CN1275621A - Human lambda crystallin and its coding sequence, preparation process and use - Google Patents

Abstract

The present invention provides a human lambda crystallin cDNA sequence, said cDNA coded protein is a homologue of rabbit lambda crystallin. Said invention also relates to polypeptide coded by said nucleotide sequence, application of these polynucleotide and polypeptide and the production method of the described polynucleotide and polypeptide.

Description

Human lambda crystallin and and encoding sequence thereof, and method for making and purposes

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 human lambda crystallin and cDNA sequence, this cDNA encoded protein is the homologue of rabbit lambda crystallin.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.

Vertebrate lens is a very special organization, though several water-soluble proteins (being crystallin) are only arranged, these albumen account for about 60% of whole lens weight in wet base.In lens, the α crystallin exists the most general, also has β/γ crystallin in addition.In birds, reptiles and other more rudimentary vertebrates, some crystallins and some enzyme are same or similar.For example, in the lens of birds and reptiles, exist the ε crystallin of (accounting for total lenticular proteic 23%) to be actually serum lactic dehydrogenase (Wistow, G., et al. (1987) Science 236:1554-1556) in a large number.δ and γ crystallin have homology with argininosuccinate lyase (Wistow, G.et al. (1988) Annu.Rev.Biochem.57:479-504), α Hydratase, phosphoenolpyruvate respectively.And ρ crystallin and aldehyde and aldose reductase (Carper, D.et el. (1987) FEBS Lett.220:209-213) and prostaglandin F synthetic enzyme (Watanabe, K.et al. (1988) Proc.Natl.Acad.Sci.U.S.A.85:11-15) relevant.

People such as Mulders are separated to the new albumen of a 35kDa from the rabbit lens, and record its cDNA sequence, are referred to as lambda crystallin.This albumen accounts for the 7-8% of total crystallin, and with 3-hydroxyl acyl-CoA desaturase certain homology (Mulders, W.M.etal. (1988) J.Biol.Chem.263:15462-15466) is arranged.

Yet, before the present invention, still do not have report about human lambda crystallin and.

An object of the present invention is to provide a kind of new polynucleotide, new lambda crystallin of this polynucleotide encoding, the present invention is named as human lambda crystallin and cDNA.

Another object of the present invention provides a kind of new human lambda crystallin and, and this albumen is named as human lambda crystallin and.

A further object of the present invention provides a kind of method of utilizing recombinant technology to produce described new people's lambda crystallin polypeptide.

The present invention also provides this people's the lambda crystallin nucleotide sequence and the application of 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 human lambda crystallin and, shows at least 70% homology from the nucleotides sequence of Nucleotide 10-1065 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 10-1065 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 10-1065 position.

In another aspect of this invention, provide a kind of isolating human lambda crystallin and 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, the method that provides a kind of generation to have the active polypeptide of human lambda crystallin and, this method comprises:

(a) nucleotide sequence that coding is had an active polypeptide of human lambda crystallin and operationally is connected in expression regulation sequence, form the human lambda crystallin and expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 10-1065 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 human lambda crystallin and;

(c) under the condition that is fit to expressing human lambda crystallin polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate and have the active polypeptide of human lambda crystallin and.

In a specific embodiments of the present invention, isolating polynucleotide total length of the present invention is 1227 Nucleotide, and its detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 10-1065 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 human lambda crystallin and to term " human lambda crystallin and (or polypeptide) encoding sequence ", as 10-1065 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.3.This degenerate sequence is meant, is arranged in the encoder block 10-1065 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 10-1065 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 ID NO.3 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 10-1065 position.Also term also comprise with SEQ ID NO.3 in from the homology of nucleotide sequence at least 70% of Nucleotide 10-1065 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have variant form with proteic, the SEQ IDNO.4 sequence of human lambda crystallin and identical function.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 " human lambda crystallin and polypeptide " refers to have the active SEQID NO.4 of human lambda crystallin and polypeptide of sequence.This term also comprises having and variant form people's shape identical function, 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 human lambda crystallin and.

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 human lambda crystallin and DNA hybridization and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-human lambda crystallin and polypeptide to obtain.The present invention also provides other polypeptide, as comprises human lambda crystallin and polypeptide or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention has also comprised the soluble fragments of human lambda crystallin and polypeptide.Usually, this fragment have the human lambda crystallin and peptide 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 human lambda crystallin and or polypeptide.The difference of these analogues and natural human lambda crystallin polypeptide 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, " human lambda crystallin and conservative property variation polypeptide " refers to compare with the aminoacid sequence of SEQ ID No.4, has 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 human lambda crystallin and polypeptid coding sequence and segmental antisense sequences thereof.This antisense sequences can be used for suppressing the expression of human lambda crystallin and in the cell.

The present invention also comprises a kind of nucleic acid molecule that can be used as probe, and this molecule has 8-100 of human lambda crystallin and polypeptid coding sequence, preferably 15-50 continuous nucleotide usually.This probe can be used for whether existing in the test sample nucleic acid molecule of the human lambda crystallin and of encoding.

The present invention also comprises the method that detects the human lambda crystallin and nucleotide sequence, 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 human lambda crystallin and polypeptide, and can be positioned at the both sides or the centre of this encoding sequence.Primer length is generally 15-50 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 human lambda crystallin and 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 human lambda crystallin and gene product or fragment.Preferably, refer to that those can combine with human lambda crystallin and gene product 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 human lambda crystallin and, comprise that also those do not influence the antibody of human lambda crystallin and function.The present invention also comprise those can with modify or without the human lambda crystallin and 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 human lambda crystallin and gene product of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing human lambda crystallin 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 T Cell Hybridomas, Elsevier, N.Y., 1981).Antibody of the present invention comprises the antibody that can block the human lambda crystallin and function and the antibody that does not influence the human lambda crystallin and function.Each antibody-like of the present invention can utilize the fragment or the functional zone of human lambda crystallin and gene product, 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 human lambda crystallin and gene product; 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).

Human lambda crystallin and 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.When sequence is longer, usually needs to 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 with its human cloning carrier, changes cell over to again, 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, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.

In one embodiment of the invention, the cDNA nucleotide sequence of human lambda crystallin and is so to obtain, with people's tire brain λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A:5 '-GGCCCAACCATGGCGTCCTCCGC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B:5 '-GGCAGACTACCGGCCTGCACCAC-3 ' (SEQ ID NO.2) is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 68 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains is about the purpose fragment of 1.2kb.

In addition, because lambda crystallin of the present invention has the natural acid sequence that is derived from the people, therefore, compare with the albumen of the same clan that derives from other species (as rabbit), estimate to have higher active and/or lower side effect (for example in the intravital immunogenicity of people lower or do not have) being applied to man-hour.

In the accompanying drawings, Fig. 1 is the homology comparison diagram of the aminoacid sequence of human lambda crystallin and of the present invention and rabbit lambda crystallin.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.

Embodiment 1

The clone and the mensuration of the cDNA sequence of human lambda crystallin and

1. primer amplification

With people's tire brain λ gtllcDNA library (available from Clontech company) is template, with a pair of oligonucleotide is primer---A:5 '-GGCCCAACCATGGCGTCCTCCGC-3 ' (SEQ ID NO.1) is a forward primer, oligonucleotide B:5 '-GGCAGACTACCGGCCTGCACCAC-3 ' (SEQ ID NO.2) is a reverse primer, carries out PCR.The PCR condition be 93 ℃ 4 minutes, carried out 35 circulations in 1 minute with 93 ℃ 1 minute, 68 ℃ 1 minute and 72 ℃ thereupon, last 72 ℃ were extended 5 minutes.The PCR segment that electrophoresis detection obtains is about the purpose fragment of 1.2kb.

2.PCR the order-checking of product

Above-mentioned pcr amplification product AB 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 1227bp, detailed sequence is seen SEQ ID NO:3, and wherein open reading frame is positioned at 10-1065 position Nucleotide.

Derive the aminoacid sequence of human lambda crystallin and according to the cDNA sequence that obtains, totally 351 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 human lambda crystallin and of the present invention, in Non-redundant GenBank+EMBL+DDBJ+PDB database and Non-redundant GenBank CDStranslations+PDB+SwissProt+Spupdate+PIR database, carry out nucleic acid and albumen homology retrieval with blast program.Found that the lambda crystallin mRNA (gb|M22743) with rabbit on nucleotide level has higher homology, identity is 83%.Homologous protein is more also supported the correct of this sequence, the lambda crystallin of human lambda crystallin and derivation Argine Monohydrochloride and rabbit (sp|P14755) identity reaches 83% on amino acid levels, and similarity reaches 90% (the lambda crystallin homology of people and rabbit is relatively seen Fig. 1).The same with the rabbit lambda crystallin, human lambda crystallin and and 3-hydroxyl acyl-CoA desaturase also have certain homology, reach 33%, 31% respectively as the identity with 3-hydroxyl acyl-CoA desaturase of Archaeoglobus fulgidus (gi|2649379), Streptomycescoelicolor (emb|CAA44233), similarity is 53%, 55%.

According to the structures shape function, the Biological Principles that the structural similitude function is relevant can be inferred the biological function of lambda crystallin of the present invention according to the function of PKI gene in the known different plant species and proteins encoded thereof.

People such as Mulders are separated to the new albumen of a 35kDa from the rabbit lens, and record its cDNA sequence, are referred to as lambda crystallin.This albumen accounts for the 7-8% of total crystallin, and certain homology is arranged with 3-hydroxyl acyl-CoA desaturase, the rabbit lambda crystallin has the NAD binding site of inferring, further specify it and have some enzymic activity (Mulders, W.M.etal. (1988) J.Biol.Chem.263:15462-15466).

Human lambda crystallin and of the present invention is 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's lambda crystallin 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's lambda crystallin end with the lambda crystallin of rabbit exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of inventor's lambda crystallin, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

Because human lambda crystallin and of the present invention is one of important component of eye lens, thus the human lambda crystallin and abnormal expression and or activity may substantial connection be arranged with some ophthalmic diseasess unusually.Therefore, inventor's lambda crystallin nucleic acid (encoding sequence or antisense sequences) can be introduced into cell, with expression level that improves human lambda crystallin and or the overexpression that suppresses human lambda crystallin and.Human lambda crystallin and albumen of the present invention or its active polypeptide fragment can be applied to patient, with treatment or alleviate because of human lambda crystallin and disappearance, 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

The expression of human lambda crystallin and in intestinal bacteria

In this embodiment, with pcr amplification product A/B among the embodiment 1 is template, use PCR Oligonucleolide primers to increase the cDNA sequence of coding human lambda crystallin and, obtain human lambda crystallin and cDNA as inserting fragment corresponding to 5 of this dna sequence dna ' and 3 ' end.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5 '-TCAAGTCGACATGGCGTCCTCCGCGGCCG-3 ' (SEQ ID NO.5), this primer contains the restriction enzyme site of SalI restriction enzyme, is 19 Nucleotide of the human lambda crystallin and encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5 '-TTGGAAGCTTTCACTGGGGCTGCACTTG-3 ' (SEQ ID NO.6), this primer contains the part encoding sequence of restriction enzyme site, translation termination and the human lambda crystallin and of HindIII restriction enzyme.

The restriction enzyme site of the restriction enzyme on the primer is corresponding to bacterial expression vector pQE-9 (Qiagen Inc., Chatsworth, CA) restriction enzyme digestion sites on, this plasmid vector coding antibiotics resistance (Ampr), 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 HindIII 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 cDNA fragment of extracting plasmid sequence verification human lambda crystallin and 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 people 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 human lambda crystallin and from solution.With 6M Guanidinium hydrochloride (pH5.0) wash-out human lambda crystallin and 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 39KDa.

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 human lambda crystallin and in eukaryotic cell (Chinese hamster ovary celI strain)

In this embodiment, with pcr amplification product A/B among the embodiment 1 is template, uses the PCR Oligonucleolide primers corresponding to 5 of this dna sequence dna ' and 3 ' end to increase the cDNA sequence of coding human lambda crystallin and, obtains human lambda crystallin and cDNA as inserting fragment.

5 ' Oligonucleolide primers sequence of using in the PCR reaction is:

5′-TCACAAGCTTATGGCGTCCTCCGCGGCCG-3′(SEQ?ID?NO.7)

This primer contains the restriction enzyme site of HindIII restriction enzyme, is 19 Nucleotide of the human lambda crystallin and encoding sequence that begun by initiator codon after this restriction enzyme site;

3 ' end primer sequence is:

5’-TTGGGGATCCTCACTGGGGCTGCACTTG-3’(SEQ?ID?NO.8)

This primer contains the part encoding sequence of the restriction enzyme site of BamHI restriction enzyme, translation termination and human lambda crystallin and.

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 (fl 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 HindIII and BamHI digestion pcDNA3 carrier and insertion fragment, 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 cDNA fragment of extracting plasmid sequence verification human lambda crystallin and 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.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 50mM Tris HCl (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 50mMTris á HCl (pH8.0) equilibrated DEAE-Sepharose post again, is that elutriant carries out gradient elution with 50mMTris á HCl (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 39kDa.

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 human lambda crystallin and gene translation product with it.Found that antibody can precipitate with albumen of the present invention 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 is denomination of invention (ii): human lambda crystallin and and encoding sequence thereof, and (iii) sequence number of method for making and purposes: information (i) sequence signature of 8 (2) SEQ ID NO.1

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.1:GGCCCAACCA TGGCGTCCTC CGC 23 (2) SEQ ID NO.2

(A) length: 23 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: the information of SEQ ID NO:2:GGCAGACTAC CGGCCTGCAC CAC 23 (2) SEQ ID NO.3: (i) sequence signature:

(A) length: 1227bp

(B) type: nucleic acid

(C) chain: strand

( D ) ： ( ii ) ：cDNA ( iii ) ：SEQ ID NO.3GGCCCAACCA TGGCGTCCTC CGCGGCCGGC TGCGTGGTGA TCGTTGGCAG AATTAAAACT 60CTGTACCCAT TGAACAACAG CTGCTCATTT CCCCCAGCCC CAACCATGGC GTCCTCCGCG 120GCCGGCTGCG TGGTGATCGT TGGCAGTGGA GTCATTGGGC GAAGCTGGGC CATGCTGTTT 180GCCAGTGGAG GCTTCCAGGT GAAACTCTAT GACATTGAGC AACAGCAGAT AAGGAACGCC 240CTGGAAAACA TCAGAAAGGA GATGAAGTTG CTGGAGCAGG CAGGTTCTCT GAAAGGCTCC 300CTGAGTGTGG AAGAGCAGCT GTCACTCATC AGTGGTTGTC CCAATATCCA AGAAGCAGTA 360GAGGGTGCCA TGCACATTCA GGAATGTGTT CCAGAAGATC TAGAACTGAA GAAGAAGATT 420TTTGCTCAGT TAGATTCCAT CATTGATGAT CGAGTGATCT TAAGCAGTTC CACTTCTTGT 480CTCATGCCTT CCAAGTTGTT TGCTGGCTTG GTCCATGTGA AGCAATGCAT CGTGGCTCAT 540CCTGTGAATC CGCCATACTA CATCCCGCTG GTTGAGCTGG TCCCCCACCC GGAGACGGCC 600CCTACGACAG TGGACAGAAC CCACGCCCTG ATGAAGAAGA TTGGACAGTG CCCCATGCGA 660GTCCAGAAGG AGGTGGCCGG CTTCGTTCTG AACCGCCTGC AATATGCAAT CATCAGCGAG 720GCCTGGCGGC TAGTGGAGGA AGGAATCGTG TCTCCTAGTG ACCTGGACCT TGTCATGTCA 780GAAGGGTTGG GCATGCGGTA TGCATTCATT GGACCCCTGG AAACCATGCA TCTCAATGCA 840GAAGGTATGT TAAGCTACTG CGACAGATAC AGCGAAGGCA TAAAACATGT CCTACAGACT 900TTTGGACCCA TTCCAGAGTT TTCCAGGGCC ACTGCTGAGA AGGTTAACCA GGACATGTGC 960ATGAAGGTCC CTGATGACCC GGAGCACTTA GCTGCCAGGA GGCAGTGGAG GGACGAGTGC 1020CTCATGAGAC TCGCCAAGTT GAAGAGTCAA GTGCAGCCCC AGTGAATTTC TTGTAATGCA 1080GCTTCCACTC CTCTCATTGG AGGCCCTATT TGGGAACACT GCAAGCCCTT AATCAGCCCT 1140CTGTGACATA GGTAGCAGCC CACGGAGATC CTAAGCTGGC TGTCTTGTGT GCAGCCTGAG 1200TGGGGTGGTG CAGGCCGGTA GTCTGCC 1227 ( 2 ) SEQ ID NO.4： ( i ) ：

(A) length: 351 amino acid

(B) type: amino acid

( D ) ： ( ii ) ： ( xi ) ：SEQ ID NO.4Met Ala Ser Ser Ala Ala Gly Cys Val Val Ile Val Gly Arg Ile 15Lys Thr Leu Tyr Pro Leu Asn Asn Ser Cys Ser Phe Pro Pro Ala 30Pro Thr Met Ala Ser Ser Ala Ala Gly Cys Val Val Ile Val Gly 45Ser Gly Val Ile Gly Arg Ser Trp Ala Met Leu Phe Ala Ser Gly 60Gly Phe Gln Val Lys Leu Tyr Asp Ile Glu Gln Gln Gln Ile Arg 75Asn Ala Leu Glu Asn Ile Arg Lys Glu Met Lys Leu Leu Glu Gln 90Ala Gly Ser Leu Lys Gly Ser Leu Ser Val Glu Glu Gln Leu Ser 105Leu Ile Ser Gly Cys Pro Asn Ile Gln Glu Ala Val Glu Gly Ala 120Met His Ile Gln Glu Cys Val Pro Glu Asp Leu Glu Leu Lys Lys 135Lys Ile Phe Ala Gln Leu Asp Ser Ile Ile Asp Asp Arg Val Ile 150Leu Ser Ser Ser Thr Ser Cys Leu Met Pro Ser Lys Leu Phe Ala 165Gly Leu Val His Val Lys Gln Cys Ile Val Ala His Pro Val Asn 180Pro Pro Tyr Tyr Ile Pro Leu Val Glu Leu Val Pro His Pro Glu 195Thr Ala Pro Thr Thr Val Asp Arg Thr His Ala Leu Met Lys Lys 210Ile Gly Gln Cys Pro Met Arg Val Gln Lys Glu Val Ala Gly Phe 225Val Leu Asn Arg Leu Gln Tyr Ala Ile Ile Ser Glu Ala Trp Arg 240Leu Val Glu Glu Gly Ile Val Ser Pro Ser Asp Leu Asp Leu Val 255Met Ser Glu Gly Leu Gly Met Arg Tyr Ala Phe Ile Gly Pro Leu 270Glu Thr Met His Leu Asn Ala Glu Gly Met Leu Ser Tyr Cys Asp 285Arg Tyr Ser Glu Gly Ile Lys His Val Leu Gln Thr Phe Gly Pro 300Ile Pro Glu Phe Ser Arg Ala Thr Ala Glu Lys Val Asn Gln Asp 315Met Cys Met Lys Val Pro Asp Asp Pro Glu His Leu Ala Ala Arg 330Arg Gln Trp Arg Asp Glu Cys Leu Met Arg Leu Ala Lys Leu Lys 345Ser Gln Val Gln Pro Gln 351 ( 2 ) SEQ ID NO.5 ( i )

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.5:TCAAGTCGAC ATGGCGTCCT CCGCGGCCG 29 (2) SEQ ID NO.6

(A) length: 28 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.6:TTGGAAGCTT TCACTGGGGC TGCACTTG 28 (2) SEQ ID NO.7

(A) length: 29 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: information (i) sequence signature of SEQ ID NO.7:TCACAAGCTT ATGGCGTCCT CCGCGGCCG 29 (2) SEQ ID NO.8

(A) length: 28 bases

(B) type: nucleic acid

(C) chain: strand

(D) topological framework: linearity is molecule type (ii): oligonucleotide (xi) sequence description: SEQ ID NO.8:TTGGGGATCC TCACTGGGGC TGCACTTG 28

Claims (10)

1. isolated dna molecular is characterized in that it comprises: coding has the nucleotide sequence of the active polypeptide of human lambda crystallin and,

Show at least 70% homology from the nucleotides sequence of Nucleotide 10-1065 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 10-1065 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 10-1065 position.

4. isolating human lambda crystallin and 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. a generation has the method for the active polypeptide of human lambda crystallin and, it is characterized in that this method comprises:

(a) nucleotide sequence that coding is had an active polypeptide of human lambda crystallin and operationally is connected in expression regulation sequence, form the human lambda crystallin and expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 10-1065 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 human lambda crystallin and;

(c) under the condition that is fit to expressing human lambda crystallin polypeptide, the reconstitution cell in the culturing step (b);

(d) isolate and have the active polypeptide of human lambda crystallin and.

9. energy and the described human lambda crystallin and polypeptid specificity of claim 4 bonded antibody.

10. a probe molecule is characterized in that, it contains 8-100 continuous nucleotide in the described dna molecular of claim 1.

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