M. etc., 71:576,1983).

In some years recently, protein engineering and engineered development made to hormone with they acceptor combine and the structural research of their active initiations becomes possibility, and understood the reason of various genetic diseasess thus.

Cunningham etc. come the binding site of growth hormone receptor is studied by using protein engineering method to prepare a large amount of human growth hormone varients, and therefore identify the zone that participates in tethelin and its receptors bind, this zone has formed by N-terminal (2-19) amino-acid residue, zone (the Cunningham that C-terminal (54-74) amino-terminal end and C-terminal (167-191) amino-acid residue are formed, B.C. etc., Science 243:1330,1989).

In addition, Uchida etc. have prepared amino-acid residue by the different metathetical tethelin varient of process, measure their differentiation activity thus to 3T3-F 442A cell, therefrom gain enlightenment is aminoacid sequence 62-67 zone for the bioactive performance (Uchida etc. that are extremely important, Biochem.Biophys.Res.Commun., 172:352,1990).

Recently, the crystallography research to the combination of human growth hormone conjugated protein with it (part of receptor protein) obtains a noticeable discovery (De Vos A.M. etc., Science 255.306,1992); Its hypothesis tethelin combines with growth hormone receptor continuously in one way, this mode is that the functional zone 1 of first locational tethelin combine with first growth hormone receptor, then second functional zone 2 of tethelin combine with second growth hormone receptor, thereby formed the dimer of growth hormone receptor, so the signal of tethelin is passed in the cell.

Though other interested be the zone 1 of human growth hormone with different from the amino-acid residue in zone 2, the binding site of receptor protein has shown the common amino-acid residue.But also recognize the tethelin varient that produces by protein engineering competitively with receptors bind (Fuh G. etc., Science, 256:1677,1992).

The latest developments of genetic analysis have made identification cause the aberrant gene of a large amount of genetic diseasess to become possibility.Gene for the tethelin that causes nanism also is like this.Because its physiologically active of tethelin performance is to mediate by the acceptor on the cytolemma, therefore the gene unconventionality relevant with tethelin can be divided into two classes roughly, and promptly unusual the and tethelin of acceptor gene itself is unusual.

Because tethelin is present on the euchromosome, so it is by the known form of taking recessive inheritance.In order to allow these unusual phenotypic expressions, therefore require the allelotrope of parental generation to produce simultaneously unusually.

In the past, reported the growth retardation that many cases produce owing to the complex combination of the mutant of parental generation growth hormone gene, whole disappearance for example, excalation and base substitution.When one of parental generation for just often, known mutations type tethelin is present in cell endocrine granules inside.

Yet, the analysis of the molecular level of the mutant tethelin that produces for the missense mutation in the organism with and in organism, role is not carried out detailed research yet.Existing known is not the effective ways that suppress the tethelin over-effect.

The inventor finds to have the high serum-concentration that the 5 years old boy who suffers from nanism who postpones the stone age demonstrates tethelin, and the IGF-1 that in induction experiment, keeps lower level, though he demonstrates the serum-concentration that increases tethelin, this is found and ensuing other further result of study becomes the present invention.

As if these endocrinological discoveries may consistent with the phenomenon of noticing in tethelin insensitivity syndromes (Rosen bloom, A.L., Acta Pediatr.Scand. (Suppl), 383:117,1192).

Yet continuous administration tethelin has caused the obvious raising of patient's growth, and this has got rid of the possibility that it is diagnosed as Laron type syndromes, because Laron type nanism is owing to the unusual of growth hormone receptor causing.

The inventor uses the Nb2 bioanalytical method to find that the growth hormone of serum of being found is a kind of tethelin of non-activity type in the child who suffers from this class defective, it is the sort of not resemble normal child's excretory, and by using isoelectrofocusing that this hormone is accredited as mutant tethelin.

Find that this mutant tethelin replaced arginine residues (R → C) (Fig. 1) with cysteine residues on the codon 77 of tethelin.The metathetical site is positioned on the second α spiral of tethelin, with the site 1 of receptors bind after (Cunningham, B.C. etc., Science, 254:821,1991).Substituted halfcystine has been considered to form a new disulfide linkage, and makes the molecular changes electric charge of generation, and causes conformational change, has the tethelin active mutant tethelin that has reduced thereby produced.

In the intracellular signal transduction of tethelin, be considered to extremely important (Argetsinger by the dimerisation of part bonded growth hormone receptor and the phosphorylation of the tyrosine residues in their protein, L.S. etc., Cell, 74:237,1993:Silva, C.M. etc., J.Biol Chem.269:27532,1994).

Growth hormone binding protein is positioned at born of the same parents' ectodomain, and plays tethelin holder (Herington, A.C. etc., Acta Endocrinol (Copenh), 124:14,1991) in the serum in vivo.

Find mutant tethelin to the affinity of tethelin bind receptor about 6 times (Fig. 7 and 8) than wild-type, the functional zone 1 and 2 that this means mutant tethelin demonstrate the different affinity from the acceptor of wild-type.The biological property of mutant tethelin is the active obvious reduction of cell signalling that the phosphorylation by acceptor forms, although it is big to the affinity of receptor protein.

After using wild-type tethelin in continuous three days for patient, do not cause remarkable reaction to IGF-1, and when in the time of an elongated segment, use with rise antagonist be used for suppressing the combining of endogenous mutant secretion of growth hormone and it and acceptor the time, it is found in the plasma concentration that increases IGF-1 and the promotion g and D and works.

Therefore, these discoveries make the inventor reach a conclusion, i.e. mutant tethelin, and when being applied to the patient who suffers from the gigantosoma that caused by the tethelin excessive secretion and acromegaly, the work that can play antagonist is in order to suppress their hypertrophy.

Finished the present invention on the basis of new discovery in the above, and relate to the mutant human growth hormones albumen of (1) aminoacid sequence that a kind of Fig. 1 of having gives, (2) has the deoxynucleotide of the base sequence of encode such amino acid sequences, (3) all has the mutant growth hormone protein of certain aminoacid sequence, wherein this amino-acid residue part is partly replaced, insert or lack extremely to a certain degree, but do not cause that it demonstrates to the enhanced affinity of growth hormone receptor and keep its active characteristic feature forfeiture of tethelin that has reduced, and (4) their application.

Mutant human growth hormones of the present invention is because its endogenic identity, organism is not produced any side effect, yet their induced growths is slow, and can find application thus, sick also untappedly go out therapeutical agent for these in the past as the active drug of treatment gigantosoma and acromegaly.Mutant tethelin demonstrates and exceeds about 6 times receptor affinity, and can be equal to or less than the medicine that the dosage that adopts in the nanism treatment is used as the treatment gigantosoma.

According to prior art, can new mutant tethelin DNA of the present invention be carried out the excalation of Nucleotide easily also feasiblely, thereby insert or replace to produce and have the enhanced receptor affinity, but the tethelin varient of essentially no hormonal activity, it illustrates by sequence shown in Fig. 2 and 3.By using protein engineering, not only can discern the binding site of mutant tethelin and acceptor, and can produce the peptide of this binding site in addition, thereby utilize its medicine as treatment gigantosoma and acromegaly.

New mutant tethelin of the present invention can pass through the hormone coding DNA chain to reproducible plasmid, follows with the plasmid transformant and cultivate these host cells to produce.These host cells comprise bacterium, yeast and zooblast.

Prokaryotic cell prokaryocyte, for example bacterium is suitable for the clone of deoxynucleotide.For example, come from the gene that colibacillary plasmid pBR322 contains anti-penbritin or tsiklomitsin, the method for the transformant of a practicable identification generation is provided.In addition, bacterial plasmid contains the promotor that works and handle in the expression of they oneself protein matter.

Except that prokaryotic cell prokaryocyte, the eukaryotic cell that comprises yeast also is an available, at yeast belong (Saccharomyces), can adopt plasmid YRp7 (Stinchcomb etc., Nature, 282:39,1979) in the expression of a bacterial strain of zymic usually.

Also can use zooblast as host cell, their clone comprises for example HeLa cell, CHO (Chinese hamster ovary) cell, COSM6 and COS-7, polyomavirus thus, adenovirus 2, the promotor of cytomegalovirus and monkey disease poison plays favourable work in order to control the plasmid expression (Thomsen etc. of these clones, PNAS, 81:659,1984).

Animal can be used mutant tethelin or its varient immunity, thereby produce their antibody.In addition, but can be with animal with the currently known methods immunity with the monoclonal antibody of preparation from the cell of secreting specificity antibody.

According to the present invention, can easily prepare mutant tethelin and its varient in a large number, and can provide they the better understanding on molecular level, this makes might develop treatment of diseases relevant with tethelin and diagnostic reagent.This comprises the preparation of gene therapy medicine, and it provides the essential methods of treatment for these diseases.

The Nucleotide of mutant tethelin or the proteinic Nucleotide of binding site can be incorporated into and comprise from retrovirus, in the appropriate carrier of the virus vector of adenovirus etc., this provides they possibilities as the medicine of the gene therapy of gigantosoma and acromegaly.

One example is described to illustrate in greater detail the present invention, wherein below with reference to accompanying drawing

Fig. 1 be the mutant tethelin of gained among the embodiment 1 aminoacid sequence (on 77 positions, R → C) and this proteinic base sequence of coding.

Fig. 2 be among the embodiment 1 gained pass through replace that (53 positions, C → A) produces aminoacid sequence and this proteinic base sequence of coding of the mutant tethelin of sudden change.

Fig. 3 be among the embodiment 1 gained pass through replace that (165 position C → A) produce aminoacid sequence and this proteinic base sequence of encoding of the mutant tethelin of sudden change.

Fig. 4 a is the gene structure of mutant tethelin, the primer design of pcr amplification; Indicate by frame in five exxon sites, and the PCR primer is indicated by arrow;

Fig. 4 b wherein demonstrates at codon 77 arginine and changes into halfcystine for the photo of the dna sequence dna that shows mutant tethelin, and it is determined by using PCR that genomic dna and RNA are carried out the direct sequence analysis.

Fig. 5 is for changing into 53 or 165 locational halfcystines the schema of the construction cDNA of L-Ala, and the oligonucleotide with following sequence is used as primer:

f1：5′ACAGAAACAGGTGGGGGCAA3′

f2：5′AATAGACTCTGAGAAAGCGGG3′

f3：5′GTCCATGTCCTTCCTGAAGGCGTAGAG3′

Use primer f1 and R2 to carry out pcr amplification, 53 suddenling change in the position, (C → A) uses primer f1 and the R3 (C → A) that 165 suddenlys change in the position simultaneously.The cDNA downstream part for preparing the normal growth hormone respectively after digesting with Restriction Enzyme HinfI and NIaIII then is bonded on the PCR product with ligase enzyme respectively.

Fig. 6 is the figure of isoelectrofocusing (IEF) result of mutant and wild-type tethelin in the demonstration serum.Serum sample (150-300ul) is carried out isoelectrofocusing in 1% HPMC-4% An Folin (PH3.5-8.0), separate, merge the sample fraction, and analyze the immunocompetence of tethelin (■), the pH gradient that forms during IEF is represented with (◆).Be considered to cause iso-electric point to reduce pH0.16 by replace arginic sudden change with halfcystine.The peak of wild-type and mutant tethelin is represented with white and black arrow respectively.

A; Propositus (patient a: boy) b: father

Fig. 7 a is for showing how wild-type and mutant tethelin suppress ¹²⁵The human growth hormone of I-mark and IM-9 cell bonded figure.

The cultivation ultimate density is 1-3 * 10 ⁷The cell of/ml (IM-9), the while adds wild-type and mutant tethelin: 0.8ng/ml[according to the funtcional relationship of their add-on-concentration with the concentration that increases ¹²⁵I]-human growth hormone (Du ' pont, USA) (0.33uCi/ml), the total solution of 250ul, 30 ℃ of mark.Cultivate after 4 hours, collection, washed cell detect the radioactivity that is bonded on the cell.

Fig. 7 b for show to suppress [ ¹²⁵I]-human growth hormone of mark and the bonded figure of growth hormone binding protein.

Descend to incite somebody to action at 4 ℃ [ ¹²⁵I]-human growth hormone (0.6uCi/ml) of mark, (Mab 263 for recombinant human somatropin conjugated protein (0.6nM) and antigrowth hormone acceptor mouse clonal antibody; AGEN, Australia) cultivated 16 hours (1: 100,000), increases wild-type and mutant tethelin concentration separately simultaneously, then adds 10% anti-mouse IgG (goat) antibody (50ul), 1% normal mouse serum (50ul) and 5%PEG (300ul).Under 4 ℃, reaction soln is cultivated 4 hours and centrifugal again, detect precipitation (pellet) by γ-counter, express the mean value of measuring for three times.

Fig. 8 a is for showing the electrophoretogram photo that depends on the tyrosine phosphorylation effect of wild-type and mutant tethelin in the IM-9 cell.

Under 37 ℃, there is and lacking 100ng/ml human growth hormone (swimming lane 1 and 2); There is wild-type tethelin (swimming lane 3; 10ng/ml, swimming lane 4; 100ng/ml), mutant tethelin (swimming lane 5; 10ng/ml, swimming lane 6; 100ng/ml) and the mutant tethelin of 10ng/ml fixed concentration, increase the wild-type tethelin (swimming lane 7 of concentration; 10ng/ml, swimming lane 8; 25ng/ml, swimming lane 9; 50ng/ml, swimming lane 10; Respectively the IM-9 cell was handled 15 minutes in the time of 100ng/ml).Precipitate the stain remover lysate of these cells and by using the Western engram analysis with horseradish peroxidase bonded same antibody to detect with the tyrosine-specific antibody mediated immunity of phosphorylation.The molecular weight form that with the kilodalton is unit is shown in the left side.

" arrow " symbol refers to that tyrosine that the effect by tethelin produces is by the protein band of phosphorylation.

Fig. 8 b is the bar block diagram of the optical density(OD) detected result of the tyrosine immunoblotting assay of the anti-phosphorylation of demonstration p120.

The optical density method determines that tyrosine is by the amount of the p-120 of phosphorylation (the IM-9 cell is in the news and is called JAK2).The intensity of optical density(OD) is with respect to being represented by the resulting value of handling as no tethelin of contrast.The mean value that shown is from the income value of three independent experiments (± SEM), statistical study is finished by student t-test.Embodiment 1

Experiment is to carry out with the blood sample of once mentioning above taking from that demonstrates the 5 years old boy who suffers from nanism who postpones the stone age below: the hormone assay method

The concentration that the radioimmunoassay kit that use is produced by Pharmacia of Sweden comes tethelin in the serum analysis, method (Tanaka T. etc. with the Tanaka that revises a little etc., J.Cli.Endocrinol Metab., 51:1058,1980) biological activity of mensuration tethelin, in the Nb2 bioanalytical method, (NIDDK-resists-hORK-IC5 with people's prolactin (hPRL) rabbit anti-serum thus; NIH) with 100,000 times extent of dilution add with suppress people's prolactin in and the activity of stimulating growth.By these methods, measured and analyzed the tethelin in the patient that suffers from nanism and normal subjects's in contrast the serum.Isoelectrofocusing

The method (Tsventnitsky V. etc., Biochem.J., 307:239,1995) of use Tsventnitsky etc. is carried out isoelectrofocusing; With the pH gradient of pH3.0-8.0 serum sample is carried out isoelectrofocusing to separate with 1% HMPC (Vltra tears)-4% An Folin damping fluid, therefrom collect the analysis that different fractions is used for immunocompetence tethelin.Combining anteserum sample from 10 normal subjectses is used as contrast.The separation of mutant growth hormone gene and genetic analysis

Isolation of genomic DNA from peripheral blood leucocyte (Gross-BellardM. etc., Eur.J.Biochem., 36:32,1973) and by PCR method amplification (Fig. 4).What be called F3 is oligonucleotide; 5 ' TATGAATTCCTCTGCCTGCCCTGCCTCAAGAG3 ', GAD:5 ' CTAACACAGTCTCTCAAAGT3 ', GSD:5 ' ACTTTGAGAGACTGTGTTAG3 ', GAE:5 ' TGGAGTGGCAACTTCCAGGG3 ' and GHS1:5 ' CTCAGGGTCCTGTGGACAGCTCACCTAGCTGCA3 ' are used as the promotor that genomic dna increases.

Carry out pcr amplification by following method: at 92 ℃, F3-GAD and GHS1-GAD are finished 3 minutes sex change first, then carry out 35 times by under 92 ℃, sex change in 1 minute, under 60 ℃, 2 minutes renaturation under 72 ℃, stretched the circulation of forming in 2 minutes, last circulation extends in to be carried out under 72 ℃ 7 minutes, and for GSD-GAE, will be by under 92 ℃, sex change in 1 minute, under 60 ℃, under 2 minutes renaturation and 72 ℃, stretched the circulation of forming in 2 minutes to repeat 35 times, only in the most last circulation stretching, extension was carried out 7 minutes.

Extract amplified production, then subclone to pBS SK (+) (Stratagene, USA) or pT7blue (Novagem, USA) and with the 373ADNA sequenator (Perkin Elmer USA) checks order.Mutational site in the further evaluation patient dna (Arg → Cys), and (Gibco BRI USA) carries out resulting PCR product direct dna sequencing to get rid of any wrong reaction that is taken place in the PCR reaction to use the duDNA cycle sequencing test kit.Found that patient dna has replaced arginine residues (Fig. 1) by cysteine residues on 77 positions.RNA analyzes

(Flow Lab., USA) isolated lymphocytes are separated whole RNA (Maniafes T etc., Cold SpringHarborLaboratory Press, 1982) with ordinary method by using MPRM Ficoll-Hypaque.With the synthetic cDNA (Martynoff G. etc., Biochem Biophys, Res.Commn., 93:645,1980) of lug RNA, synthetic cDNA is used for the cDNA of PCR reaction with the amplification growth hormone gene.Below under the condition, GHS2; 5 ' TGGACAGCTCACCTAGCTGCA3 ', GHAS1; 5 ' GGATTTCTGTTGTGTTTCCT3 ', GHS3; 5 ' TTGACACCTACCAGGAGTTT3 ' and GHAS3; 5 ' CTAGAAGCCACAGCTGCCCT3 ' is used as Oligonucleolide primers and carries out pcr amplification.

For GHS2-GHAS1, at 92 ℃, sex change was carried out 3 minutes, will be by under 92 ℃, sex change 1 minute under 68 ℃, under the renaturation 1.5 minutes and 72 ℃, is stretched the circulation of forming in 1.5 minutes and is repeated 40 times, does not wherein the most once circulate stretching, extension was carried out 7 minutes.

For GHS3-GHAS3, carry out sex change first, then will be by under 92 ℃, sex change 1 minute, under 68 ℃, renaturation 1.5 minutes and under 72 ℃, stretch 40 circulations forming in 1.5 minutes and repeat 40 times, wherein last circulation is carried out stretching, extension 7 minutes, and amplified production is carried out the base order-checking.

The structure of wild-type and mutant growth hormone cDNA

Human growth hormone by two types of pcr amplifications is the cDNA of wild-type and mutant, wherein use from human growth hormone produce the pituitary adenoma cell preparation the cDNA library (Clontech, USA).The base of the accuracy of the structure of the growth hormone cDNA that each has been discerned by DNA checks order and confirms.

For the Oligonucleolide primers that is used for PCR method, GHS1 is used as adopted primer, and 5 ' TAAGAATTCGAGGGGTCACAGGGATGCCACCCC3 ' is used as antisense primer.

Carry out PCR below under the reaction conditions: sex change was first carried out under 92 ℃ 3 minutes, will be by 92 ℃ of following sex change 1 minute, 48 ℃ of following renaturation 1.5 minutes and stretch the circulations of forming in 1.5 minutes down at 72 ℃ and repeat 40 times, wherein last circulation is carried out stretching, extension 7 minutes.

Use transformant (Transformer) MT (C/ontech, USA) cDNA of structure mutant tethelin.In order to remove the signal sequence of growth hormone cDNA, use have adopted primer (5 ' GCGGATCCTTCCCAACCATTCCCTTATC3 ') and the GHAS1 that contain the BamH1 site of manually mixing to carry out pcr amplification as antisense primer.Measure resulting cDNA to confirm sudden change (Fig. 1) by direct determination of alkali base sequence.The expression and the functional analysis of wild-type (normally) and mutant tethelin

Each expression vector that produces wild type and saltant growth hormone comprises the dna sequence dna that contains the promoter operator PLOL that comes from lambda phage; The N that contains the transcriptional antitermination factor N-albumen that can produce in conjunction with host cell utilizes the site and the mRNA of wild type and saltant growth hormone can be attached to ribosome bind site on the ribosomes of host cell inside, ATG initiation codon and required gene is inserted into dna sequence dna (Japanese Patent Publication No.87780/1994) with the Restriction Enzyme site in the carrier in the bacteriophage of ATG initiation codon ATG.

When importing expression vector during host cell, heating under the repressor fail temperature contains in the suitable host cell of thermo-labile repressor C1, intestinal bacteria for example, and make it express wild-type and mutant tethelin respectively.This expression product is positioned at N-terminal, and methionine residues derives from initiator codon, but can produce sophisticated wild-type and mutant human growth hormones (Japanese unexamined patent publication number 500003/1982) with this methionine residues of specificity aminopeptidase cancellation.

Cultivate transformant, centrifugal or filtration cell suspension, collecting cell, then by physics and the molten born of the same parents of chemical process with segregation mutant tethelin.

Purification step is finished in combination by currently known methods, and these methods comprise the fractional separation with ammonium sulfate etc., gel permeation chromatography, and ion exchange chromatography uses the affinity chromatography of antibody and efficient forward or backwards chromatography.

In order to prepare the small amount of sample that is used to test, wild-type and mutant tethelin are cloned into the BamHI-EcoRI site (pGEXKG) of plasmid respectively, and then are incorporated in the DH5 α cell.Also in the clone that the glutathione-S-transferase that provides with Pharmacia of Sweden merges, prepare expression product.

Thought already that the intramolecular crosslinking between two halfcystines in the recombination mutation type tethelin that obtains by top method was occurring in three kinds of different types, i.e. normal type (53-165) and two kinds of mutants (53-77) and (77-165).Therefore, will be by step shown in Figure 5 from the lymphocytic cDNA of the patient displacement that suddenlys change, thus produce the cDNA that is wherein replaced by alanine residue at 53 or 165 locational halfcystines.In this case, can produce the cDNA that is wherein replaced by serine residue at 53 or 165 locational halfcystines.

With these genes at expression in escherichia coli producing two kinds of mutant tethelin, wherein a pair of halfcystine go up in 77 and 165 positions (Fig. 2) and 53 and 77 positions (Fig. 3) respectively form crosslinked.

Measure the biological activity separately of wild-type and mutant tethelin with IRMA and Nb2 bioanalysis system.The Nb2 bioanalysis is carried out under the situation of the serum that has or do not exist to come the patient who does not detect tethelin or galactogogue in the comfortable serum.Being added in the sample recombinant human somatropin conjugated protein (rhGHBP) until ultimate density separately is 0.1,0.5 or lnM.

In expressing people's lymphoblast clone IM-9 of growth hormone receptor, study competitive combination by a step receptor assay method (Lesniak, M.A. etc., J.Biol.Chem., 249:1661,1974).

By using immunoprecipitation to study wild-type and mutant tethelin combines with the direct of rhGHBP.

Detect the tyrosine phosphorylation effect that depends on tethelin in the IM-9 cell with the method (Silva, M.D. etc., Endocrinology, 132:101,1993) of silva etc.(RC20:TranscutionLaboratories USA), with the naked eye detects the combination of antibody to the monoclonal antibody of the tyrosine of the anti-phosphorylation of use in immunoprecipitation and Western engram analysis method with the ECL test kit of Amersham CO of USA production.

Isoelectrofocusing shows except that known wild-type (normally) tethelin, and mutant tethelin also is present in propositus (patient's) the serum (Fig. 6).

Whether has biological reactivity in order to measure the mutant growth hormone gene, thereby expression mutant and wild-type growth hormone gene obtain product in the expression vector cell transformed that contains the promotor operator gene that comes from lambda phage, also these genes are expressed in gst fusion protein plastome simultaneously to obtain product.

By the analysis in the IRMA cell, find that mutant and wild-type tethelin all have immunoreactivity.Also measured their biological activity by the Nb2 bioanalysis.

Although in the NB2 bioanalysis, two kinds of materials are found the biological activity that demonstrates similar level in serum free medium, and the biological activity of mutant tethelin is reduced to half less than wild-type tethelin in patients serum's substratum.Expect that growth hormone binding protein causes the interferential possibility in Nb2 bioanalysis system, it is conjugated protein to add recombinant human growth hormone in analyzing substratum.

Discovery is when the recombinant human growth hormone that has 0.5nM and lnM is conjugated protein, the biological activity of mutant tethelin and immunoreactive ratio obviously are reduced to 0.45 ± 0.05 (P＜0.05) and 0.22 ± 0.08 (P＜0.05) respectively, and these protein concns are equivalent to the protein-bonded concentration of effective physiology in the peripheral blood.

In the IM-9 cell [ ¹²⁵I]-mode that the human growth hormone and the combining of growth hormone receptor of mark is found to depend on concentration changes by the displacement conversion that becomes mutant tethelin from wild-type.Displacement with mutant tethelin is 10 at protein concn ^-11-10 ^-9Demonstrate salient in the M scope.

The IC that they obtain at 0.84 ± 0.30nm and 0.86 ± 0.41nm place respectively separately ₅₀Be worth almost equal.

Yet, be 10 at protein concn ^-1110 ^-9During the scope of M, can not carry out (Fig. 7) reposefully with the displacement of mutant tethelin.

In addition, in the IM-9 cell [ ¹²⁵I]-human growth hormone of mark and recombinant human somatropin protein-bonded combine to be found also change by the displacement conversion that becomes mutant tethelin from wild-type in the mode that depends on concentration.

Mutant tethelin has the IC of 0.12 ± 0.02nM ₅₀(3 mensuration, mean value ± SE), be starkly lower than the respective value of wild-type tethelin 0.68 ± 0.08nM proves for protein-bonded affinity to exceed about 6 times of wild-type kind.

By by means of the Western engram analysis, compared the tyrosine phosphorylation effect that depends on tethelin in the growth hormone receptor in the IM-9 cell that uses between wild-type and the mutant tethelin.

With recombinant type tethelin and wild-type tethelin, be called normal growth hormone both and promote the true opposite of tyrosine phosphorylation, mutant tethelin self not only can not cut any ice to the phosphorylation of tyrosine, and obviously suppresses the phosphorylation by the wild-type inducing growth hormone.Suppress the phosphorylation of tyrosine even can added fashionable observing (Fig. 8) simultaneously with 1: 10 concentration when mutant tethelin and wild-type tethelin.

Claims (14)

1, the mutant human growth hormones albumen of aminoacid sequence that a kind of Fig. 1 of having gives.

2, the deoxynucleotide of base sequence that a kind of Fig. 1 of having gives, the described aminoacid sequence of its coding claim 1.

3, a kind of mutant human growth hormones albumen as claimed in claim 1 is characterised in that described mutant human growth hormones has growth hormone receptor enhanced affinity, and the tethelin activity that has also kept reducing.

4, a kind of have the protein of aminoacid sequence according to claim 1, its amino-acid residue part is partly replaced, insert or disappearance to a certain degree, but do not cause it to demonstrate the active characteristic feature forfeiture of tethelin that the enhanced affinity of growth hormone receptor and maintenance have been reduced.

5, the protein of aminoacid sequence that a kind of Fig. 2 of having gives and its deoxynucleotide of encoding.

6, the protein of aminoacid sequence that a kind of Fig. 3 of having gives and its deoxynucleotide of encoding.

7, the described proteinic peptide of a kind of claim 1, it comprises the binding site of a described proteic receptor protein, and the deoxynucleotide of the aminoacid sequence of the described peptide of encoding.

8, a kind of deoxynucleotide with base sequence of the described proteinic aminoacid sequence of coding claim 4.

9, a kind of have as claim 2,5,6, the 7 or 8 described expression plasmids that are arranged in the deoxynucleotide in promotor downstream separately.

10, a kind of by using the biomass cells that transforms as plasmid as described in the claim 9 to express the product that produces.

11, a kind of antibody, it and claim 1, the mutant human growth hormones albumen described in 4,5 or 6 produces and interacts.

12, a kind of as claimed in claim 11 be the antibody of monoclonal antibody.

13, a kind of medicine that is used for the treatment of gigantosoma or acromegaly, it comprises a kind of as claim 1,4,5,6 or 7 described albumen or peptides as activeconstituents.

14, a kind of gene therapy medicine that is used for, it uses as claim 2, the deoxynucleotide described in 5,6,7 or 8.

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