CN1127528A - Modified alpha-lactalbumin - Google Patents

Abstract

Modified alpha -lactalbumin, for example of bovine or human origin, comprising fewer phenylalanine residues than wild type alpha -lactalbumin may be used as a dietary component for sufferers of hyperphenylalaninemia. Preferably all of the phenylalanine residues are replaced. The modified alpha -lactalbumin may be expressed in the mammary gland of non-human host animals so as to accumulate in, and if desired be separated from, their milk.

Description

The alpha-lactalbumin of modifying

The present invention relates to modifying protein useful in the hyperphenylalaninemia invalid diet.

Because people's hyperphenylalaninemia disease of causing of genetic cause is at Phenylalanine hydroxylase PAH:E.C.1.99.1.2) locus, the locus of encoding D HPR (dihydropteridine reductase) and at BH ₄Two results that locus is undergone mutation of (the biological butterfly cry of certain animals of tetrahydrochysene) route of synthesis.In all hyperphenylalaninemia patients, be not lower than 3% and in the patient of Caucasia, may be similar to 1% what the PHA locus was undergone mutation.Because more than half patients has serious PAH defective (typical phenylketonuria or " PKU ") among the hyperphenylalaninemia patient that the PAH sudden change causes.This liver enzyme is responsible for phenylalanine is transformed into tyrosine.

Shown in top diagram, the PAH defective causes phenylalanine and its organic acid metabolite to accumulate in tissue and blood, if do not control, will cause serious and irreversible backwardness in baby and children.

Global PKU sickness rate scope is 11 of about 1000,000 philtrum to Japan from Hibernian per 5,000.Conservative on average being estimated as in per 1,000,000 life to world wide PKU sickness rate has 50 examples.The PKU sickness rate of the U.S. is approximately in the newborn infant of all selective examination has 1 in per 10,000.

In a single day ill baby makes a definite diagnosis, and beginning dietetic treatment in early days is crucial to reduce blood phenylalanine levels.The general objects of treatment is that blood phenylalanine levels is remained on below the 1000 μ mol/l (16.5mg/dl) and preferably below 600 μ mol/l.Nearest neuroscience and study of behaviour evidence show that therapeutic goal in the future can be lower: the phenylalanine value maintain as far as possible near normal value (＜120 μ mol/l, or＜1.98mg/dl).

In theory, the frequent blood of process is monitored and is adhered to using the phenylalanine restrictive diet can reach biochemical ideally control PKU (Trahms, " Nutritional Care in MetabolicDisorders/in Food; Nutrition and Diet Therapy.8th Edition (1992), Mahan﹠amp; Arlin, eds., W.B.Sanders Co., Philadelphia, Pennsylvania; Kitagawaet al, Enzyme 38 321-327 (1987); But in fact seldom obtain this ideal results and Owada et al.Acta Paediatr.Jpn.30 405-409 (1988)).Particularly the design of nutrition treatment is around using the prescription that reduces phenylalanine content.Usually obtainablely be used to suffer from the baby of PKU and children's prescription is the free amino group acid mixture that 95 or 100% no phenylalanine protein hydrolyzate or adding add phenylalanine on a small quantity or not.Normally a kind of additional amino acid of tyrosine, the tyrosine level keeps below normal mean value in the phenylketonuria, PKU people of treatment because worry can make.

The conformability of diet is important in the ill baby of the early stage prevention of life and children's intelligence are blunt not only, and particularly to suffer among the pregnant woman of hyperphenylalaninemia disease the grownup in later stage also be important.In fact untreated pregnant woman offspring gets complication such as backwardness and microcephalus.Minority also has congenital heart disease and birth weight low.These consequences are very unfortunate, because in most of the cases baby itself does not suffer from the hyperphenylalaninemia disease.Also have report to treat or do not have the big children of hyperphenylalaninemia of phenylalanine dietary control with advancing age and seldom, teenager and grownup's IQ reduce gradually, and difficulty of learning and behavior have obstacle.Therefore, especially from nearest report, clearly PKU has represented a kind of disease lifelong and that stride generation, need to carry out continuously and all the life dietary control (Scriver et al., TheMetabolic Basis of Inherited Disease, Scriver, C.R., Beaudet.A., Sly.W., Valle.D., (eds), Tth edit.McGrawo Hill, in press, Chapter 27; Joint PAO/ WHO/UNU Expert Consulltation, Energy and Protein Re-quirements, WHO Tech.Rept.Ser.No.724, WHO, Geneva, Switzerland (1985); Levy H.L.Treatment of genetic diseases, Ed., R.J.Desnick, Churchill-Livingston, NY, (1991)).

Patient's PUK diet program has 3 covers.At first, provide all indispensable amino acids except that phenylalanine to make baby and children's normal growth and growth, if the adult then is used to keep.Usually in the amino acid composition that limits the prescription form, add VITAMIN and inorganics.For the baby, baseline amino acid pattern should be complementary with the human milk (Nayman et al.32 1279-1289 (1979) t and Joint PAo/WHO/UNU Expert Consultation, Energy andProtein Requirements, WHO Tech.Rept.ser.No.724, WHO, Genera, Switzerland (1985)), but the phenylalanine content exception, it should not exist for the human milk lower or fully.For children and adult, except that phenylalanine, the minimum formulation amino acid pattern should adapt to the form of these groups patient needs of PAO/WHO/UNU suggestion.

Secondly, the food that limits phenylalanine content is added in the basic diet of amino acid prescription as natural additive.Because these foods contain the phenylalanine of different levels, what of the selection of food and consumption provide more or less handiness and palatability for comprehensive diet.The phenylalanine levels that is allowed only is to be suitable for growth and/or to keep needed.For example, all the maximum tolerance level of the phenylalanine of taking in childhood is 500mg/ days, and acceptable scope is 200-500mg/ days.

According to Young ﹠amp; (Am.J.Clin.Nutr.45 1323 (1987) and the U.S. paediatrics research institute nutrition council (Pediatrics 57 783 (1976)) estimate that human absolute phenylalanine need be shown in following table 1 to Pellett: show 1:mg/ days mg/kg/ days mg/g protein baby 25-90 preprimary children ^*200-500 69 63 bigger children ^*200-500 22 22 young grownups 14 19

* patient PKU who almost completely lacks from the PAH activity estimates.

The third composition of PKU diet is: behind the needs that satisfied indispensable amino acid and phenylalanine, must guarantee enough calorific value food.Food such as fruit, vegetables, fat and the oil of this available lower protein, Hi CHO obtain.

Although above mentioned general treatment prescription is providing indispensable amino acid in theory, the restriction phenylalanine is taken in and obtained enough biochemistry control aspects is effective, but for some VITAMIN, inorganics and amino acid, its nutrition adequacy and/or bioavailability are shady (Acosta et al.J.Inher.Metab.Dis.5 107 (1982)).And make them suffer the painful of the flat and high infiltration burden of taste and smell and cause complaisance poor.To having required emphasis of food allergy and regulation and control in life to containing lower or not containing the needs of the foodstuff products that is easier to accept of phenylalanine.

It is desirable to, these products answer mouthfeel good, and smell is good, and when using them, the selection of food and the design of menu have maximum handiness.Ideal product or prescription should have height palatability and low (preferably not having) phenylalanine: these are to produce complaisance and make the food that contains low albumen phenylalanine reach the crucial quality that more normal diet maximum is taken in.The phenylalanine content food with high biological value low or that lack fully will be a kind of very valuable diet composition, because the more and natural food that contains phenylalanine volume can be accepted.

The technology of studying this problem with new and more efficient methods is provided now.Use site-directed mutagenesis, the combination of protein engineering and recombinant DNA technology can be through making the relative dna codon mutation change specific known protein matter to reduce or eliminate phenylalanine residue.

Recent years, we are to recombinant DNA and genetic technique, and the understanding of protein engineering has had sizable expansion.This knowledge hierarchy makes us can select particular proteins, and according to its known array, 26S Proteasome Structure and Function suddenlys change.Then, the DNA of encoding such proteins can be from suitable source clone, and bulging becomes required composition, and order-checking is expressed to confirm its change and to be cloned on the suitable carriers.

Cloned recent years coding various medicinal go up and nutrition on the dna sequence dna of important protein matter.These comprise Regular Insulin, plasminogen activator, α ₁-antitrypsin, blood factor VIII and IX, N,O-Diacetylmuramidase, α-opalescin and lactoferrin.Can derive its protein sequence and confirm from these dna sequence dnas with polypeptide collection of illustrative plates and Edman edman degradation Edman.

Utilize protein X-radiocrystallography and multidimensional nuclear mag-netic resonance spectroscopy can measure the three-dimensional structure of numerous protein.This technology with high functional machine and advanced program coupling connection, the change that can simulate amino-acid residue with it to protein structure and the issuable influence of function (Nilsson et al, Curr.Opin.in Strus.Biol.2 569-575 (1992); Presta, L.G., Curr.Opin.iro Struc.Biol.2 593-596 (1992); Cech, T.R., Curr.Opin.in Struc.Biol.2 605-609 (1992); Pickersgill andGoodenough, Trends in Food Sci.and Tech.5 122-126 (1991)).

For the protein that will modify and the modification that will carry out, the mutant DNA sequence that the scheme of having set up can be used for separating this protein DNA of coding and produces the amino acid needed change of coding.This class scheme comprises carries out subclone to containing the amino acid whose gene region that will change to some extent.Fragment with subclone is used for site-directed mutagenesis then.Can use this technology for accurate target site and required modified types.Can be in the most accurate site the single base of dna sequence dna be become in other 3 kinds of bases any with the method for the oligomerization nucleosides primer that is used for the mutagenized dna sequence.So, the diverse amino acid of this DNA codified (McPherson, M.J., Directed Mutagenesis, Oxford Univ.Press, NY (1991); Carter, P., Biochem.J.237 1-7 (1986); Nickoloff and Deng, Anal.Biochem.220 81-88 (1992)).

When selecting suitable protein to be used for the phenylalanine replacement, should be taken into account several aspects.At first, the protein as food must be able to obtain in a large number.Therefore, must obtain high-caliber expression, and obtain and be easy to purifying easily as fruit product and will have advantage.Secondly, the more and more exquisiteness although the protein simulation becomes, the 26S Proteasome Structure and Function result behind a plurality of amino acid changes are still supposition.Because bigger polypeptide contains more amino acid, the increase of size reflects that usually the number of phenylpropyl alcohol benzoic acid residue is bigger, thereby needs more mutagenesis to replace them.At first, smaller polypeptides is the target of better operation.In principle, can select in the cell or secretary protein.Yet this selection is subjected to the restriction that expression system is selected.

When consider modifying which kind of protein, must consider various factors, comprise proteinic structure, the problem of physiology and trophology, and whether be easy to from host's synthetic cell, extract this protein.

As for configuration aspects, amino acid whose relative position and amino acid whose physics and chemical feature are controlled protein such as activity in the protein, the feature of specificity and stability.Proteinic structure and its function are closely related.In general, when proteinic amino acid and conformation thereof were changed, important was not change its function and guarantee that it correctly processes and if necessary talk about from cell secretion and come out.

The physiology aspect depends on that selected protein characteristic and it are a kind of enzyme or a kind of structure, adjusting, transportation or food proteins.A potential problem is the proteinic 26S Proteasome Structure and Function feature of sudden change influence, to cause host cell or the sustain damage in some way of its function.

On trophology, protein is chemically needing complete equipilibrium as far as possible.Therefore, before possible sudden change is proposed any imagination, must analyze natural protein, according to aminoacids content, digestibility and supersensitivity are estimated its nutritive potential.

The easiness of extracting is somewhat dependent upon production method.Which kind of pollution substance this will determine to exist and therefore use which kind of extracting method.

The topmost aspect of the present invention is to select α-opalescin as modifying object, fully sets forth all these variations and inconsistent consideration realizes the present invention.

According to a first aspect of the invention, provide the α-opalescin of a modification, compared, do not comprised phenylalanine residue or comprise still less phenylalanine residue with wild-type α-opalescin.Be different from people α-opalescin varient, Phe wherein-80 is replaced by Methionin.

In the human milk, α-opalescin is main whey protein, has (accounting for 29%-Heine et al of gross protein, J.Nutr.121 277-283 (1991)) with the level of～2.5g/l.This is a kind of spheric calcium-binding protein, and relative molecular weight is 14,200, in nearly all Mammals milk discovery is arranged all.

α-opalescin is of high nutritive value, and its indispensable amino acid accounts for 63% of sum.Ox (Vilotte et al, Biochimic 69 609-620 (1987)) and people's alpha-lactalbumin (Hallet al, Biochem, J.242 735-742 (1987)) all contain 123 amino-acid residues, each monomer only has 4 phenylalanines and 4 tyrosine residuess.Alpha-lactalbumin becomes the part of our diet with the form of people, cow, sheep, goat and camel milk for thousands of years.The function of alpha-lactalbumin is dual.It mainly acts on is a kind of composition as the lactose synthetase system.This synthetic enzyme is synthetic lactose in lactating mammary gland.Second component that alpha-lactalbumin serves as a kind of more specific protease system with the substrate specificity of modifying UDP β 1,4-galactosyltransferase (E.C.2.4.1.22).Secondly, α-opalescin serves as a kind of source of nutrition.

9 kinds of alpha-lactalbumin sequences measured fully (Mackenzie and White, adv.in Prot.Chem.41 173-315 (1991); Vilotte et al, Gene 112 251-255 (1992)), the amino acid composition of 13 kinds of α-opalescins is disclosed (in Prot.Chem.41 173-315 (1991) for Mackenzie andWhite, adv).Acharya etc. (J.Mol.Biol.208 99-127 (1989)) disclose the crystalline structure of baboon α-opalescin under 1.7 in 1989, recently, the crystalline structure of people's α-opalescin also discloses (Acharya et al, J.Mol.Biol.221 571-581 (1991)) under 1.7 .

α-opalescin and C-type N,O-Diacetylmuramidase forms from a common precursor differentiation, and this is generally accepted (Hall and Campbell, Essays in Biochem.22 1-26 (1986) The Biochemical Soc).

Alpha-lactalbumin is a quite little secretory protein, and it has 4 phenylalanine residues.Because it is the damp protein of a kind of breast, so it is suitable for expressing in transgene mammal mammary gland more satisfactoryly, as described below, selects this mammary gland as expression system.α-opalescin is suitable for satisfying structure discussed above fully, physiology, the needs of nutrition and the property gathered in the crops aspect.

Structurally, 4 phenylalanine residues of people and cattle mature protein are respectively 3,31,53 and 80 positions and 9,31,53 and 80 positions.These two kinds of proteinic protein engineerings be studies show that: according to its size and space, almost arbitrary in principle residue all can be used to replace phenylalanine.Yet Phe-31 and 80 positions relate separately to lactose synthetase reaction and calcium coupling collar, therefore must be more careful when consideration is carried out suitable replacement to these positions.

Known α-opalescin can with the galactosyltransferase of other kind bring into play function (Khatra et al, Eur.J.Biochem 44 537-560 (1974); Powell andBrew, Biochem.J.142 203-209 (1974)).

On trophology, when selecting suitable protein to modify, should consider aspect 3: aminoacids content, digestibility and antigenicity.Because this food will be edible for baby and grownup, so its nutritional quality and indispensable amino acid level must be high as far as possible.In general in milk protein matter, it is caseic 51.4% that indispensable amino acid accounts for, account for complete in milk protein matter 52.5% and account for 58.8% of milk albumin matter.Yet in α-opalescin, indispensable amino acid accounts for 63.2% (Heine et al, J.Ntr., 121 277-283 (1991)) of sum.The more important thing is that α-opalescin has high-load Methionin and halfcystine and high-load especially tryptophane (account for total amino acid 5.9%).Measure intravital true digestibility and have some problems, yet the digestibility that studies show that α-opalescin that can carry out usually is the same with casein high and better than β-lactoglobulin (BLG).Main milk albumin matter BLG does not find in the human milk, and thinks that it is a kind of potential antigen in the susceptibility crowd.Closely similar and these similaritys expection of ox α-opalescin and people's α-opalescin may reduce the antigenicity of ox alpha-lactalbumin.Compare with other milk protein matter, this is seemingly correct, and (Pediatrics 32 425-443 (1963) for Goldman, A.S.; Strobel, S., Hum.Nutr.:Appli.Nutri.40A (1) 45-54 (1986)).

Phenylalanine α-opalescin product that reduce or that do not have phenylalanine can preferably be produced in the galactophore of transgenic animal of lactation, and natural protein wherein may still exist.This may make purifying become difficult, unless amino acid whose replacement itself has changed proteinic characteristic.Yet, in the lactation animal milk, the alpha-lactalbumin of modifying and the peculiar advantage of expression thereof are: if the expression of lactation natural animal α-opalescin is very low for the alpha-lactalbumin of modified is expressed, expression perhaps natural or the wild-type form is prevented from or when suppressing, purifying may be unnecessary from natural α-opalescin.Stop or suppress the effect that natural form is expressed by using embryonic stem cells that lactation animal transgenosis can be reached.Purifying and when keeping the change that proteinic structure must guard, a kind of possibility is the outer aminoacid sequence of additionalamount, is beneficial to purifying as add a tail (Smith et al, Gene 32 321-327 (1984)) at C-terminal if desired.Extra aminoacid sequence should have a kind or the multiple protein that causes and be easier to isolating feature; For example, extra aminoacid sequence can make protein be separated according to electric charge.Poly-Arg or poly-Arg/Lys tail can change electric charge and be enough to obtain more effective and more cheap extraction.

Consider the phenylalanine content of other milk protein matter (as casein), the same with example probably in actually operating to natural alpha-lactalbumin, preferred partial purification α-opalescin of going out to modify at least from this proteinoid.

Particularly useful aspect the palatability of the present invention's protein composition in improving PKU patient's diet and the complaisance thereof.This can be suitable by selecting, and the protein of called after α-opalescin realizes that it can reach the standard that has the height nutritive value and can solve all problems discussed above.This protein DNA of clones coding then, sudden change to be removing at least some (but preferred whole) phenylalanine residues, expresses in can mass production making the system that is enough to supply world market.

Can derive from any suitable animal as the α that modifies the basis-milk-protein branch.Preferred people and lactation animal, as cow, sheep, goat and camel.It is best that the α-opalescin that originates from people or ox is likely.The both have similar nutritive value and human use they existing several thousand.

Ox or people's alpha-lactalbumin are particularly suitable for the starting point as the sudden change modification, because people's class gets used to eating this two kinds of protein, and they are closely similar, and 72% homology is arranged on amino acid levels.Depend on the consideration of structure and/or nutrition aspect, with some or all phenylalanine residues of one or more aminoacid replacement.The preferred amino acids that can select is as shown in table 2.On in 4 phenylalanine positions any one, available these amino acid whose any combinations replace.Preferably replace,, fail on 3 or 2 positions therein although the present invention also covers the replacement that carry out a position in all these positions.

Table 2

Preferred phenylalanine replaces

Human ox Phe 3/9 Tyr, Leu, Arg, Met, Trp Tyr, Ser, LeuPhe 31 Leu, Ile, Trp, Tyr Leu, Ile, Trp, TyrPhe 53 Tyr, Leu, Met, Trp Tyr, Leu, Met, TrpPhe 80 Tyr, Met, Trp, Leu Tyr, Met, Trp, Leu

If the selection of substituted amino acid only is according to energy minimization and structural considerations, Tyr so ₃-Leu ₃₁-Tyr ₅₃-Tyr ₈₀People's alpha-lactalbumin and Tyr ₉-Leu ₃₁-Tyr ₅₃-Tyr ₅₃-Tyr ₈₀The milky white egg of ox α be preferred (or having) than four replacements replacement mutant still less.If also consider nutritional need, so Tyr _3/9-Tyr ₃₁-Tyr ₅₃-Tyr ₈₀People and Niu α-opalescin (or having than four replacements replacement mutant still less) are the mutant of selecting.The best further method that replaces of decision is to see at other kind α-opalescin and occupy the amino acid of corresponding position in the vertebrates lysosome, because checking the enterable database of the public shows: the N,O-Diacetylmuramidase of alpha-lactalbumin and vertebrates kind has closely similar structure and thinks that they originate from identical ancestral gene, according to these data, preferred Tyr ₃-Tyr ₃₁-Leu ₅₃-Leu ₈₀People's alpha-lactalbumin (or having) than four replacements replacement mutant still less.

Can replace some or all phenylalanines, for the purpose of structure, can be preferably one or more constant in them.If do not carry out the consideration of any structure aspect, can replace at least one, 2,3 or 4 crude benzene alanine residues, it is many more preferred more to replace number.The natural variation body (J.Dairy Res.59 425-429 (1992) for May-nard, F.) of people's alpha-lactalbumin that the known Phe80 of existence is replaced by Methionin; This varient itself is not a part of the present invention, although in the prescription of the present invention and method that it can be used for below will discussing.

In order to help purifying, can further modify α-opalescin, for example, can add a poly-Ary or Arg/Lys tail at this proteinic C-end.This will change this proteinic electric charge and can carry out quite cheap large-scale purification to it.It also has the advantage of improving this proteinaceous nutrient quality.

Can use a cover technology that is called protein engineering now to produce the alpha-lactalbumin of modification of the present invention through recombinant DNA technology.

Can obtain the equivalent (co-ordinates) (Acharya et al, J.Mol.Biol.208 99-127 (1989)) of baboon α-opalescin from the Brookhaven database.The proteinic equivalent of human protein and cow can't obtain at present, but thinks that the proteinic three-dimensional structure of baboon can provide acceptable template to the simulation of people and Niu wild-type and mutant ' alpha '-opalescin.

At first can consider amino acid whose influence of displacement in amino acid type level.Therefore, aromatics should replace aromatics, and electric charge replaces electric charge, and shape replaces shape or the like.

The first step is the state that is determined in the three-dimensional structure of natural supposition each residue in 4 Phe residues.They are: Phe-3 (people) or Phe-9 (ox) are at molecular surface; Phe-31 is exposed to the surface; Phe-53 is in the intramolecule chamber, and Phe-80 major part is by embedding.Be that these 4 residues separate fully easily, so that have reason to suppose that the interaction between these sites may be very little so if the change that imports is most of conservative.

The model of setting up shows that arbitrary basically residue all can replace Phe-3/9 and 31, because the both is at molecular surface.The Phe residue of two embeddings can be with the very little residue of peripheral protein matter activity is replaced.And Leu and Met can be used for replacing Phe, but polarity or charged group destroy based structures probably, so superiority is relatively poor.

Can obtain some guidances from document, but they are not to be exclusively used in α-opalescin or purpose of the present invention (as Bordo and Argos (J.Mol.Biol.217 721-729 (1991)), Lesk and Bosevell (Curr.Opih.Struct.Biol.2242 (1992)), Singh and Thornton (" Atlas of Protein Side-Chain Interactions " .Vols 1and 2, IRZ Press.Oxford, and computer program (the program SYBYL of TriposAssociates for example England (1992)), ^TM)), these instruct when estimating arbitrary other that may need to carry out and modify may be useful.

Following description has been carried out brief summary to this position:

3: be surface residue, the most of residues of receivability: Tyr, Leu, Arg.Met.Trp (order that progressively increases by relative energy), most preferably Tyr.

9: be surface residue, the most of residues of receivability.The residue that most of α-opalescins have a Ser. to select for use in this position is: Tyr.Ser.Ile.

31: be exposed to the surface, so the arbitrary residue of receivability.Yet this zone is important to lactose synthetase reaction and hydrophobic interaction may be important.Therefore, if retentive activity is more important, Leu and may be Ile most preferably is Trp then herein.

53: most of by embedding, Tyr.Leu, Met.Trp are the orders that progressively increases by energy.

80: fully by embedding.Tyr, Met, Trp, Leu are orders herein.Be calcium coupling collar zone, therefore, change herein may be very important herein.Tyr is the most close same form residue.

C end: be positioned at the surface, therefore quite expose.Adding some residues herein may significantly not influence its structure, but can improve its separation characteristic.

Can in people, ox or other α-opalescin, use arbitrary combination of these replacements of advising.

Although the α-opalescin according to modification of the present invention can prepare with any method (comprising chemosynthesis) in principle, the general method of selecting comprises the use of recombinant DNA technology, and wherein protein is expressed from the corresponding nucleic acids sequence.

According to a second aspect of the invention, provide a kind of method for preparing the α-opalescin of modification recited above, this method comprises successive amino acid coupling is associated in together, and/or connects few-and/or many-peptide.For preferably, indicate as top, successive amino acid is coupled to together preferably mediates with rrna.Now, within the general knowledge scope that many expression method those skilled in the art fully grasp." molecular cloning " the 2nd edition of Sambrook etc. particularly, cold spring harbor laboratory publishes (1989) can provide reference.

Nucleic acid itself useful in this class is expressed forms a part of the present invention.According to the 3rd aspect of the present invention, a kind of nucleic acid of isolating or reorganization is provided, its coding is compared the α-opalescin that does not comprise the less modification of phenylalanine residue or phenylalanine residue with wild-type α-opalescin.

Consider the degeneracy of genetic codon, many different nucleotide sequence codifieds arbitrary particular proteins of the present invention.All these sequences are included in the scope of the present invention, although preferred actually following nucleotide sequence:

(a) its codon is corresponding with the codon of wild type gene as far as possible;

(b) selection of its codon is consistent with the preferred codon of expressive host; Or

(c) its codon is according to standard (a) and (b) the compromise selection.

The recombinant DNA of α-opalescin of modifying according to coding of the present invention both had been not limited to cDNAs and also had been not limited to the proteinic natural gene group sequence that coding is modified, although these sequences may be preferred.Can use " minigene " method of WO-A-9005188, some (but not being whole) introns that wherein are present in natural gene are present in the construct, and it is imported host cell.

According to this aspect of the invention, recombinant DNA often is a kind of carrier format.The present invention is not limited to arbitrary specific carrier form, and for example, it can be plasmid, cosmid or phage.The carrier that contains enough adjusting sequences (comprising promotor) of the effective coupling connection of the α-opalescin sequence of modifying with coding is useful as expression vector the time.The carrier that does not comprise the adjusting sequence of effective coupling connection is useful as cloning vector the time.

According to recombinant DNA of the present invention also can be the construct form that is applicable to the preparation transgenic animal, for example through microinjection or import the construct of animal through homologous recombination.

The 4th aspect of the present invention be successive Nucleotide is coupled to together and/or with the widow-and/or polymerized nucleoside couple together the preparation nucleic acid of the present invention, in the complete those skilled in the art's ken of this method.

According to the 5th aspect of the present invention, provide a kind of host of containing top described recombinant DNA.This host can be a kind of cloning host, and wherein normally a kind of bacterium as intestinal bacteria, perhaps also can be a kind of expressive host.In expressive host, the DNA of the alpha-lactalbumin that coding is modified can allow to express with composing type or under adjusting condition with enough adjusting sequence (comprising promotor) the coupling connection and this host that are used to express effectively.Can use large-scale expressive host in the present invention, comprise bacterium (particularly E.coli), yeast is (as Saccharomyces cerevisiae, Hansenula polymorpha and Pischia pas-toris), insect cell (as Spodoptera frugiperda) and mammal cell line (as BHK and Chinese hamster ovary celI system).However, most preferred expressive host is an animal, particularly inhuman placental mammals, its kind comprises the DNA of the alpha-lactalbumin that coding modifies in being, the adult female performance that this kind is is expressed α-opalescin of modifying so that accumulate the α-opalescin of modification in its milk in mammary gland.If α-opalescin of modifying is from the kind different with host animal, this host transformed is transgenic animal strictly speaking; For example: the cow of the DNA of people's α-opalescin that carrying encodes modifies or the transgenic animal that bull is people's gene.However, according to the present invention, not all host animal all is transgenic animal, because it fully in range of possibility of the present invention and in some cases, a kind of host animal (for example ox) of given kind preferably is provided, and it carries the DNA of α-opalescin (being the ox alpha-lactalbumin) of the modification of coding identical type in this example.Conventionally revise or can prepare this class " similar transgenosis " animal through the technology that has formed and being used to of will continuing development prepares transgenic animal is carried out through gene therapy technology (this technology be formed for replace or additional gene version) with another gene version.

The mammary gland expression system has high expression level, low cost, correct processing and the advantage that easily obtains.The a few thing person has produced ox and people's α-opalescin (Vilotte et al, Eur.J.Biochem.186 43-48 (1989) from the transgenic animal of lactation; Hochi etal, Mol.Reprod and Devel.33 160-164 (1992); Sozdier et al, FEBSLetters 297 (1,2) 13-18 (1992)) and demonstrated the high-caliber protein of production.For the mammary gland (WO-A-8801648) of lactation, the surface (Stinnakre et al, FEBS Letters 284 (1) 19-22 (1991)) of α-directing heterologous gene of opalescin promotor energy.

Other transgenic animal are included in the female milk of growing up the proteinic transgene mammal of expressing heterologous and describe in the literature.For example, WO-A-8800239 and WO-A-9005188 described the production of transgene mammal, as sheep, it can express the medicinal valuable protein of, and comprises factors IX and α-antitrypsin.All methodologies of listing above and other publication all can be made amendment to produce according to transgenosis of the present invention or similar genetically modified animal.Preferably in the present invention use known lactation animal (for example cow, sheep, goat or or even camel or pig), but whether host's selection is at first depended on convenient, and do not depended on arbitrary requirement of the present invention itself as the host.

Can prepare with arbitrary method easily according to transgenic animal of the present invention or other host.Therefore, the present invention is not limited to be used for arbitrary specific method of its preparation.For example, can by top WO-A-8800239 and WO-A-9005188 is described prepares transgenic animal or prepare their (as described in WO-A-9003432) with the embryonic stem cells technology with microinjection.

Wild-type alpha-lactalbumin expression of gene should stop or make at least and weakens among the host, so that simplify purifying.

According to the 6th aspect of the present invention, a kind of food that is applicable to the hyperphenylalaninemia patient is provided, this food comprises at least a α-opalescin that does not contain phenylalanine residue or phenylalanine residue modification still less of comparing with wild-type α-opalescin.

Modify α-the opalescin preferable feature as mentioned above.

This food can (and normally) comprise other composition.The protein of modifying protein or polypeptides in combination low separately or with L-amino acid and/or other or that do not contain phenylalanine will be applicable to baby, children and the grownup who suffers from PKU.For baby and very young children, seem and taste the infant formulas of the normal complete protein of Ying Gengxiang with α-opalescin low or that do not have a phenylalanine as the prescription on protein basis, and therefore should be than the easier tolerance of special diet prescription of general protein hydrolysate or aminoacid mixture.

The special diet food or the fill-in that are used for baby and very young children's the α-opalescin that contains modification according to the present invention can contain other composition, as fat source, and VITAMIN, inorganics and other nutritional factor of carbohydrate source and the level of fitting out.Except the alpha-lactalbumin of modifying, any L-amino acid (as L-tyrosine) when extra nitrogenous source is all right can improve the total necessary amino acid masses of product after the adding, make its nutritional sufficiency.Under any circumstance, the total phenylalanine content of finished product generally is no more than 80mg, preferably is no more than 25mg/100g powder rice Equivalent, does not perhaps have phenylalanine.Arbitrary low Phe or do not have the Phe polypeptide, as also being suitable for the total quality of amino acid collection of illustrative plates that obtains with raising as extra nitrogenous source for coming from caseic polypeptide (CDP) (from the cheese processing process, rennin (rennet) decomposes breast and rises protein (α-casein) and CDP is discharged in the whey portion).Also can add L-tyrosine and non-essential amino acid, the amount of adding reaches amount in human milk and normal child's diet and/or higher more abundant to guarantee nutrition than this amount.

Special diet food or additive that the alpha-lactalbumin that contains modification is used for baby and young child can be the liquid that can eat immediately, or with the form of powder or concentrated liquid, become edible immediately form through adding water and stirring.(60-75kcal/ml) edible immediately liquid formulations or reprovision powder generally contain 1.2 to 3.0g to every 100ml, and preferably approximately 1.3 is to 1.5g protein; 2.2 to 4.0g, preferably approximately 3.6g, fat or fats mixt; And 6 to the 9g carbohydrate.

For most of babies, lactose generally is preferred carbohydrate source, but the corn solids syrup, and sucrose or other sugar also can be used for suffering from the special diet food of the baby of PKU and young child.

In addition, but contain the α of modification-opalescin and generally contain inorganics so that the calcium of receiving amount on the trophology, phosphorus, potassium, sodium to be provided as low Phe or the special diet food that do not have a Phe protein source, chlorine, magnesium, iron, copper, zinc, manganese, molybdenum, selenium, chromium, fluorine and iodine, and add the VITAMIN of q.s, and as vitamin A, D, E, B1, B2, B6, B12, C, nicotinamide, pantothenic acid, folic acid, vitamin K, vitamin H and born of the same parents' alkali.The nutritional factor such as the carotene that also should add other, middle sulfonic acid, inositol, carnitine and Nucleotide.

Being included in above-mentioned every kind of nutritive ingredient amount in the special diet food and its acceptable chemical species provides in following code: U.S.'s infant formulas regulations of the United States Federal's laws and regulations on the management (181-184 part) and 1980, the United States Federal's food, medicine and makeup regulations part 2, the IV chapter, the 192nd part, on September 26th, 1980 formulated, public's method 96-359 of revision on October 7th, 1985.

The baby who the instruction (official of European Economic Community magazine, No.L 175/35,1991) and the nutrition committee of pharmacopeia of infant formulas and other prescription is advised by the EC council provides similar but coarse suggestion with children's food international standard.In conjunction with FAO/WHO codex alimentarius outline, CAC/RS, 72/74,1967.Minimum value, maximum value and the preferred value of suggestion have been shown to show.

Table 3

Table 3

Example (1980) the every 100ml* of U.S. infant formulas system			The preferable range or the every 100ml of approximation of the alpha-lactalbumin infant formulas of modifying
				Nutrition	Minimum value	Maximum value
Protein (gm)	????1.2	????3.0					(1.3-2.0 the alpha-lactalbumin of modification)
			Fat (gm) linolic acid (mg)	????2.2 ????200	????4.0 ????-	???????3.4-3.8 ?????????330
Carbohydrate (gm)	????-	????-					???????6.8-7.4
			VITAMIN:
A(IU) (μg)	????166.7 ????50	????500 ????150					?????????200 ?????????60
			D(IU) (μg)	????26.7 ????0.67	????66.7 ????1.67	?????????40 ?????????1
E(ID) (mg)	????0.5 ????0.33	?????- ?????-					?????????0.95 ?????????0.64
			K(μg)	????2.7	?????-	?????????5.5
B 1(VitB1) (μ g)	????26.7	?????-					?????????67
			B 2(riboflavin) (μ g)	????40.0	?????-	?????????100
B 6(pyridoxol) (μ g)	????23.3	?????-					?????????42
			B 12(μg)	????0.1	?????-	?????????0.13
Nicotinic acid (μ g)	????166.7	?????-					?????????500

Table 3 (continuing)

The every 100ml* of U.S.'s infant formulas regulations (1980)			The preferable range or the every 100ml of approximation of α-opalescin infant formulas of modifying
				Nutrition	Minimum value	Maximum value
Folic acid (folacin) (μ g)	????2.7	???-					????????5.0
			Pantothenic acid (μ g)	????200	???-	????????210
Vitamin H (μ g)	????1.0	???-					????????1.5
			Vitamins C (xitix) (mg)	????5.3	???-	????????5.5
Choline (mg)	????4.7	???-					????????10
			Inositol (mg)	????2.7	???-	????????3.2
Inorganics:
				Calcium (mg)	????40	???-	????????42
Phosphorus (mg)	????20	???-	????????28
				Magnesium (mg)	????4.0	???-	????????4.5
Iron (mg)	????0.1	???2.0	??????0.8-1.2
				Zinc (mg)	????0.3	???-	????????5.0
Manganese (μ g)	????3.3	???-	????????15
				Copper (μ g)	????40	???-	???????20-47
Iodine (μ g)	????3.3	???50	????????6.0
				Sodium (mg)	????13	???40	???????15-20
Potassium (mg)	????53	???133	???????56-60
				Chlorine (mg)	????37	???100	??????375-400

* calculate according to 66.67 Kcal/100ml

Therefore, for baby and very young children, the ideal special diet replenishes also should be similar to the human milk, except its phenylalanine content should be no more than 80mg/100g prescription or solids.In whole infancy, the Childhood and adhere to that low-phenylalanine diet treatment is indispensable pubescence.Daily demand amout is shown in following table 4:

Table 4

Table 4

The baby of beta-oxybutyria * *, children, teenager are to selecting the approximate daily demand amout of nutrition in the trouble benzene
									Nutrition	Unit	The year order
???0＜6mo.	????6-12mo.	??1＜4yr.	???4＜7yr.	????7＜11yr.	????11＜15yr.	?????15＜19yr.
							Energy	???kcal/kg			???145-95	????135-80	?????-	??????-	???????-	???????-	????????-
	???kcal/day	?????-	??????-	???1，300	????1，700	??????2，400			??2，00-2，700	???2，100-1，800
								(scope)					(900-1，800)	(1，300-2，300)	?(1，650-3，700)	?(1，500-3，700)	(1，200-3，900)
Protein	?????g/kg	????2.5	?????2.2	?????-	??????-	???????-			???????-	????????-
								?????g/day			?????-	??????-	?????25	?????30	???????35	?????45-50	??????45-55
Carbohydrate	?????g/day	????????????????????????kcal×0.35－0.35＋4<——kcal×0.50－0.60＋4——>
									Fat	?????g/day	????????????????????????kcal×0.50＋9????4<——kcal×0.35＋9——>
Phenylalanine	?????mg/kg	???70-20	????50-15	???40-15	????33-15	?????30-15	?????30-15	??????30-10

* all known indispensable amino acids (except that phenylalanine), indispensable fatty acid, inorganics and VITAMIN should be supplied with q.s.

* takes from: the modern nutrition of health and disease: M.Shils, and V.Young edits; 1988, the 7th edition, Lea and Febiger Philadelphia.

Big-age-child, teenager and pregnant woman's diet, the recipe of particularly suffering from PKU should contain low Phe or not have the Phe prescription, and its add-on should surpass 50% of low phenylalanine food gross calorific value.This prescription can be designed to be applicable to each age group especially, promptly considers to have the daily intaking amount of suggestion, or forms a kind of versatile formulation also with special description of use and to each category extent of dilution difference.This prescription is different from the baby and the unusual prescription of young child, contains the nutritive ingredient of following RDI level (approximation) in about 500-1000kcal or 500-1000ml, and is as shown in table 5.

Table 5

Table 5

Daily intaking amount (the RDI of suggestion s)
					Nutrition	Unit of measure	Children below 4 years old	Grownup and the children more than 4 years old	The pregnant woman
Vitamin A	????μgRE	????400	????875	????800
					Vitamins C	????mg	????40	????60	????70
Calcium	????mg	????800	????900	????1200
					Iron	????mg	????10	????12	????30
Vitamins D	????μg	????10	????6.5	????10
					Vitamin-E	????mgα-TE	????6.0	????9.0	????10
Vitamin K	????μg	????15	????65	????65
					VitB1	????mg	????0.7	????1.2	????1.5
Riboflavin	????mg	????0.8	????1.4	????1.6
					Nicotinic acid	????mgNE	????9.0	?????16	????17
Vitamin B6	????mg	????1.0	????1.5	????2.2
					Folate	????μg	????50	????180	????400
Vitamin B12	????μg	????0.7	?????2.0	????2.2
					Vitamin H	????μg	????20	????60	????65
Pantothenic acid	????mg	????3.0	????5.5	????5.5
					Phosphorus	????mg	????800	????900	????1200
Magnesium	????mg	????80	????300	????320
					Zinc	????mg	????10	????13	????15
Iodine	????μg	????70	????150	????175
					Selenium	????μg	????20	????55	????65
Copper	????mg	????0.9	????2.0	????2.5
					Manganese	????mg	????1.3	????3.5	????3.5
Fluorochemical	????mg	????1.0	????2.5	????3.0
					Chromium	????μg	????50	????120	????13
Molybdenum	????μg	????38	????150	????160
					Muriate	????mg	????1000	????3150	????3400
Protein	????g	????16	????50	????60

For children and grownup's (comprising the pregnant woman), the target of special dietary supplement formula is to make prescription have palatability and is the high quality composition that makes us aftertaste in the diet.This prescription should contain all indispensable amino acids (except that phenylalanine) and VITAMIN and inorganics, particularly those not high VITAMIN and inorganicss of content in the low albumen food (as fruits and vegetables).These nutritions (isolating protein is outer) are calcium, iron, folic acid, magnesium and trace mineral.

Be used to suffer from the children of PKU, grownup and pregnant woman's representative formula contains but is not limited to following preferred nutrition scope, and is as shown in table 6.

Table 6

Table 6

The suggestion daily intaking amount
					Nutrition	Unit of measure	Preferred range in every liter	For the %RDI in grownup and the every 500ml daily intaking amount of the children more than 4 years old	For the %RDI in the every 500ml daily intaking amount of pregnant woman
Vitamin A	????μgRE	????600-800	????35-45	????40-50
					The main plain C of dimension	????mg	????50-100	????40-85	????30-60
Calcium	????mg	????500-1000	????30-55	????20-40
					Iron	????mg	????5-30	????20-125	????8-50
Vitamins D	????μg	????6-10	????45-75	????30-50
					Vitamin-E	???mgα-TE	????6-10	????35-55	????30-50
Vitamin K	????μg	????20-100	????15-75	????16-75
					VitB1	????mg	????0.5-1.2	????20-50	????16-40
Riboflavin	????mg	????0.8-1.4	????30-50	????25-45
					Nicotinic acid	????mgNE	????10-15	????30-45	????30-45
Vitamin B6	????mg	????1-2	????35-65	????20-45
					Folate	????μg	????100-400	????30-110	????12-50
Vitamin B12	????μg	????1-2	????25-50	????20-45
					Vitamin H	????μg	????20-50	????15-40	????10-40
Pantothenic acid	????mg	????3-5	????30-45	????30-45
					Phosphorus	????mg	????800-1000	????45-55	????35-40
Magnesium	????mg	????100-300	????15-50	????16-45
					Zinc	????mg	????5-15	????20-60	????16-50
Iodine	????μg	????50-150	????15-50	????14-40
					Selenium	????μg	????20-50	????20-45	????16-40
Copper	????mg	????1-2	????25-50	????20-40
					Manganese	????mg	????1-3	????15-40	????14-40
Fluorochemical	????mg	????1-3	????20-60	????16-50
					Chromium	????μg	????10-100	????4-40	????40-385
Molybdenum	????μg	????50-100	????15-35	????16-30
					Muriate	????mg	????1000-3000	????15-50	????14-45
Protein	????g	????30-60	????30-60	????25-50

Low phenylalanine or the α-opalescin that does not have a phenylalanine also are very suitable for adding in the milk beverage, for the big-age-child of PAH defective, and the pregnant woman, teenager and grownup use.

As selection, α-the opalescin of this modification or the dietetic food that contains the alpha-lactalbumin of modification replenish and can be used in the food as bread, ice-creams and frosting mixture, as the functional protein source, they can also not contain phenylalanine, or are that phenylalanine content reduces at least.

The milk of transgenic animal recited above or similar transformed host animal according to the present invention through further modifying or not modifying applicable to as food, the milk when host's wild-type α-opalescin expression of gene weakens or stoped especially.The proteinic further processing of modifying comprised removing be used to regulate or the amino acid tail of other purposes, for example, before it adds in the prescription, process.

According to the 7th aspect of the present invention, the method of getting food for the hyperphenylalaninemia patient is provided, this method comprises to the hyperphenylalaninemia patient provides one or more food, contains the alpha-lactalbumin than the modification still less of wild-type alpha-lactalbumin phenylalanine residue in this food.

And the alpha-lactalbumin preferable feature of modification is as described in top first aspect related content of the present invention.

This food preferable feature is as described in top second aspect relative section of the present invention.More at large, the feature of each aspect of the present invention is pressed the needs of other each side, and has done necessary correction in detail.

Dosage of taking and time are handled by patient oneself consideration, but generally abide by the doctor, nutritionist or other MA's guidance.

The present invention illustrates with following non-restrictive example.Mentioned some accompanying drawings in an embodiment, wherein:

Fig. 1 discusses in embodiment 1, has shown genome λ clone's the restriction map of 2 overlappings of people's alpha-lactalbumin gene.

Fig. 2 discusses in embodiment 1, has shown people's alpha-lactalbumin transgenic constructs.

Fig. 3 discusses in embodiment 1, has shown that the SDS-PAGE of people's alpha-lactalbumin transgenic mice and non-transgenic mouse skimming milk analyzes.

Fig. 4 discusses in embodiment 1, has shown the Western blot of people's alpha-lactalbumin transgenic mice milk and people's alpha-lactalbumin standard substance.

Fig. 5 discusses in embodiment 2, has shown the sequence of ox α-opalescin PCR primer.

Fig. 6 discusses in embodiment 2, has shown the position of ox alpha-lactalbumin PCR primer and product.

Fig. 7 discusses in embodiment 3, has shown the sequence of the ox alpha-lactalbumin PCR primer that is used for mutagenesis.

Fig. 8 discusses in embodiment 4 and 5, has shown to be used for Phe PCR scheme that replaces and the clone's scheme that is used for transgenic constructs PKU1 to 4.

Fig. 9 discusses in embodiment 4, has shown the Western analysis of PKU1 to 4 transgenic mice milk and control mice milk and ox alpha-lactalbumin standard substance.

Figure 10 discusses in embodiment 4, has shown that milk and the ox α-opalescin standard substance diluent with 2 PKU1 transgenic mices of Western engram analysis strain carries out quantitatively.

Figure 11 discusses in embodiment 5, has shown the PCR scheme of Phe replacement and clone's scheme of transgenic constructs PKU5 and PKU1K.

Figure 12 discusses in embodiment 6, has shown the PKU5 that is used for ox α-opalescin and expresses at the COS cell clone's scheme to 10 constructs;

Figure 13 has discussed, has shown the radioautograph with the immunoprecipitation material of the COS cell conditioned medium liquid of pC-BOVA and pC-PKU5 to 10 construct transfection in embodiment 6.

Embodiment 1

The people alpha-lactalbumin is in transgenic mice

The clone of clone and expressing human alpha-lactalbumin gene:

The dna sequence dna of people's alpha-lactalbumin is open (Hall et al., Biochem.J.242735-742 (1987)).End user's sequence, design PCR primer clones 2 small segments from the human gene group DNA, a 5 ' end at this gene, another is at its 3 ' end.Their subclones are screened commodity (Stratagene) λ genomic library to the pUC18 carrier and as probe.Isolate 2 recombinant phages (4a and 5b.l) (Sambrook et al., " molecular cloning " second edition, cold spring harbor laboratory (1989)) that contain the alpha-lactalbumin gene with the method for having set up.Restriction map shows: two clones contain whole encoding sequences of alpha-lactalbumin gene, but difference is 5 ' different with 3 ' sequence amount (Fig. 1) of existing.

Clone 5b.1 place being shown the sequential analysis of son and the part order-checking of clone 4a shows: these clones are identical with disclosed sequence.

Transgenic constructs (Fig. 2):

PHA-1 is by people's alpha-lactalbumin coding region, from go up to pUC18 with 7kb EcoRI/SalI fragment cloning the 1 clone 5b.1 that produces～5 ' flank of 1.8kb and the 3 ' flank of 3kb form.

PoBHA (sheep β-lactoglobulin, people's alpha-lactalbumin) is made up by 4 dna fragmentations:

(1) 4.2kg SalI/EcoRV fragment contains sheep β-lactoglobulin promotor (WO A-9005188);

(2) 74bp synthetic oligonucleotide, corresponding with the base 15-77 of the sequence of the BclI connector of 8bp and people's alpha-lactalbumin, as tack BglI fragment;

(3) from the fragment of 6.2kg BglI/PstI people α-opalescin of 1 clone 4a, comprise the BglI site at base 77 places and the zone between 3 ' the flank XhoI site;

(4) expression of pSL1180 (Pharmacia) people's alpha-lactalbumin in transgenic mice of shearing with PstI and SalI:

Two constructs of called after pHA-1 (containing people's alpha-lactalbumin gene and flanking region) and pOBHA (containing the people's alpha-lactalbumin gene by the control of sheep beta lactoglobulin promotor) are expelled in the mouse embryo, and bring up into transgenic animal.

The expression level scope of people's α-opalescin in these mouse milk is from detecting～3mg/ml.Table 7 has been listed the brief summary of transgenic protein relative quantity.Use is with the SDS-PAGE of Coomassie blue stain, and isoelectrofocusing is observed the Western trace with the antibody (Sigma) of the anti-people's alpha-lactalbumin of commodity and taken from the skimming milk of these animals with a layer folding focus analysis.The result of these analyses shows: compare with people α-opalescin standard substance (Sigma), transgenic protein has appropriate size, pI and antigenicity.

Fig. 3 and 4 has shown the result of several mouse.Fig. 3 has shown the transgenic mice milk of degreasing and the not SDS of transgenosis control mice milk-PAGE analysis.Fig. 4 has shown the Western trace of these milk and people's alpha-lactalbumin standard substance.

Table 7

Transgenic mice juice is analyzed

Mouse Coomassie blue Western trace 205.19 pHA1--204.10 pHA1 ++ ++ 204.7 pHA1 ++++++230.15.3 pHA1 +++n.d.230.15.5 pHA1 +++n.d.230.15.6 pHA1 +++n.d.230.21.5 pHA1 +++n.d.230.21.1 pHA1 ++ n.d.235.15 OBHA-n.d.235.19 OBHA ++ ++ 236.6 OBHA ++ ++ 234.1 OBHA++ 234.4 OBHA ++ ++ 234.14 OBHA++ (table 7 has shown through compare the relative level of people's α-lactoalbumin in transgenic mice milk that estimates with the protein standard items on coomassie gel and Western trace.

-????＝＜0.5mg/ml

+????＝～0.5-1mg/ml

++???＝～1-2mg/ml

+++??＝～2-3mg/ml

N.d.=does not detect)

Embodiment 2

The ox alpha-lactalbumin in mouse the clone and cloning by expression in alpha-lactalbumin:

3 kinds of known varients are arranged in the ox alpha-lactalbumin, and wherein Type B is the most general a kind of.From the difference of the A varient of Bos (Bos) nomadicus f.d.indicus and B varient on residue 10: the Glu among the A is replaced to the Arg among the B.Obtain (McKenzie ﹠amp as yet with sequence area from the C varient of Bos (Bibos) javanicus; White, Advances in Protein Chemistry 41 173-315 (1991)).

Use PCR primer shown in Figure 5 clened cows α-opalescin gene (coding N type) from genomic dna〉below SEQ ID NOs listed these primers.

Ba-2????SEQ?ID?NO：1

Ba-7????SEQ?ID?NO：2

Ba-8????SEQ?ID?NO：3

Ba-9????SEQ?ID?NO：4

In all PCR reactions, the DNA source is Holstein-Friesian cow blood.

The promoter region length of using primer Ba-7 to combine amplification with primer Ba 1 is 2.05kb.With this BamHI/EcoR fragment cloning to Bluescript (on the pBA-P2).

Use primer Ba-9 to combine whole ox alpha-lactalbumin genes that amplification comprises 300bp3 ' flanking region with primer Ba-2.These primers comprise BamHI Restriction Enzyme recognition site, and the direct subclone of 3kb fragment that this site allows to increase produces construct pBA-G3 to the BamHI site of pUC18.

To be connected on the EcoRV/BamHI fragment of BA-G3 from the BamHI/EcoRV fragment of clone pBA-P2, produce construct pBA (Fig. 6).

Because the Taz polysaccharase lacks proofreading activity, the ox alpha-lactalbumin DNA that therefore guarantees amplification and the identical necessity of disclosed ox alpha-lactalbumin gene.All exons and 2 promoter fragments have been carried out sequential analysis.Relatively the ox alpha-lactalbumin shows that outward son and the disclosed sequence of Vilotte demonstrate 3 places and change:

(1) exon I+759: C becomes A; 5 ' non-coding region.The existing report of this change (Hoehi et al., Mol.Reprod.and Devel.33 160-164 (1992)).

(2) exon I+792: CTA becomes CTG; The both is leucic codon.

(3) exon II+1231: GCG becomes ACG; Become Threonine by L-Ala.This is the symbol of the more general B form of this protein.

Although can not get rid of mispronouncing of sequence in the pcr amplification process, top mismatch is likely and produces owing to the source of ox DNA is different.The expression of ox alpha-lactalbumin in transgenic mice:

PBA construct (Fig. 6) is injected into mice embryonic and brings up out 9 transgenic animal.Carry out the milk analysis by embodiment 1 is described.Compare through the SDS-PAGE gel analysis of Coomassie blue stain and with the alpha-lactalbumin normal content and to show: the expression level scope from can not detect～0.5-1, g/ml.

Embodiment 3

The SEQ ID NOs of site-directed mutagenesis PCR primer:

Design following SEQ ID NOs as the PCR primer, be used for present embodiment and embodiment subsequently:

PKU-1????????????????SEQ?ID?NO：5

PKU-2????????????????SEQ?ID?NO：6

PKU-2L???????????????SEQ?ID?NO：7

PKU-3????????????????SEQ?ID?NO：8

PKU-4????????????????SEQ?ID?NO：9

PKU-5????????????????SEQ?ID?NO：10

PKU-6????????????????SEQ?ID?NO：11

PKU-7????????????????SEQ?ID?NO：12

PKU-8????????????????SEQ?ID?NO：13

PKU-9????????????????SEQ?ID?NO：14

PKU-10 SEQ ID NO:15 replaces the Phe residue:

4 phenylalanines (Phe) residue of ox alpha-lactalbumin appears in preceding 3 exons of this gene.

According to the protein simulation, the trophology aspect, the amino acid variation body that is present in different types of natural alpha-lactalbumin or lysozyme gene is designed for cover 7 oligonucleotide (PKU- PCR primer 1,2,2L, 3,4,7 and 8) that Phe replaces.The sequence of these primers (with coding strand or noncoding strand complementation), the amino acid variation of expection and each mutagenesis point are as shown in Figure 7.These oligonucleotide serve as PCR primer and mutagenic primer.PKU primer 1 is used in combination with 2 or 2L, and 7 are used in combination with 8, produces to contain the 435bp amplified fragments (A) that preceding 2 Phe replace. PKU primer 3 and 4 or 9 and 10 produces PCR product (B) (see figure 8) of the 601bp that contains back 2 Phe replacement.

Use site EcoRI/BamHI and BamHI/XbaI subclone PCR product to check order respectively.PvuI site (being transformed into the PKU primer 2,2L, 3,7 and 8) produces single restriction site and does not change this district's amino acids coding between amino acid 31 and amino acid 53.The amino acid tail of C-terminal:

Adding extra amino acid at alpha-lactalbumin gene C end can separate from by source property alpha-lactalbumin protein matter in the life-span.Purpose is whether the amino acid of measuring increase influences genetically modified expression.Poly-Arg tail:

Primer PKU-5 and 6 (seeing Fig. 7 and 8) is used to increase 3 ' end of ox alpha-lactalbumin gene to produce the PCR product C of 0.44kb.Primer PKU6 contains the coding region of 6 one Arg residues, is right after after last natural Leu amino acid of ox alpha-lactalbumin sequence and produces single S alI and SnaBI site.Single S alI/SnaBI site can be digested and be inserted poly-Arg or the poly-Arg/Lys tail of connector to produce required random length.Natural terminator codon is subsequently～3 ' sequence of 30bps, and end up with BspMI Restriction Enzyme site.

Embodiment 4

Ox alpha-lactalbumin construct in vivo

Assembling and be expressed in the construct of expressing in the galactophore of transgenic animal:

5 constructs have been prepared altogether: natural ox alpha-lactalbumin gene (pNARG-H) and 2 ox alpha-lactalbumin genes (PKU-1 is to PKU-4) that have and do not contain the sudden change of poly-Arg tail with poly-Arg tail.Phe mutant in these constructs is aspect protein simulation and trophology.The design of construct is listed in Fig. 8.

Make up pBARG-H from 5 dna fragmentations:

(1): from pBA, SstI is to the fragment of the 2.04kg of HpaI;

(2): from pBA, HpaI is to the fragment of the 1.25kg of HindIIII;

(3): from the fragment of PCR product C HindIII to the 0.44kb of BspMI;

(4): from the fragment of pNA BspMI to the 0.51kb of BglII;

(5): with the carrier pSL 1180 of SstI and Bg1II digestion.

Make up PKU-1 from 6 dna fragmentations

(1): from the fragment of pBA SstI to the 2.04kg of HpaI;

(2) from the fragment of PCR product A (the PKU primer is to 1 and 2) HpaI to the 0.46kb of PvuI.

(3): from the fragment of PCR product B (primer is to 3 and 4) PvuI to the 0.60Kb of BsaBI;

(4): from the fragment of pBA BsaBI to the 0.22kb of HindIII;

(5): from the fragment of pBA HindIII to the 0.95kb of BglII;

(6): with the carrier PSL 1180 of SstI and BglII digestion;

PKU-2 makes up in the mode identical with PKU-1, except wherein fragment 5 from pBARG-H and contain poly-Arg tail.

PKU-3 makes up in the mode identical with PKU-1, uses PKU primer 1 except fragment (2) (PCP product A) and increases in conjunction with 2L.

PKU-4 makes up in the mode identical with PKU-3, except fragment (5) from pBARG-H and contain poly-Arg tail.

Aminoacid replacement and plasmid PKU-1 thereof that above-mentioned mutagenesis produces are displayed in Table 8 to PKU-4.

People's alpha-lactalbumin 3 ' flanking region:

9.3kb BamHI fragment from λ clone 4a (Fig. 1) contains 3 ' flanking region of people's alpha-lactalbumin gene and all comprise the suitable target of screening before it is implanted as the transgenosis embryo in all PKU construct.The fragment of this 9.3kb is inserted into (see figure 8) on the single BglII site that all exists in all above-mentioned constructs.

Digest the DNA that to be used for microinjection through shearing from the carrier sequence with BamHI.Be used for the preparation of DNA of microinjection and the production of transgenic mice and carry out (Hogan et al. by disclosed method, " Manipulating the Mouse Embryo:A Laboratary Man-Hal ", Cold Spring Harbor laboratory Press (1986)).Table 8: the aminoacid replacement that in transgenic constructs, occurs:

Replace Arg tail people 3 ' flank plasmid

Position 9 30 53 80

Tyr，Tyr，Tyr，Tyr????-???????????+??????????pPKU-1

Tyr，Tyr，Tyr，Tyr????+???????????+??????????pPKU-2

Tyr，Leu，Tyr，Tyr????-???????????+??????????pPKU-3

Tyr，Leu，Tyr，Tyr????+???????????+??????????pPKU-4

Wild type++ pBARGh do not have the expression of ox alpha-lactalbumin in transgenic mice of phenylalanine:

The transgenosis person of foundation animal is at natural gift lactation juice in postpartum the 10th, and its milk carried out Western Blots analyze (see figure 9).Whole constructs (the ox alpha-lactalbumin construct of wild-type ox alpha-lactalbumin pBARG-H and 4 mutagenesis (: KU1-4)) can both in milk, express alpha-lactalbumin protein in the justacrine.Stronger above band (see figure 9) is likely what the glycosylation varient formed, and it also is present in (data does not show) among pBARG-H.Table 9 has shown the proteinic relative quantity of no phenylalanine ox alpha-lactalbumin in these transgenic mice milk.For carrying out quantification of protein, the serial dilutions of milk sample is carried out Western Blots and compared with ox α-opalescin standard substance.Figure 10 has shown PKU-1 transgenic mice sample has been used this program.

In conjunction with the technology of having set up (as ELISA, PI, order-checking and circular dichroism) protein is further analyzed.Sound out this proteinic size and electric charge and folded situation with these methods.

Table 9

The expression of PKU1-4 in transgenic mice milk

The alpha-lactalbumin protein of construct in transgenosis milk

pBARG-H????????????????????0-0.50mg/ml

PKU-1??????????????????????0-0.25mg/ml

PKU-2??????????????????????0-0.50mg/ml

PKU-3??????????????????????0-0.10mg/ml

PKU-4??????????????????????0-0.50mg/ml

RNA analyzes:

Use the specific probe of ox alpha-lactalbumin that the total RNA of transgenosis mammary gland is carried out the Northern engram analysis.As desired, the animal sample that all has detectable recombinant protein level all shows ox alpha-lactalbumin mRNA and expresses.Between the transgenic protein of mRNA amount and existence, there is not clear and definite dependency.

In order to confirm that expressed protein comes from the alpha-lactalbumin gene of sudden change, extract ox alpha-lactalbumin mRNA and the order-checking that exists among the RNA with RT-PCR method clone.The result confirms to transcribe out mRNA from PKU1-4 construct, and therefore the protein of translation should be no phenylalanine.

Embodiment 5

Mutagenesis under the people's alpha-lactalbumin promotor control

The expression in vivo of ox alpha-lactalbumin

Embodiment 1,2 shows with 4 data: the genetically modified expression of people's alpha-lactalbumin is compared quite high with natural ox alpha-lactalbumin transgenosis, and this has reflected in the endogenous and the difference of human gene expression level.Because this may be owing to the difference of 5 ' control region.So the sequence of the alpha-lactalbumin gene of choosing replaces 5 ' district of the transcription initiation site of ox alpha-lactalbumin.

Prepared 2 constructs, called after PKU-5 and PKU-1H, the aminoacid replacement that wherein mixes is as shown in table 10.The design of construct is listed in Figure 11.

PKU-5: first clone step in, with 3 fragment subclones to the EcoRI/BamHI site of pUC18:

(1) use PKU primer 7 in conjunction with 8 EcoRI that obtain through pcr amplification to PvuI fragment (Segment A, Figure 11);

(2) use PvuI that PKU primer 9 and 10 obtains through pcr amplification to the fragment of BsaBI (fragment B, Figure 11).

(3) from the BsaBI of pBA fragment to HindIII.

Final construct comprises 6 dna fragmentations:

(1) contains the fragment of the SalI of people's alpha-lactalbumin promotor from N clone 4a (Fig. 1) to the 3.7kb of KpnI;

(2) oligonucleotide of synthetic 152bp comprises people's alpha-lactalbumin sequence from the KpnI site to AVG and the ox alpha-lactalbumin sequence from AUG to the HapI site;

(3) from the HpaI of the first subclone step 1.25kb fragment to HindIII;

(4) from the 0.95kb fragment of pBA from HindIII to BglII;

(5) from the 3.7kb fragment of people's alpha-lactalbumin gene 3 ' flank from BamHI to XhoI of λ clone 4a (Fig. 1);

(6) Bluescript KS-carrier of shearing with SalI and BamHI.

PKU-1H to be making up with PKU and identical mode, except fragment (3) from PKU-1 (embodiment 4).

Table 10

Aminoacid replacement in the transgenic constructs

Replace people's promoter plasmid position 9 30 53 80

Tyr，Tyr，Tyr，Tyr????+????????????pPKU—1H

Ser, Tyr, Leu, the expression of Leu+pPKU-5 in transgenic mice:

Two construct: KU-1H and PKU-5 are expelled in the mouse embryo.Up to the present the transgenic animal of PKU-5 construct have been obtained.Bring up these animals and be used for breeding to analyze its milk.

Embodiment 6

The ox alpha-lactalbumin of mutagenesis expression transient expression construct in vivo:

In order to analyze the sudden change on a large scale of alpha-lactalbumin gene, set up a vivoexpression test.Wherein, with natural ox with the sudden change ox alpha-lactalbumin Ji Jibianmaqu ( embodiment 2,3,4 and 5: sequence location 756 to 3030; Vilotte et al., Biochemie 69, and 609-620 (1987)) be cloned among the COS fibrocyte expression vector pcDNA 3 (Invitrogen) to produce pC-BOVA and pC-PKU-5 to-10.

With reference to Figure 12, it is as follows to list its structure in detail:

(1) will contain ox alpha-lactalbumin sequence from position 756 to the position synthetic oligonucleotide 831 the HpaI site be cloned on the HindIII/BamHI site of carrier pCDNA3;

(2) coding region of ox alpha-lactalbumin gene is inserted to the fragment between the BamHI with HpaI, produce construct pC-BOVA;

(3) HpaI among pC-BOVA is changed into to the 1.25kb fragment of HindIII contain HpaI that Phe replaces and arrive-10 to produce construct pC-PKU-5 to the 1.25kb fragment (Figure 11) of HindIII.

The existing of restriction site PpmMI between the amino acid 9 and 30 and the AvrII between amino acid 53 and 80 (Figure 11) allows to produce 1 or 2 substituted PKU constructs in 4 phenylalanines, and be as described in Table 11.

Table 11

Be present in the aminoacid replacement in the COS cell construction body

Replace plasmid

9????30???53???80

Phe，Phe，Phe，Phe????pC-BOVA

Ser，Tyr，Leu，Leu????pC-PKU-5

Phe，Tyr，Leu，Phe????pC-PKU-6

Tyr，Phe，Phe，Tyr????pC-PKU-7

Phe，Phe，Phe，Tyr????pC-PKU-8

Phe，Phe，Phe，Leu????pC-PKU-9

Ser, Phe, Phe, the expression of Phe pC-PKU-10 in the COS cell:

With 10 μ g plasmid transfection cells, after 72 hours, on the protein labeling ³⁵S-methionine(Met) and ³⁵S-halfcystine.Collect supernatant liquor and through on polyacrylamide gel, analyzing alpha-lactalbumin behind the immunoprecipitation.

Figure 13 has shown that the transient expression in the COS cell causes ox alpha-lactalbumin secretion natural and sudden change.The expression level that is become aminoacid replacement (pC-PKU 10) generation of Ser by Phe9 equates with wild-type ox alpha-lactalbumin.Phe30 is replaced by Tyr and Phe53 is replaced by Leu that (pC-PKU6), Phe90 are replaced by Tyr that (behind the pC-PKU8), expression level descends.PC-PKU5,7 and 9 be expressed under this pilot system detection level.The aminoacid replacement of this system's definable high expression level in transgenic animal.

Embodiment 7

How to use the proteinic milk that contains modification

For the children that suffer from phenylketonuria, need a kind of Special food that does not have phenylalanine to replenish.Because in the conformability of diet in those early years is crucial, the therefore preferred delicious and arbitrary treatment prescription of diet conformability enhanced.

When design phenylketonuria children's diet, must determine the protein of every day, caloric value and amino acid need (comprising phenylalanine) to keep the needs of life-span and growth, calculating replenishes protein and the caloric value that provides by special diet, and what whole food decision needs satisfy but do not exceed the phenylalanine needs then.The supply of this phenylalanine realizes through milk and other general food of adding measured quantity under the normal circumstances.

Diet every day of the bent type of phenylketonuria children should comprise 3 parts/day special fill-in, and this fill-in contains the alpha-lactalbumin of modification and other nutrition in the previously described table 3.General suggestion is restrictive phenylalanine diet, and it is clocklike adjusted to satisfy the age, and the needs of growing and keeping make to give birth to make with continuity.

Embodiment 8

The protein that preparation/separation is modified from milk

For great majority (if not all) special formulation goods, need from the alpha-lactalbumin matter of modifying, separate and the normal alpha-lactalbumin of purifying.For example, for children, teenager and pregnant woman, prescription zero or minimum Phe should have maximum diet handiness and whole probably certainly with dietetic treatment PKU.

Therefore design adds poly-Arg or Arg/Lys tail at the C end, so as can be more effective with separate more at an easy rate.This design can change the proteinic electric charge of modification so that from normal alpha-lactalbumin it is separated.This separation should available laboratory and the whey of commercial quantities separates and then carries out liquid chromatography technology and realize, back one separation may be with the basis of molecule charge differences.Liquid chromatography has shown permission separation and protein purification and other composition from the whey of milk.

Other technology also can be used, and identifies especially for laboratory or bench scale.These technology are including, but not limited to SDS-PAGE chromatogram and radioimmunoassay.

Embodiment 9

The prescription of modifying protein

Can prepare some special diet prescriptions according to the present invention and be used for the treatment of hyperphenylalaninemia.These prescriptions are unique in the useful prescription of institute, because wherein a kind of protein completely (alpha-lactalbumin of modification) is the amino acid source of dominant protein and no Phe, and in general formulation, the N source is all with the L-amino acid whose form or the casein protein of extensive hydrolysis.

Dominant complete protein characteristic has strengthened the diet conformability in these new treatments prescriptions, because its taste and palatability have had great improvement, pure L-amino acid and peptide mixt typically taste are very poor and be difficult to stand.Therefore the protein of modifying is used for lasting till that since infancy the dietetic treatment of the whole life course of period of pregnancy and Adulthood has advantage at a series of prescriptions of production.

These prescriptions that contain the alpha-lactalbumin source of identical modification can change protein content altogether, add L-amino acid to improve nutritive value, and adding the nonprotein source provides energy, as add sugar, corn steep liquor and/or fat, and VITAMIN, inorganics and seasonings (if suitable).Clearly, the special diet prescription that is used for the baby should be attempted to be complementary with human milk's composition or its physiological effectiveness, and should contain the fatty calorific value of high percentage ratio thus.Be used for children and adult prescription and should contain more gross protein and less fat, if any, should adding VITAMIN and the inorganics different with the required ratio of baby with young child.Get whole food and make up to be designed to have the diet group of identical phenylalanine levels with the prescription of these non-baby's groups, therefore the different diet group of convertible use further increases the diet diversity, thereby has strengthened its conformability.

Sequence description: (1) physical data (A) is used for the applicant of all designated states except that US

(A) title: medical protein company limited

(B) street: Orchard Brae House 30 Queesferry Road

(C) city: Edinburgh

(E) country: Britain

(F) postcode (number): EH4 2HG (i) only limits to contriver/applicant of US:

(A) name: Colman, Alan

(B) street: 65 Cromwell Lane

(C) city: Coventry

(E) country: Britain

(F) postcode (number): CN4 8AQ

(A) name: Wright, Gordon

(B) street: 168 ESKhill

(C) city: Penicuik

(D) state: Midlothian

(E) country: Britain

(F) postcode (number): EH26 8DQ

(A) name: SAWYER, Lindsey

(B) street: 14 MORNINGSIDE PARK

(C) city: EDINBURGH

(E) country: Britain

(F) postcode (number): EH10 5HB

(A) name: Rigden, Daniel John

(B) street: 38 Denver Avenue

(C)fdym：Crewe

(D) state: Cheshire

(E) country: United Kindom

(F) postcode (number): CW2 7PX is the invention exercise question (ii): the α-opalescin of modification is sequence number (ii): 15 (iv) computer-reader forms

(A) media types: Floppy disk

(B) computer: IBM PC compatibility

(C) operating system: PC-DOS/MS-DOS

(D) software: Patent In Releose#1.0, Version#1.25

(EPO) (2) SEQ ID NO:1 data

(i) sequence signature:

(A) length: 27 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:1:GCGGATCCAC AACTGAAGTG ACTTAGC 27 (2) SEQ ID NO:2 data

(i) sequence signature:

(A) length: 35 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:2:GATGGATCCT GGGTGGTCAT TGAAAGGACT GATGC 35 (2) SEQ ID NO:3 data (i) sequence signatures:

(A) length: 43 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style is molecule type (ii): cDNA (xi) sequence description: SEQ ID NO:3:GCAGGCGAAT TCCTCAAGAT TCTGAAATGG GGTCACCACA CTG 43 (2) SEQ ID NO:4 data (i) sequence signatures:

(A) length: 33 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style is molecule type (ii): cDNA (xi) sequence description: SEQ ID NO:4:GAGGATCCAA TGTGGTATCT GGCTATTTAG TGG 33 (2) SEQ ID NO:5 data

(i) sequence signature:

(A) length: 46 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:5GCTGAATTCG TTAACAAAAT GTGAGGTGTA TCGGGAGCTG AAAGAC 46 (2) SEQ ID NO:6 data

(i) sequence signature:

(A) length: 58 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO.6GCGGATCCGA TCGCTTGTGT GTCATAACCA CTGGTATGGT ACGCGGTACA GACCCCTG 58 (2) SEQ ID NO:7 data

(i) sequence signature:

(A) length: 58 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:7GCGGATCCGA TCGCTTGTGT GTCATAACCA CTGGTATGGA GCGCGGTACA GACCCCTG 58 (2) SEQ ID NO:8 data

(i) sequence signature:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: cDNA

(xi) SEQ ID NO:8 sequence description GCGGATCCGA TCGTACAAAA CAATGACAGC ACAGAATATG GACTCTACCA GATAAATAAT 60AAAATTTGG 69 (2) SEQ ID NO:9 data

(i) sequence signature:

(A) length: 34 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: cDNA

(vi) sequence description: SEQ ID NO:9:GCTCTAGATC ATCATCCAGG TACTCTGGCA GGAG 34 (2) SEQ ID NO:10 data

(i) sequence signature:

(A) length: 24 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:10:GCTGAAGCTT CACTTACTTC ACTC 24 (2) SEQ ID NO:11 data

(i) sequence signature:

(A) length: 65 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style

(ii) molecule type: cDNA

(xi) sequence description: SEQ ID NO:11:GCGGATCCAA AGACAGCAGG TGTTCACCGT CGACGACGCC TACGTAACTT CTCACAGAGC 60CACTG 65 (2) SEQ ID NO:12 data (i) sequence signatures:

(A) length: 46 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style is molecule type (ii): cDNA (xi) sequence description: SEQ ID NO:12:GCTGAATTCG TTACAAAAT GTGAGGTGAG CCGGGAGCTG AAAGAC 46 (2) SEQ ID NO:13 data (i) sequence signatures:

(A) length: 54 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style is molecule type (ii): cDN A (xi) sequence description: SEQ ID NO:13:GCGGATCCGA TCGCTTGTGT GTCATAACCA CTGGTATGAT ACGCGGTACA GACC 54 (2) SEQ ID NO:14 data (i) sequence signatures:

(A) length: 69 base pairs

(B) type: nucleic acid

(C) chain: strand

(D) topology: line style is molecule type (ii): cDNA (xi) sequence description: SEQ ID NO:14:GCGGATCCGA TCGTACAAAA CAATGACAGC ACAGAATATG GACTCCTCCA GATAAATAAT 60AAAATTTGG 69 (2) SEQ ID NO:15 data (i) sequence signatures

(A) length: 34 base pairs

(A) type: nucleic acid

(C) chain: strand

(D) topology: line style is molecule type (ii): cDNA (vi) sequence description: SEQ ID NO:15:GCTCTAGATC ATCATCCAGC AGCTCTGGCA GGAG 34

Claims (28)

1. the alpha-lactalbumin of a modification, it is compared with the wild-type alpha-lactalbumin and is not contained phenylalanine residue or phenylalanine residue still less, and it is different from people's alpha-lactalbumin varient that Phe-80 is replaced by Methionin.

2. according to the alpha-lactalbumin of the modification of claim 1, contain an amino acid tail at its C end and be beneficial to purifying.

3. according to the alpha-lactalbumin of the modification of claim 2, C end tail wherein is poly-Arg or Arg/Lys.

4. according to the alpha-lactalbumin of claim 1,2 or 3 modification, it is the ox alpha-lactalbumin of modifying.

5. according to the ox alpha-lactalbumin of the modification of claim 4, wherein Phe9 is by Tyr, and Ser or Leu replace.

6. according to the ox alpha-lactalbumin of the modification of claim 4 or 5, Phe wherein ₃₁By Leu, Ile, Trp or Tyr replace.

7. according to the ox alpha-lactalbumin of the modification of claim 4,5 or 6, Phe wherein ₅₃By Tyr, Leu, Met or Trp replace.

8. according to each the ox alpha-lactalbumin of modification of claim 4 to 7, wherein Phe ₈₀By Tyr, Met, Trp or Leu replace.

9. according to the alpha-lactalbumin of claim 1,2 or 3 modifications that require, it is people's alpha-lactalbumin of modifying.

10. people's alpha-lactalbumin of the modification that requires according to claim 9, wherein Phe ₃By Tyr, Leu, Arg, Met or Trp replace.

11. according to people's alpha-lactalbumin of claim 9 or 10 modifications that require, Phe wherein ₃₁By Leu, Ile, Trp or Tyr replace.

12. people's alpha-lactalbumin of the modification that requires according to claim 9,10 or 11, wherein Phe ₅₃By Tyr, Leu, Met or Trp replace.

13. according to people's alpha-lactalbumin of the modification of each requirement of claim 9 to 12, wherein Phe ₈₀By Tyr, Met, Trp or Leu replace.

14. a method for preparing according to the alpha-lactalbumin of the modification of each requirement of claim 1 to 13, this method comprise and successive amino acid is coupled to together and/or connects few-and/or many-peptide.

15. a coding does not contain phenylalanine residue or the phenylalanine residue nucleic acid than the isolating or reorganization of the alpha-lactalbumin of wild-type alpha-lactalbumin modification still less.

16. the nucleic acid of the isolating or reorganization of an alpha-lactalbumin that is coded in the modification of each requirement in the claim 1 to 13.

17. the recombinant DNA that requires in claim 15 or 16, it is a kind of carrier format.

18. a method for preparing the nucleic acid that require according to claim 15 or 16, this method comprise and successive Nucleotide is coupled to together and/or connects widow-and/or many-Nucleotide.

19. a host, it contains claim 15,16 or 17 recombinant DNAs that require.

20. the host who requires in the claim 19, it is a kind of inhuman viviparous Mammals, its kind contains the DNA of the alpha-lactalbumin that coding modifies in being, the adult female performance that this kind is is expressed the alpha-lactalbumin of modification in mammary gland, the alpha-lactalbumin of modification is accumulated in milk.

21. the host mammal that requires in the claim 20, it is cow (or bull), sheep, goat, camel or pig.

22. the host who requires in the claim 19,20 or 21, wherein host's wild-type alpha-lactalbumin expression of gene is obstructed or is weakened.

23. a food that is applicable to the hyperphenylalaninemia patient, this food does not contain phenylalanine residue, or contains the alpha-lactalbumin of at least a modification, and the phenylalanine residue that this alpha-lactalbumin comprises than wild-type alpha-lactalbumin still less.

24. according to the food of claim 23, the alpha-lactalbumin of the wherein alpha-lactalbumin of Xiu Shiing, or at least a modification is that each is desired in the claim 2 to 13.

25. according to claim 23 or 24 desired food, it is that a kind of baby of being applicable to or children's hyperphenylalaninemia patient's recipe is filled or fills a prescription.

26. according to the food of claim 23 or 24, it is the milk according to claim 19,20 or 21 desired female mammals.

27. give the phenylketonuria patient method of feed for one kind, this method comprises to the patient takes a kind or the multiple food that contains the alpha-lactalbumin of modification, this alpha-lactalbumin does not contain phenylpropyl alcohol and contains sour residue, or the propylhomoserin residue wild-type alpha-lactalbumin of benzene still less.

28. according to the method for claim 27, food wherein is that each is desired for claim 23 to 26.

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