CN1261104A - Human oxido-reductase subunit and its coding sequence, preparing process and application - Google Patents
Human oxido-reductase subunit and its coding sequence, preparing process and application Download PDFInfo
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Abstract
The prestent invention discloses the cDNA sequnce of a new human reduced NADH: ubiquinone oxidoreductase 14 KDa subunit (NADH-CoQ14KDa subunit). The protein coded with the sequence is the homolog of ox's NADH-CoQB14.5b subunit. The polypeptide coded by said nucleotide sequence, the application of said polynucleotide and polypeptide, and the process for preparing said polynucleotide and said polypeptide are also disclosed.
Description
The present invention relates to the genetically engineered field, particularly, the present invention relates to a kind of new human gene nucleotide sequence.More particularly, the present invention relates to people's nicotinamide adenine dinucleotide reduced-CoQ oxide-reductase 14 kilodalton subunits (NADH:ubiquinone oxidoreductase 14 kDa subunit, abbreviate " NADH-CoQ 14 kDa subunits " as) the cDNA sequence, this albumen is the homologue of ox NADH-CoQ B14.5b subunit.The invention still further relates to by this nucleotide sequence coded polypeptide the application of these polynucleotide and polypeptide, and the production method of described polynucleotide and described polypeptide.
Plastosome NADH: ubiquinone oxide-reductase enzyme (NADH:ubiquinone oxidoreductase) (being also referred to as " composite I ") is the proteolytic enzyme mixture of subunit more than.It is positioned at mitochondrial inner membrane, is the first step of plastosome oxidative phosphorylation electron transfer chain.It accepts to derive from the electronics of NADH, and by a series of electron carrier it is transferred on the ubiquinone (Coenzyme Q10 99.0).The electron carrier of composite I inside then comprises FMN and 6 iron-sulphur bunch (N-1a, 1b, 2-5) (Ohnishi, T.et al., Mitochondrial iron-sulfur flavohydrogenases.In:Capaldi, R.A.:Menbrane Proteins in Energy Transduction.New York:Dekker (pub.), 1979; Ragan, C.I.et al.Structure of NADH-ubiquinone oxidoreductasereductase (Complex I) .Curr.Top.Bioenerg.15:1,1987).Composite I can be divided into 3 major portions: flavoprotein part, iron-sulphur protein part, hydrophobin part.
Flavoprotein part part has comprised that vitamin B2 phosphate (FMN), 6 iron ions and 3 are respectively 51,24, polypeptide (Galante, Y.M.et al.Arch.Biochem.Biophys.192:559-568,1979 of 10kDa; Ragan, C.I.et al.Biochemstry, 21:2518-2524,1982).Wherein NADH binding site and FMN are positioned at (Chen, S.﹠amp on the polypeptide of 51Kda; Guillory, R.J.J.Biochem.Chem.256:8318-8323,1981).Iron-sulphur protein has partly comprised 9 or 10 iron ion (Ragan, C.I.et al.Biochemstry, 21:2518-2524,1982) and the albumen of a 15KDa, the latter's effect seemingly combines with ubiquinone (being ubiquinone) and gives ubiquinone (Suzuki, H ﹠amp electron transport; Ozawa, T., Biochem.Biophys.Res.Commu.138:1237-1242), MTND6 albumen also may be positioned at this part (Chomyn, A.et al.Science, 234:614,1986).The hydrophobin part has then comprised iron-sulifide protein, and they are considered to be likely electron donor (Ohnishi, T.et al.J.Biol.Chem., 260:2782-2788,1985 of ubiquinone; Ohnishi, T.et al.Biochem.Biophys.Res.Commun.56:775-782,1974).
Research to composite I is carried out the most detailedly in the cor bovinum plastosome.The primary structure of its 41 subunits is illustrated.Wherein 7 protein protomers are by the chondriogen group coding, and all the other are then by nuclear gene group coding (Walker, J.E.et al.Methods of Enzyme, 260:14-34,1995).In the people, research to each subunit of composite I (being made of at least four ten above subunits equally) is relatively slow, except that gene by the plastosome coding, the nuclear gene group gene that minority coding composite I is only arranged is by more careful research, as 8kDa, 10kDa, 24kD and the 51kDa subunit etc. of composite I.
In various mitochondrial myopathy patients with different clinical diseases, the defective of composite I is very common.To the research of philtrum mutant composite I equally based on the gene in the plastosome.Up to now, found a large amount of sudden changes relevant with the composite I function in Mitochondrial Genome Overview, these sudden changes comprise (Wallace, D.C., et al.Am.J.Hum.Genet., 57:201-223,1995 such as point mutation, disappearance; Schon, E.A., et al.J.Bioenerg.Biomebr., 29:131-149,1997; Cooper, J.M., et al.Biochimica et Biophysica.Acta., 1101:198-203,1992).Rational idea is that the nuclear gene of coding composite I subunit also may cause mitochondrial disease, this guess has obtained certain experiment support equally: found 75-, 13,24 or disappearance (the Moreadith et al.J.Clin.Invest. of 20kDa subunit in some mitochondrial disease patients, 79:463-467,1987; Schapira et al. Molecular basis of mitochondrial myophathies:polypeptideanalysis in complex-I deficiency Lancet I (8584), 1988 500-503; Slipetz et al.Am.J.Hum.Genet., 48:1121-1126,1991).Yet contacting directly between nuclear gene and composite I functional defect still remains further to be inquired into.
Arizmendi in 1992, people such as JM report have recorded two cor bovinum plastosome complex subunit B14.5a and B14.5b, and (FEBS lett, 1992,313 (1): 80-84), these two proteic nucleotide sequences of encoding are also checked order.
In the people, except that 7 plastosome subunits of coding composite I, only the minority nuclear gene has been carried out detailed research on the nucleotide sequence level, found 10 composite I subunit nuclear genes (GenBankAccession No.AF035840 that checked order so far altogether, AF013160, AF038406, AF044959, AF067139, HS17379, AFO20352, AF020351, HSU65579, HSU64028).Therefore, the nuclear gene of further seeking other subunit of coding composite I for the mutual relationship between these subunits of detail knowledge and in whole mixture role, and then to the genesis mechanism of illustrating some relative disease with significant.
Therefore, this area presses for other subunits of finding and isolating the NADH-CoQ oxydo-reductase.
An object of the present invention is to provide a kind of new polynucleotide, a subunit of this polynucleotide encoding NADH-CoQ oxide-reductase, people NADH-CoQ oxide-reductase subunit gene of the present invention is named as people NADH-CoQ 14 kDa subunits.
Another object of the present invention provides a kind of new people NADH-CoQ oxide-reductase subunit, and this albumen is named as people NADH-CoQ 14 kDa protein subunits.
A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people NADH-CoQ 14 kDa subunit polypeptides.
The present invention also provides the application of this people NADH-CoQ 14 kDa subunit nucleic acid sequences and polypeptide.
In one aspect of the invention, a kind of isolated dna molecular is provided, it comprises: coding has the nucleotide sequence of the active polypeptide of people NADH-CoQ 14 kDa protein subunits, shows at least 70% homology from the nucleotides sequence of Nucleotide 51-410 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 51-410 position.Preferably, described sequence encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.4.More preferably, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 51-410 position.
In another aspect of this invention, provide a kind of isolating people NADH-CoQ 14 kDa protein subunit polypeptide, it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.Preferably, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.
In another aspect of this invention, provide a kind of carrier, it contains above-mentioned isolated DNA.
In another aspect of this invention, provide a kind of described carrier transformed host cells.
In another aspect of this invention, provide a kind of generation to have the method for the active polypeptide of people NADH-CoQ 14 kDa protein subunits, this method comprises:
(a) nucleotide sequence that coding is had an active polypeptide of people NADH-CoQ 14 kDa protein subunits operationally is connected in expression regulation sequence, form people NADH-CoQ 14 kDa protein subunit expression vectors, show at least 70% homology from the nucleotides sequence of Nucleotide 51-410 position among described nucleotide sequence and the SEQ ID NO.3;
(b) change the expression vector in the step (a) over to host cell, form the reconstitution cell of people NADH-CoQ 14 kDa protein subunits;
(c) under the condition that is fit to expressing human NADH-CoQ 14 kDa protein subunit polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate and have the active polypeptide of people NADH-CoQ 14 kDa protein subunits.
In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 450 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 51-410 position Nucleotide.
In the present invention, " isolating ", " purifying " or " pure substantially " DNA are meant, this DNA or fragment have been arranged in the sequence of its both sides and have separated under native state, refer to that also this DNA or fragment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.
In the present invention, refer to encode has the nucleotide sequence of the active polypeptide of people NADH-COQ 14 kDa protein subunits to term " people NADH-CoQ 14 kDa protein subunit (or polypeptide) encoding sequences ", as 51-410 position nucleotide sequence and degenerate sequence thereof among the SEQ IDNO.3.This degenerate sequence is meant, is arranged in the encoder block 51-410 position Nucleotide of SEQ ID NO.3 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, thus with SEQ ID NO.3 in 51-410 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.4 of also encoding out.This term also comprises can be under the moderate stringent condition, more preferably under the height stringent condition, with among the SEQ IDNO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 51-410 position.Also term also comprise with SEQID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 51-410 position, preferably at least 80%, more preferably at least 90%, at least 95% nucleotide sequence best.
This term also comprises encoding to have variant form with proteic, the SEQ ID NO.4 sequence of people NADH-CoQ 14 kDa subunit identical functions.These variant forms comprise (but being not limited to): several (are generally 1-90, preferably 1-60, more preferably 1-20,1-10 best) disappearance, insertion and/or the replacement of Nucleotide, and several (are generally in 60 to hold interpolation 5 ' and/or 3 ', preferably being in 30, more preferably is in 10, is in 5 best) Nucleotide.
In the present invention, " pure substantially " protein or polypeptide are meant that it accounts at least 20% of the total material of sample at least, preferably at least 50%, more preferably at least 80%, and at least 90% (by dry weight or weight in wet base) best.Purity can be measured with any suitable method, as measure the purity of polypeptide with column chromatography, PAGE or HPLC method.Substantially pure polypeptide is substantially free of the component of following it under the native state.
In the present invention, term " people NADH-CoQ 14 kDa protein subunit or polypeptide " refers to have the active SEQ ID of people NADH-CoQ 14 kDa protein subunits NO.4 polypeptide of sequence.This term also comprises having and variant form people NADH-CoQ 14 kDa subunit identical functions, SEQ ID NO.4 sequence.These variant forms comprise (but being not limited to): several (are generally 1-50, preferably 1-30, more preferably 1-20,1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20, preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the reactive derivative of people NADH-CoQ 14 kDa protein subunits.
The variant form of this polypeptide comprises: homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of people NADH-CoQ 14 kDa subunit DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-people NADH-CoQ 14 kDa subunit polypeptides to obtain.The present invention also provides other polypeptide, as comprises people NADH-CoQ 14 kDa subunit polypeptides or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of people NADH-CoQ 14 kDa subunit polypeptides.Usually, this fragment have people NADH-CoQ 14kDa subunit polypeptide sequence at least about 10 continuous amino acids, usually at least about 30 continuous amino acids, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, best at least about 100 continuous amino acids.
Invention also provides the analogue of people NADH-CoQ 14 kDa protein subunits or polypeptide.The difference of these analogues and natural human NADH-CoQ 14 kDa subunit polypeptides can be the difference on the aminoacid sequence, also can be the difference that does not influence on the modified forms of sequence, perhaps haves both at the same time.These polypeptide comprise natural or the inductive genetic variant.The induce variation body can obtain by various technology, as by radiation or be exposed to mutagenic compound and produce random mutagenesis, also can pass through site-directed mutagenesis method or the biological technology of other known moleculars.Analogue also comprises having the analogue that is different from the amino acid whose residue of natural L-(as D-amino acid), and has non-natural analogue that exist or synthetic amino acid (as β, gamma-amino acid).Should be understood that polypeptide of the present invention is not limited to the above-mentioned representational polypeptide that exemplifies.
(the not changing primary structure usually) form of modification comprises: the chemically derived form such as the acetylize or carboxylated of the polypeptide that body is interior or external.Modification also comprises glycosylation, carries out glycosylation modified and polypeptide that produce in the procedure of processing as those in the synthetic and processing of polypeptide or further.This modification can be carried out glycosylated enzyme (as mammiferous glycosylase or deglycosylating enzyme) and finishes by polypeptide is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (as Tyrosine O-phosphate, phosphoserine, phosphothreonine).Thereby also comprise the polypeptide that has been improved its anti-proteolysis performance or optimized solubility property by modifying.
In the present invention, " people NADH-CoQ 14 kDa subunit conservative propertys variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, there are 10 at the most, preferably at the most 8, more preferably at the most 5,3 amino acid is replaced by similar performance or close amino acid and is formed polypeptide at the most best.These conservative property variation polypeptide preferably carry out the amino acid replacement according to table 1 and produce.
Table 1
| Initial residue | Representational replacement | The preferred replacement |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val；Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu；Phe；Ile | Leu |
| Phe(F) | Leu；Val；Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr；Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The present invention also comprises people NADH-CoQ 14 kDa subunit polypeptide encoding sequences and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of people NADH-CoQ 14 kDa subunits in the cell.
The present invention also comprises a kind of nucleic acid molecule that can be used as probe or primer, and this molecule has 8-100 of people NADH-CoQ 14 kDa subunit polypeptide encoding sequences, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of coding people NADH-CoQ 14 kDa subunits.
The present invention also comprises the method that detects people NADH-CoQ 14 kDa subunit nucleotide sequences, and it comprises with above-mentioned probe and sample and hybridizing whether detection probes combination has taken place then.Preferably, this sample is the product behind the pcr amplification, and wherein the pcr amplification primer is corresponding to the encoding sequence of people NADH-CoQ 14 kDa subunit polypeptides, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 20-50 Nucleotide.
In the present invention, can select various carrier known in the art for use, as commercially available carrier.Such as, select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide operationally is connected in expression regulation sequence then, can form protein expression vector.
As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, as Chinese hamster ovary celI, COS cell etc.
On the other hand, the present invention also comprises people NADH-CoQ 14 kDa subunit DNA or the polypeptide of its fragment coding has specific polyclonal antibody and monoclonal antibody, especially monoclonal antibody.Here, " specificity " is meant that antibody capable is incorporated into people NADH-CoQ 14 kDa subunit gene product or fragments.Preferably, refer to that those can combine with people NADH-CoQ 14 kDa subunit gene products or fragment but nonrecognition and be incorporated into the antibody of other irrelevant antigen molecule.Among the present invention antibody comprise those can in conjunction with and suppress the molecule of people NADH-CoQ14 kDa protein subunit, comprise that also those do not influence the antibody of people NADH-CoQ 14 kDa protein subunit functions.The present invention also comprise those can with modify or without the people NADH-CoQ 14 kDa subunit gene product bonded antibody of modified forms.
The present invention not only comprises complete mono-clonal or polyclonal antibody, but also comprises having immunocompetent antibody fragment, as Fab ' or (Fab)
2Fragment; Heavy chain of antibody; Light chain of antibody; Genetically engineered strand Fv molecule (people such as Ladner, U.S. Patent No. 4,946,778); Or chimeric antibody, as have the murine antibody binding specificity but still keep antibody from people's antibody moiety.
Antibody of the present invention can be prepared by the known various technology of those skilled in that art.For example, the people NADH-CoQ 14 kDa subunit gene products of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human NADH-CoQ 14 kDa subunits or its have antigenic segmental cell and can be used to immune animal and produce antibody.Antibody of the present invention also can be monoclonal antibody.This type of monoclonal antibody can utilize hybridoma technology to prepare that (see people such as Kohler, Nature 256; 495,1975; People such as Kohler, Eur.J.Immunol.6:511,1976; People such as Kohler, Eur.J.Immunol.6:292,1976; People such as Hammerling, In Monoclonal Antibodies and TCell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block people NADH-CoQ 14kDa subunit function and the antibody that does not influence people NADH-CoQ 14 kDa subunit functions.Each antibody-like of the present invention can utilize the fragment or the functional zone of people NADH-CoQ 14 kDa subunit gene products, obtains by the routine immunization technology.These fragments or functional zone can utilize recombinant methods or utilize Peptide synthesizer synthetic.Can come immune animal and produce with the gene product of producing in the prokaryotic cell prokaryocyte (for example E.Coli) with the unmodified form bonded antibody of people NADH-CoQ 14 kDa subunit gene products; With posttranslational modification form bonded antibody (as the albumen or the polypeptide of glycosylation or phosphorylation), can come immune animal and obtain with the gene product that produces in the eukaryotic cell (for example yeast or insect cell).
People NADH-CoQ 14kDa subunit Nucleotide full length sequence of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention about nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or by the prepared cDNA storehouse of ordinary method well known by persons skilled in the art as template, amplification and must relevant sequence.Also can carry out twice or pcr amplification repeatedly, and then the fragment that each time amplifies is stitched together by proper order.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, and people NADH-CoQ 14 kDa subunit fragments length especially of the present invention are shorter.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.
In an example of the present invention, the cDNA nucleotide sequence of people NADH-CoQ 14 kDa subunits is so to obtain, with people's liver λ gt11cDNA library (available from Clontech company) is template, with oligonucleotide is primer---A1:5 '-CCTTGTAGTTCGTGGTCTGAGAC-3 ' is a forward primer, oligonucleotide A2:5 '-AGCAGGTATCAGTGAAACTGGAG-3 ' is a reverse primer, carries out PCR.Obtain the full length cDNA sequence of SEQ ID NO.3 after the order-checking.
The oxidative phosphorylation reacting cells of respiratory chain obtains the main path of energy in the plastosome.Wherein, composite I is the first step of this approach.Have been found that the defective and the multiple disease-related of composite I; There is document also to think, relation (J.Bioenerg.Biomebr., 20 (3): 365-382 that composite I also may be certain with having of aging; Biochimica et Biophysica.Acta., 1101:198-203,1992).The sudden change of forming each subunit of composite I brings in various degree influence usually can for the function of composite I, thus with some disease-related.Great majority have all been found many different mutant forms in seven genes of plastosome coding composite I subunit, some sudden change wherein is found and disease-related, these diseases comprise Leber hereditary ophthalmic nerve disease (Leber ' shereditary optic neuropathy, LHON), Parkinsonism (PD) (Cooper, J.M., et a1.Biochimica et Biophysica.Acta., 1101:198-203,1992) and presenile dementia (AD) etc.The nuclear gene of coding composite I subunit also may cause mitochondrial disease, this imagination has obtained certain experiment support equally: found 75-, 13,24 or disappearance (the Moreadith etal.J.Clin.Invest. of 20kDa subunit in some mitochondrial disease patients, 79:463-467,1987; Schapira et al.Molecular basis of mitochondrialmyophathies:polypeptide analysis in complex-I deficiency Lancet I (8584), 1988 500-503; Slipetz et al.Am.J.Hum.Genet., 48:1121-1126,1991).
Gene of the present invention is estimated to be a subunit of coding people composite I.On the one hand, as a part of forming composite I, it must bring into play unique effect to the function of whole mixture, as the gene of other known coding complex subunit, its sudden change certainly leads to certain influence for the function of mixture integral body, thereby causes the generation of disease; On the other hand, the present invention also provides a kind of new approach for the effect of nuclear gene in integral body of further illustrating composite I.
In the accompanying drawings,
Fig. 1 is the nucleotide sequence homology comparison diagram of people NADH-CoQ 14 kDa subunits (NADHS14) of the present invention and cor bovinum plastosome composite I B14.5 subunit (BOS14.5).Wherein, identical Nucleotide marks with " | ".
Fig. 2 is the homology comparison diagram of the aminoacid sequence of people NADH-CoQ 14 kDa subunits (NADHS14) of the present invention and cor bovinum plastosome composite I B14.5 subunit (BOS14.5).Wherein, identical amino acid marks with " | " between two sequences, and similar amino acid marks with " ".Similar amino acid is: A, S, T; D, E; N, Q; R, K; I, L, M, V; F, Y, W.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The clone and the mensuration of the cDNA sequence of people NADH-CoQ 14 kDa subunits
1. primer amplification
With people's liver λ gt11cDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A1:5 '-CCTTGTAGTTCGTGGTCTGAGAC-3 ' (SEQ ID NO:1) is a forward primer, oligonucleotide A2:5 '-AGCAGGTATCAGTGAAACTGGAG-3 ' (SEQ ID NO:2) is a reverse primer, carries out PCR.The PCR condition of A1/A2 be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 64 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.Electrophoresis detection obtains the purpose fragment of about 450 base pairs.
2.PCR the order-checking of product
Pcr amplification product A1/A2 is connected with pGEM-T carrier (Promega), transformed into escherichia coli JM103, extract plasmid with QIAprep Plasmid test kit (QIAGEN), carry out the mistake of orientations Lieque with double-stranded nested type disappearance test kit (Pharmacia) to inserting fragment, carry out Rapid identification and ordering with PCR to lacking son then.Use SequiTherm EXCEL
TMDna sequencing kit (Epicentre Technologies) checks order to disappearance of brachymemma successively, with computer software splicing order, obtain full length cDNA sequence at last, altogether 450bp, detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 51-410 position Nucleotide.
Derive the aminoacid sequence of people NADH-CoQ 14kDa subunit according to the full-length gene group sequence that obtains, totally 119 amino-acid residues, its aminoacid sequence sees SEQ ID NO:4 for details.
Embodiment 2
Homology relatively
Full length cDNA sequence and proteins encoded thereof with people NADH-CoQ 14 kDa subunits of the present invention, in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBankCDS translations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with BLAST.Found that the B14.5b subunit with the cardiod plastochondria composite I of ox has very high homology.With PCGENE software relatively, the identity of B14.5b subunit on protein level of cardiod plastochondria composite I of finding it and ox is up to 74%, and similarity reaches 82.4% (Fig. 2); And on nucleic acid level, the identity between both encoding sequences (CDS) is also up to 84.2% (Fig. 1).Therefore people NADH-CoQ 14 kDa subunit genes of the present invention are considered to the homologue of B14.5b subunit gene in human body of the cardiod plastochondria composite I of ox.According to the structures shape function, the Biological Principles that the structural similitude function is close, the function that can infer human NADH-CoQ 14 kDa subunits of the present invention from the member's of this family gene or proteic function.
As everyone knows, the oxidative phosphorylation of respiratory chain reaction is extremely important for organism in the plastosome, and this approach is the main path that cell obtains energy.Wherein, composite I is the first step of this approach.Have been found that the defective and the multiple disease-related of composite I; There is document also to think, relation (J.Bioenerg.Biomebr., 20 (3): 365-382 that composite I also may be certain with having of aging; Biochimica et Biophysica.Acta., 1101:198-203,1992).Have reason to think that the sudden change of forming each subunit of composite I brings in various degree influence usually can for the function of composite I, thus with some disease-related.
Relevant experiment has also proved this viewpoint.Great majority have all been found many different mutant forms in seven genes of plastosome coding composite I subunit, some sudden change wherein is found and disease-related, these diseases comprise Leber hereditary ophthalmic nerve disease (LHON), Parkinsonism (PD) (Cooper, J.M., et al.Biochimica et Biophysica.Acta., 1101:198-203,1992) and presenile dementia (AD) etc.
The nuclear gene of coding composite I subunit also may cause mitochondrial disease, this imagination has obtained certain experiment support equally: found 75-, 13,24 or disappearance (the Moreadith et al.J.Clin.Invest. of 20kDa subunit in some mitochondrial disease patients, 79:463-467,1987; Schapira et al.Molecular basis ofmitochondrial myophathies:polypeptide analysis in complex-I deficiency Lancet I (8584), 1988 500-503; Slipetz et al.Am.J.Hum.Genet., 48:1121-1126,1991).Yet contacting directly between nuclear gene and composite I functional defect still remains further to be inquired into.
Gene of the present invention is estimated to be a subunit of coding people composite I.On the one hand, as a part of forming composite I, it must bring into play unique effect to the function of whole mixture, as the gene of other known coding complex subunit, its sudden change certainly leads to certain influence for the function of mixture integral body, thereby causes the generation of disease; On the other hand, the present invention also provides a kind of new approach for the effect of nuclear gene in integral body of further illustrating composite I.
People NADH-CoQ 14 kDa subunits of the present invention are used for further functional study except can be used as this family's a member, also can be used for producing fusion rotein with other albumen, such as producing fusion rotein with immunoglobulin (Ig).In addition, inventor NADH-CoQ 14 kDa subunits can also merge with other members of this family or exchange fragment, to produce new albumen.For example the N end of the N of inventor NADH-CoQ 14 kDa subunits end with the B14.5b subunit of cor bovinum plastosome composite I exchanged, to produce the albumen that new activity is higher or have new features.
At the antibody of inventor NADH-CoQ 14 kDa subunits, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).
In addition, inventor NADH-CoQ 14 kDa subunit nucleic acids (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves people NADH-CoQ 14 kDa subunits or the overexpression that suppresses people NADH-CoQ14kDa subunit.People NADH-CoQ 14 kDa protein subunits of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of people NADH-CoQ 14 kDa subunit deletions, no function or unusual cause related disorders arranged.In addition, can also be with carrying out relevant diagnosis or prognosis judgement based on nucleotide sequence of the present invention or antibody.
Embodiment 3
People NADH-CoQ 14 expression of kDa subunit in intestinal bacteria
In this embodiment, with the PCR product among the embodiment 1 is template, with the cDNA sequence of coding people NADH-CoQ 14 kDa subunits use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people NADH-CoQ 14 kDa subunit cDNA as inserting fragment.
5 ' Oligonucleolide primers sequence of using in the PCR reaction is:
5′-TCAGGGATCCATGATCGCACGGCGGAACC-3′(SEQ?ID?NO.5)，
This primer contains the restriction enzyme site of BamHI restriction enzyme, is 19 Nucleotide of the people NADH-CoQ 14 kDa subunit encoding sequences that begun by initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5’-ACTGGTCGACTCAACGTATTGGATGGAAT-3’(SEQ?ID?NO.6)，
This primer contains the part encoding sequence of restriction enzyme site, translation termination and the people NADH-CoQ 14kDa subunit of SalI restriction enzyme.
The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, the CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Amp
r), a bacterium replication orgin (ori), an adjustable promotor/operon of IPTG-(P/O), a ribosome bind site (RBS), a 6-histidine mark thing (6-His) and restriction enzyme cloning site.
With SalI and BamHI digestion pQE-9 carrier and insertion fragment, will insert fragment subsequently and be connected to the pQE-9 carrier and keep open reading frame initial at bacterium RBS.Transform available from Qiagen with connecting mixture subsequently, the E.coli bacterial strain of commodity M15/rep4 by name, M15/rep4 contains the plasmid pREP4 of multiple copied, and it is expressed the lacI repressor and carries kalamycin resistance (Kan
r).Screen transformant containing on the LB culture dish of Amp and Kan, the extracting plasmid, the cDNA fragment of sequence verification people NADH-CoQ 14 kDa subunits has correctly been inserted carrier.
Incubated overnight (O/N) contains the positive transformant clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml) and Kan (25 μ g/ml).Spend the night (O/N) culture with 1: 100-1: 250 thinning ratio dilution, be inoculated into then in the large volume substratum, culturing cell grows to 600 optical density(OD) (OD
600) when being 0.4-0.6, add IPTG (" isopropylthio-") to final concentration be 1mM.By making lacI repressor inactivation, IPTG induces startup P/O to cause gene expression dose to improve.Continued culturing cell 3-4 hour, centrifugal subsequently (6000 * g, 20 minutes).The ultrasonic degradation inclusion body, collecting cell also is dissolved in cell precipitation in the Guanidinium hydrochloride of 6M.After the clarification, by containing under the condition that 6-His marker albumen combines closely making, with nickel-chelate column chromatography purifying dissolved people NADH-CoQ 14 kDa subunits from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out people NADH-CoQ 14 kDa subunits from post.Available several method is the sex change protein precipitation from Guanidinium hydrochloride.Perhaps, use the dialysis step to remove Guanidinium hydrochloride, perhaps isolated purifying protein from nickel-chelate column.Protein behind the purifying is incorporated in second post, has the linear Guanidinium hydrochloride gradient of successively decreasing in this post.Protein denaturation when being attached to this post is used Guanidinium hydrochloride (pH5.0) wash-out subsequently.At last, soluble protein is dialysed with PBS, then protein is kept in the stock solution that final concentration is 10% (w/v) glycerine.
SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is about 14KDa.
In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.
Embodiment 4
The expression of people NADH-CoQ 14 kDa subunits in eukaryotic cell (Chinese hamster ovary celI strain)
In this embodiment, with the PCR product among the embodiment 1 is template, with the cDNA sequence of coding people NADH-CoQ 14 kDa subunits use corresponding to 5 of this dna sequence dna ' and the PCR Oligonucleolide primers of 3 ' end increase, obtain people NADH-CoQ 14 kDa subunit cDNA as inserting fragment.
5 ' Oligonucleolide primers sequence of using in the PCR reaction is:
5′-TCAGGGATCCATGATCGCACGGCGGAACC-3′(SEQ?ID?NO.7)
This primer contains the restriction enzyme site of BamHI restriction enzyme, is 19 Nucleotide of the people NADH-CoQ 14 kDa subunit encoding sequences that begun by initiator codon after this restriction enzyme site;
3 ' end primer sequence is:
5’-ACTGGATATCTCAACGTATTGGATGGAAT-3’(SEQ?ID?NO.8)
This primer contains the part encoding sequence of the restriction enzyme site of EcoRV restriction enzyme, translation termination and people NADH-CoQ 14kDa subunit.
The restriction enzyme site of the restriction enzyme on the primer is corresponding to the restriction enzyme digestion sites on the expressing cho cell carrier pcDNA3, this plasmid vector coding antibiotics resistance (Amp
rAnd Neo
r), a phage replication starting point (f1 ori), a virus replication starting point (SV40 ori), T7 promotor, a viral promotors (P-CMV), a Sp6 promotor, a SV40 promotor, a SV40 tailing signal and corresponding polyA order, a BGH tailing signal and a corresponding polyA order.
With BamHI and EcoRV digestion pcDNA3 carrier, will insert fragment subsequently and be connected to the pcDNA3 carrier.Subsequently with connecting mixture Transformed E .coli DH5 α bacterial strain.Screen transformant containing on the LB culture dish of Amp, incubated overnight (O/N) contains the clone of required construction in the LB liquid nutrient medium of adding Amp (100 μ g/ml).The extracting plasmid, and the cDNA fragment of sequence verification people NADH-CoQ 14 kDa subunits has correctly been inserted carrier.
The plasmid transfection Chinese hamster ovary celI is to adopt lipofection, carries out with Lipofectin (GiBco Life).After the transfection 48 hours, through the lasting G418 pressurization screening in 2-3 week, collecting cell and cell conditioned medium are measured the expressing protein enzyme activity.Remove G418, continuous passage is cultivated; To mixing the clone cell Method of Limited Dilution, select to have the cell subclone of higher protein-active.The above-mentioned positive subclone of a large amount of according to a conventional method cultivations.After 48 hours, beginning collecting cell and supernatant are with ultrasonic degradation method smudge cells.With 50mMTrisHCl (pH7.6) solution that contains 0.05%Triton is balance liquid and elutriant, uses through the Superdex of pre-equilibration G-75 post and collects above-mentioned proteic active peak.Using 50mMTrisHCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTrisHCl (pH8.0) solution that contains 0-1M NaCl, collects above-mentioned proteic active peak.Be that dialyzate is dialysed to expressing protein solution with PBS (pH7.4) then.Last freeze-drying is preserved.
SDS-PAGE glue with 12% carries out electrophoresis, identifies that the molecular weight size of expressing protein is 14KDa.
In addition, the amino acid that reaches proteic N end and each 10 amino acid length of C end is checked order, find consistent with the sequence of SEQ ID NO.4 with ordinary method.
Embodiment 5
Preparation antibody
The recombinant protein that obtains in embodiment 3 and 4 is used for immune animal to produce antibody, and concrete grammar is as follows.Recombinant molecule is standby after separating with chromatography.Also available SDS-PAGE gel electrophoresis separates, electrophoretic band downcut from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days,, mouse is carried out peritoneal injection with booster immunization with the dosage of 50-100 μ g/0.2ml with the same antigen of non-complete Freund ' s adjuvant emulsion.Carried out booster immunization one time every 14 days, carry out at least three times.The sero-fast specific reaction that obtains is active to be assessed in the ability of external precipitation people NADH-CoQ 14 kDa subunit gene translation products with it.Found that antibody can precipitate with protein-specific of the present invention ground specifically.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table (1) general information:
(i) applicant: Fudan University
(ii) denomination of invention: a kind of human oxido-reductase subunit and encoding sequence thereof, and method for making and purposes
(iii) sequence number: the information of 8 (2) SEQ ID NO.1
(i) sequence signature
(A) length: 23 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: the information of SEQ ID NO.1:CCTTGTAGTT CGTGGTCTGA GAC 23 (2) SEQ ID NO.2
(i) sequence signature
(A) length: 23 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: the information of SEQ ID NO:2:AGCAGGTATC AGTGAAACTG GAG 23 (2) SEQ ID NO.3:
(i) sequence signature:
(A) length: 450bp
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
( ii ) ：cDNA ( xi ) ：SEQ ID NO.3：CCTTGTAGTT CGTGGTCTGA GACCAGGCCT CAAGTGGAAA CGGCGTCACC ATGATCGCAC 60GGCGGAACCC AGAACCCTTA CGGTTTCTGC CGGATGAGGC CCGGAGCCTG CCCCCGCCCA 120AGCTGACCGA CCCGCGGCTC CTCTACATCG GCTTCTTGGG CTACTGCTCC GGCCTGATTG 180ATAACCTGAT CCGGCGGAGG CCGATCGCGA CGGCTGGTTT GCATCGCCAG CTTCTATATA 240TTACGGCCTT TTTTTTTGCT GGATATTATC TTGTAAAACG TGAAGACTAC CTGTATGCTG 300TGAGGGACCG TGAAATGTTT GGATATATGA AATTACATCC AGAGGATTTT CCTGAAGAAG 360ATAAGAAAAC ATATGGTGAA ATTTTTGAAA AATTCCATCC AATACGTTGA AGTCTTCAAA 420ATGCTTGCTC CAGTTTCACT GATACCTGCT 450 ( 2 ) SEQ ID NO.4：
(i) sequence signature:
(A) length: 119 amino acid
(B) type: amino acid
(D) topological framework: linearity
(ii) molecule type: polypeptide
(xi) sequence description: the information of SEQ ID NO.4Met Ile Ala Arg Arg Asn Pro Glu Pro Leu Arg Phe Leu Pro Asp 15Glu Ala Arg Ser Leu Pro Pro Pro Lys Leu Thr Asp Pro Arg Leu 30Leu Tyr Ile Gly Phe Leu Gly Tyr Cys Ser Gly Leu Ile Asp Asn 45Leu Ile Arg Arg Arg Pro Ile Ala Thr Ala Gly Leu His Arg Gln 60Leu Leu Tyr Ile Thr Ala Phe Phe Phe Ala Gly Tyr Tyr Leu Val 75Lys Arg Glu Asp Tyr Leu Tyr Ala Val Arg Asp Arg Glu Met Phe 90Gly Tyr Met Lys Leu His Pro Glu Asp Phe Pro Glu Glu Asp Lys 105Lys Thr Tyr Gly Glu Ile Phe Glu Lys Phe His Pro Ile Arg 119 (2) SEQ ID NO.5
(i) sequence signature
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: the information of SEQ ID NO.5:TCAGGGATCC ATGATCGCAC GGCGGAACC 29 (2) SEQ ID NO.6
(i) sequence signature
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: the information of SEQ ID NO.6:ACTGGTCGAC TCAACGTATT GGATGGAAT 29 (2) SEQ ID NO.7
(i) sequence signature
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: the information of SEQ ID NO.7:TCAGGGATCC ATGATCGCAC GGCGGAACC 29 (2) SEQ ID NO.8
(i) sequence signature
(A) length: 29 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO.8:ACTGGATATC TCAACGTATT GGATGGAAT 29
Claims (14)
1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the active polypeptide of people NADH-CoQ 14kDa protein subunit,
Show at least 70% homology from the nucleotides sequence of Nucleotide 51-410 position among described nucleotide sequence and the SEQ ID NO.3; Perhaps
Described nucleotide sequence can be under the moderate stringent condition with SEQ ID NO.3 in from the nucleotide sequence hybridization of Nucleotide 51-410 position.
2. dna molecular as claimed in claim 1 is characterized in that, described sequence encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.4.
3. dna molecular as claimed in claim 1 is characterized in that, this sequence has among the SEQ ID NO.3 nucleotide sequence from Nucleotide 51-410 position.
4. isolating people NADH-CoQ 14kDa protein subunit polypeptide is characterized in that it comprises: have polypeptide or its conservative property variation polypeptide or its active fragments of SEQ ID NO.4 aminoacid sequence, or its reactive derivative.
5. polypeptide as claimed in claim 4 is characterized in that, this polypeptide is to have SEQ ID NO.4 polypeptide of sequence.
6. a carrier is characterized in that, it contains the described DNA of claim 1.
7. one kind with the described carrier transformed host cells of claim 6.
8. host cell as claimed in claim 7 is characterized in that this cell is intestinal bacteria.
9. host cell as claimed in claim 7 is characterized in that this cell is an eukaryotic cell.
10. a generation has the method for the active polypeptide of people NADH-CoQ 14kDa protein subunit, it is characterized in that this method comprises:
(a) nucleotide sequence that coding is had an active polypeptide of people NADH-CoQ 14kDa protein subunit operationally is connected in expression regulation sequence, form people NADH-CoQ 14 kDa protein subunit expression vectors, show at least 70% homology from the nucleotides sequence of Nucleotide 51-410 position among described nucleotide sequence and the SEQ ID NO.3;
(b) change the expression vector in the step (a) over to host cell, form the reconstitution cell of people NADH-CoQ 14 kDa protein subunits;
(c) under the condition that is fit to expressing human NADH-CoQ 14 kDa protein subunit polypeptide, the reconstitution cell in the culturing step (b);
(d) isolate and have the active polypeptide of people NADH-CoQ 14 kDa protein subunits.
11. method as claimed in claim 10 is characterized in that, this sequence is from Nucleotide 51-410 position among the SEQ ID NO.3.
12. energy and the described people NADH-CoQ 14 kDa protein subunit polypeptid specificity bonded antibody of claim 4.
13. a nucleic acid molecule is characterized in that, it is the antisense sequences of the described dna molecular of claim 1.
14. a probe molecule is characterized in that, it contains 8-100 continuous nucleotide in the described dna molecular of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99101361A CN1261104A (en) | 1999-01-20 | 1999-01-20 | Human oxido-reductase subunit and its coding sequence, preparing process and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN99101361A CN1261104A (en) | 1999-01-20 | 1999-01-20 | Human oxido-reductase subunit and its coding sequence, preparing process and application |
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ID=5270400
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001088153A1 (en) * | 2000-05-19 | 2001-11-22 | Biowindow Gene Development Inc. Shanghai | Novel polypeptide...a nadh ubiquinone oxidoreductase 14 and polynucleotide encoding it |
| WO2001094537A2 (en) * | 2000-05-24 | 2001-12-13 | Shanghai Biowindow Gene Development Inc. | Novel polypeptide--human nadh ubiquinone oxidoreductase 21.89 and polynucleotide encoding it |
-
1999
- 1999-01-20 CN CN99101361A patent/CN1261104A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001088153A1 (en) * | 2000-05-19 | 2001-11-22 | Biowindow Gene Development Inc. Shanghai | Novel polypeptide...a nadh ubiquinone oxidoreductase 14 and polynucleotide encoding it |
| WO2001094537A2 (en) * | 2000-05-24 | 2001-12-13 | Shanghai Biowindow Gene Development Inc. | Novel polypeptide--human nadh ubiquinone oxidoreductase 21.89 and polynucleotide encoding it |
| WO2001094537A3 (en) * | 2000-05-24 | 2004-04-08 | Shanghai Biowindow Gene Dev | Novel polypeptide--human nadh ubiquinone oxidoreductase 21.89 and polynucleotide encoding it |
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