CN1690208A - Penicillium marneffei mannose dehydrogenase and its encoding sequence and use - Google Patents

Abstract

The invention provides a new penicillium marneffei mannose-dehydrogenase expressed in penicillium marneffei and its coded sequence, and also provides the method for preparation and application of the protein and its nucleic acid. With NADP+ as a cofactor, mannose-dehydrogenase can reversibly catalyze D-mannose and D-mannose-1-phosphoric to corresponding ketone compound D-fructose and D-fructose-1-phosphoric. Long-chain mannose-dehydrogenase is secondary alcohol dehydrogenase, which has an extensive usage in agriculture, pharmacy and industry.

Description

A kind of new Penicillium marneffei MDH, its encoding sequence and application thereof

Technical field

The present invention relates to fields such as molecular immunology, molecular biology, information biology and genetically engineered, particularly, the present invention relates to a kind of Penicillium marneffei bacterium (Penicillium marneffei) mannitol dehydrogenase (MDH) and encoding sequence thereof.The present invention also provides the preparation method and the application of this mannitol dehydrogenase and nucleotide sequence thereof.

Background technology

The research of fungal gene group starts from nineteen nineties, and first fungi of finishing genome sequencing is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae, Goffeau et al.1996).In the past few years, FGI (the Fungal Genome Initiative) council has chosen 15 species that have important value in medicine and industrial or agricultural and has carried out the research of genomics from more than 150 ten thousand kinds of fungies.In June, 2003, the FGI council has increased 44 species newly, comprising the Penicillium marneffei bacterium.

(Penicillium marneffei is a kind of mould that Capponi etc. at first separated from the liver of a Chinese bamboo rat in Vietnam in 1956 PM), with the naming of the director Marneffei of its institute to the Penicillium marneffei bacterium.But after this do not attract much attention.Disalvo in 1973 is in the case of American South card finder's natural infection first, and the patient is a missionary who once travelled in South East Asia.

One autochthonal peasant finds this bacterium on one's body to Deng Zhuo continuous heavy rain in 1964 in Guangxi, but the whole world did not all also have this sick report at that time, so it is regarded as Histoplasma capsulatum.Deng Zhuo continuous heavy rain in 1984 the first has at home been reported a routine penicilliposis marneffei.

From 1984 to 1987,19 whole bodies that Pathology Deparment of Guangxi Medical University collects were sent out in the type case, had 4 examples to find that the disease that causes immune deficiency is arranged, as lymphoma, and leukemia, congenital thymic hypoplasia, SLE (the bright-colored wound of systemic red), TB (tuberculosis) etc.1999, Deng Zhuolin found that at first domestic first example merges the patient of AIDS.In the domestic in recent years newfound case, the patient who merges AIDS is more and more.Thailand's to 1998 year existing 1300 examples merge the patient of AIDS.The patient of the AIDS of discovered in recent years 70-80% infects the Penicillium marneffei bacterium later stage.

The Penicillium marneffei bacterium is conditioned pathogen more, and 25 ℃ are filamentous fungus, are non-pathogenic bacteria; 37 ℃ are the yeast form, are pathogenic bacterium.It is a kind of deep pathomycete.It invades the huge system of biting of monokaryon of human body, destroys people's immunity system, makes the immunity degradation of human body.Because it is in partial physical disturbance effect, and meta-bolites, effects such as toxin produce inflammatory reaction, cause dense swollen, changes such as granuloma.And destroy blood vessel easily and enter blood circulation, cause septicemia.The approach that enters human body generally is respiratory tract or skin.

Seminose contains the open loop polyol of six carbon atom, is the content found at occurring in nature rich saccharide the most.Plant, fungi, protozoon and bacterium can both be synthesized seminose, and store as carbon source.The D-seminose is not widely used in food and pharmaceutical industry owing to not corroding tooth and going for the diabetic subject.

Usually, mannitol dehydrogenase (MDH) NADP+ as the condition of cofactor under reversible catalysis D-seminose and D-mannose-1-phosphate be oxidized to corresponding ketone compounds D-fructose and D-fructose-1-phosphate.The mannitol dehydrogenase of long-chain is the secondary alcohol desaturase, because it is widely used in agricultural, pharmacy industry, industry, and has been widely used.It is different from other alcohol and polyol, is because it does not contain conservative tyrosine and does not rely on Zn2+ and other metal ion.

Summary of the invention

The object of the present invention is to provide a kind of new Penicillium marneffei MDH (MDH), its encoding sequence and this proteic application.

Above-mentioned purpose of the present invention is achieved by the following technical solution:

In one aspect of the invention, provide a kind of isolating polynucleotide from Penicillium marneffei bacterium (Penicilliummarneffei), it contains the polynucleotide sequence of coding Penicillium marneffei bacterium MDH, and described polynucleotide are selected from:

A) polynucleotide of polypeptide that contain the aminoacid sequence of SEQ ID NO.2 with coding have the polynucleotide of at least 70% homology,

B) coding contains the polynucleotide with the polypeptide of the aminoacid sequence of aminoacid sequence at least 70% homology of SEQ ID NO.2,

C) and a) or b) polynucleotide complementary polynucleotide, and

D) contain a), b) or the polynucleotide of at least 15 continuous bases of polynucleotide sequence c).

Preferable, described polynucleotide encoding one polypeptide, this polypeptide has the sequence shown in the SEQ ID NO.2.

Perhaps preferable, described polynucleotide contain

(i) as the nucleotide sequence of Nucleotide 112-912 position among the SEQ ID NO.1, or

(ii) in genetic code degeneracy scope corresponding at least one sequence of (i) sequence, or

(iii) be complementary to (i) or (ii) at least one sequence and randomly of the sequence hybridization of sequence

(iv) the justice that has of neutral function is suddenlyd change in (i).

Better, described polynucleotide contain the nucleotide sequence of Nucleotide 112-912 position among the SEQ ID NO.1.

In another aspect of this invention, provide a kind of isolated polypeptide, described polypeptide has sequence or its fragment with aminoacid sequence at least 70% homology shown in the SEQ ID NO.2.Preferable, described polypeptide is the polypeptide that contains aminoacid sequence shown in the SEQID NO.2.

Provide a kind of carrier more on the one hand of the present invention, described carrier contains above-mentioned isolated polynucleotide.

Provide a kind of genetically engineered host cell more on the one hand of the present invention, described host cell is with above-mentioned carrier transformed host cells.

The method that the present invention also provides a kind of production to have the active polypeptide of Penicillium marneffei bacterium MDH, this method comprises the steps:

(I) nucleotide sequence that coding is had an active polypeptide of Penicillium marneffei bacterium MDH operationally is connected in expression regulation sequence, form protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 112-912 position among described nucleotide sequence and the SEQ ID NO.1;

(II) change the expression vector in the step (I) over to host cell, form Penicillium marneffei bacterium MDH reconstitution cell;

(III) be fit to express under the condition of Penicillium marneffei bacterium MDH polypeptide the reconstitution cell in the culturing step (II);

(IV) isolate and have the active polypeptide of Penicillium marneffei bacterium MDH.

Preferable, above-mentioned production has in the method for the active polypeptide of Penicillium marneffei bacterium MDH, and described nucleotides sequence is classified among the SEQ ID NO.1 nucleotide sequence from Nucleotide 112-912 position as.

The present invention also provides a kind of antibody, described antibody be can with above-mentioned Penicillium marneffei bacterium MDH specificity bonded antibody.

The present invention also comprises a kind of probe molecule, and this probe molecule contains the 8-100 of the nucleotide sequence of Penicillium marneffei bacterium MDH, preferably 15-50 continuous nucleotide usually.This probe molecule can be used for whether existing in the test sample nucleic acid molecule of coding Penicillium marneffei bacterium MDH.

The present invention also provides and has contained described polypeptide or its continuous segmental pharmaceutical composition.

The present invention also provides described polynucleotide, polypeptide and antibody preparing diagnosis and treatment at the medicine of the microbial disease of Penicillium marneffei and the application in the test kit.

The present invention also provides and has comprised described polynucleotide, polypeptide or antibody or their continuous pulsating test kit and biochip.

The present invention also comprises the method for the nucleotide sequence that detects Penicillium marneffei bacterium MDH, 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 can be positioned at the both sides or the centre of this encoding sequence corresponding to the encoding sequence of Penicillium marneffei bacterium MDH.Primer length is generally 20-50 Nucleotide.

According to Penicillium marneffei MDH disclosed by the invention (MDH) NADP+ as the condition of cofactor under reversible catalysis D-seminose and D-mannose-1-phosphate be oxidized to corresponding ketone compounds D-fructose and D-fructose-1-phosphate.The mannitol dehydrogenase of long-chain is the secondary alcohol desaturase, and it has been widely used in agricultural, pharmacy industry and industry.

Embodiment

Polynucleotide of the present invention can be dna form or rna form.Dna form comprises cDNA, genomic dna, or the DNA of artificial chemosynthesis.DNA can be strand or double-stranded.The DNA of strand can be coding strand or noncoding strand.

In the present invention, " isolating " polynucleotide are meant, these polynucleotide or segment have been arranged in the sequence of its both sides and have separated under native state, refer to that also these polynucleotide or segment with under the native state follow the component of nucleic acid to separate, and separate with the protein of in cell, following it.

Among the present invention, term " encoding sequence of Penicillium marneffei bacterium MDH " is meant that coding has the nucleotide sequence of the active polypeptide of Penicillium marneffei bacterium MDH, as 112-912 position nucleotide sequence and degenerate sequence thereof among the SEQ ID NO.1.This degenerate sequence is meant, is arranged in the encoder block 112-912 position Nucleotide of SEQ ID NO.1 sequence, has the be encoded degenerate codon of same amino acid of one or more codons to replace the sequence that the back produces.Because the degeneracy of codon, thus with SEQ ID NO.1 in 112-912 position nucleotide sequence homology be low to moderate about 70% the degenerate sequence described sequence of SEQ ID NO.2 of also encoding out.This term also is included under the moderate stringent condition, more preferably under the height stringent condition, with among the SEQ ID NO.1 from the nucleotide sequence of the nucleotide sequence hybridization of Nucleotide 112-912 position.In addition, this term also comprise with SEQ IDNO.1 in from the homology of nucleotide sequence at least 70% of Nucleotide 112-912 position, preferably at least 80%, at least 90% nucleotide sequence more preferably.

This term also comprises encoding to have the variant form of open reading frame sequence among proteic, the SEQ IDNO.1 with Penicillium marneffei bacterium MDH 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, term " Penicillium marneffei bacterium MDH " is meant and has the active SEQ ID of Penicillium marneffei bacterium MDH NO.2 polypeptide of sequence.This term also comprises having and variant form Penicillium marneffei bacterium MDH identical function, SEQ ID NO.2 sequence.These variant forms include, but is 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, in 10, more preferably be in 5 preferably) 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 active part and the reactive derivative of Penicillium marneffei bacterium MDH.

The variant form of this polypeptide comprises: homologous sequence, allelic variant, natural mutation, induced mutation body, under high or low tight degree condition can with the coded albumen of the DNA of the DNA hybridization of Penicillium marneffei bacterium MDH and the polypeptide or the albumen that utilize the antiserum(antisera) of anti-Penicillium marneffei bacterium MDH to obtain.The present invention also provides other polypeptide, as comprises Penicillium marneffei bacterium MDH or its segmental fusion rotein.Except the polypeptide of total length almost, the present invention also provides the soluble fragments of Penicillium marneffei bacterium MDH.Usually, this fragment have Penicillium marneffei bacterium MDH 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.

Invention also provides the analogue of Penicillium marneffei bacterium MDH.The difference of these analogues and natural 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 can also comprise 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, can select various carrier known in the art for use, as commercially available carrier.

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 etc.Eukaryotic host cell commonly used comprises yeast cell.

On the other hand, the present invention comprises that also Penicillium marneffei bacterium MDH is had specific antibody, especially monoclonal antibody.Here " specificity " is meant that antibody capable is incorporated into Penicillium marneffei bacterium MDH or fragment.Preferably, refer to that those can combine with Penicillium marneffei bacterium MDH 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 Penicillium marneffei bacterium MDH, comprise that also those do not influence the antibody of MDH protein function.The present invention also comprise those can with modify or without the MDH 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, United States Patent (USP) 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 Penicillium marneffei bacterium MDH of purifying or its have antigenic fragment, can be applied to animal to induce the generation of polyclonal antibody.Similarly, expressing Penicillium marneffei bacterium MDH or its has 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 prepare (see people such as Kohler, Nature256; 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 Penicillium marneffei bacterium MDH and the antibody that does not influence its function.Each antibody-like of the present invention can utilize fragment or the functional zone of Penicillium marneffei bacterium MDH, obtains by immunological technique, and 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 Penicillium marneffei bacterium MDH; 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).

Penicillium marneffei bacterium MDH nucleotide sequence 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 relevant nucleotide sequence, especially open reading frame sequence designs primer, and with commercially available cDNA storehouse or the cDNA storehouse of press the currently known methods preparation as template, increase and must be about sequence.

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, especially fragment length more in short-term.Certainly, also can be by first synthetic a plurality of small segments, and then connect and obtain the very long fragment of sequence.

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 Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

Embodiment 1

The clone of Penicillium marneffei bacterium MDH gene

1. total RNA separates (Totle RNA isolation)

With the Penicillium marneffei bacterium, after 37 ℃ or 25 ℃ of cultivations, the centrifugal supernatant of abandoning is got thalline, add its total RNA of TRIzolReagents extracting (TRIzol Reagents, Gibco, NY, USA).Identify total RNA quality with the denaturing formaldehyde gel electrophoresis.

2.mRNA separation (mRNA isolation)

With the cellulose column of band Oligo d (T) separate mRNA among total RNA (Invitrogen, Carlsbad, CA), quantitatively.

3.cDNA the structure in library (Construction of cDNA library)

With mRNA is template, synthetic double chain cDNA under the ThermoScript II effect.Cross the fragment of post screening length＞500bp, use the phenol-chloroform extracting, ethanol sedimentation, the sterilized water dissolving is connected to pDONR ^TM222 (CA) carrier is with CloneMiner for Invitrogen, Carlsbad ^TM(Invitrogen, Carlsbad CA) pack cDNA Library Construction Kit, and electric shock transforms, and the host bacterium is used DH10B (Invitrogen, Carlsbad, CA) bacterium.Coated plate is also measured titre.

4. order-checking and database are set up (Seqencing and Database Constructing)

Select the clone who has the external source fragment to insert in the library, amplification back extracting plasmid (Qiagen, Germany), hold as 3 ' and the 5 ' universal primer of holding with T3 and T7, adopt thing fluorescent mark (Big-Dye, Perkin-Elmer, method USA) of stopping, (Perkin-Elmer carries out the EST large scale sequencing on USA) at ABI 377 sequenators.Sequencing result is removed the carrier sequence with FACTURA software, is transferred to the processing of carrying out next step on the SUN Ultra 450Server.All sequence informations are used the GCG software package again, and (Wisconsingroup, USA) BLAST in and the existing database of FASTA software search (Genebank+EMBL) are lower than 95% sequence with no homology or homology and are considered as new gene and set up database.

5. the full-length clone of gene (Cloning of Full-length cDNA)

On the new gene fragment order information basis that obtains, carry out the cDNA full-length clone, carry out in two stages:

(1) " electronic cloning " (Electronic Cloning)

Search the dbEST database with new gene fragment order as probe, with overlap＞50bp, homology is at (the Expressed Sequence Tag of the expressed sequence tag more than 98%, being called for short " EST ") sequence thinks same sequence (consensus sequence), take out and splice with AUTOASSEMBLER software, part EST can the extension probes sequence.Whether the sequence that is extended with the STRIDER software analysis has complete open reading frame (Open Reading Frame again, ORF), on Nucleotide and amino acid levels, whether homology is arranged with definite this sequence with BLAST search Genbank or SwissProt, to help how differentiate resulting full length gene integrity with other species.By the method for electronic cloning, can obtain the full length sequence of Penicillium marneffei bacterium MDH gene usually.

(2) the terminal rapid amplifying of cDNA (Rapid Amplification of cDNA Ends, RACE)

If do not obtain complete cDNA total length yet, then, in Penicillium marneffei bacterium cDNA library, carry out the long range PCR reaction at 5 ' or 3 ' end design primer of existing sequence by " electronic cloning " method.Then to PCR product cloning, order-checking.The sequence that is extended with AUTOASSEMBLER and STRIDER software analysis has or not complete ORF, as not having, repeats said process until obtaining total length.

(3)RT-PCR

For 5 ' and 3 ' end known sequences,, can consider to adopt the method for RT-PCR if the centre still has an intersegmental crack (gap) to obtain from existing public database or its data storehouse.At sequence 5 ' end design primer, 3 ' end primer adopts Oligo-dT, increases in the total RNA of Penicillium marneffei storehouse.Then product is cloned, checked order.Splice at last and obtain total length.

By being used in combination above-mentioned 3 kinds of methods, obtained the complete encoding sequence of candidate's Penicillium marneffei bacterium MDH, promptly obtain the sequence shown in the SEQ ID NO.1.

Derive the aminoacid sequence of MDH according to the full length cDNA sequence that obtains, totally 266 amino acid, its aminoacid sequence sees SEQ ID NO.2 for details.

Embodiment 2

The sequence information and the homology analysis of Penicillium marneffei bacterium gene:

The full-length cDNA of Penicillium marneffei MDH (MDH) gene that the present invention is new is 1000bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 112-912 position Nucleotide.Derive the aminoacid sequence of Penicillium marneffei MDH (MDH), totally 266 amino-acid residues according to full-length cDNA.Detailed sequence is seen SEQ ID NO.2.The full length cDNA sequence and the coded protein thereof of Penicillium marneffei MDH are carried out Nucleotide and protein homology retrieval with blast program in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR database, found that: on amino acid levels, it has 77% homogeny and 88% similarity with the 1-266 amino acids residue of spore mould (Cladosporium fulvum) mannitol dehydrogenase (NADP-dependent mannitoldehydrogenase (EC 1.1.1.138), SwissProt Accession No.Q96W29).Therefore there is higher homology in the Penicillium marneffei MDH gene with the mould mannitol dehydrogenase gene of spore on protein level, belongs to same family and both also have very high similarity on function.

Penicillium marneffei MDH 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, Penicillium marneffei MDH of the present invention can also merge with other members of this family or exchange fragment, to produce new albumen.For example proteic N end of Penicillium marneffei MDH of the present invention and the proteic N end of a mould mannitol dehydrogenase of spore are exchanged, to produce the albumen that new activity is higher or have new features.

At the antibody of Penicillium marneffei MDH of the present invention, be used to screen other members of this family, perhaps be used for affinity purification associated protein (as other members of this family).

Embodiment 3

The 26S Proteasome Structure and Function research of Penicillium marneffei MDH:

With the aminoacid sequence of Penicillium marneffei MDH the PROSITE database (network address is: retrieval motif (motif) http://expasy.hcuge.ch/sprot/scnpsitl.html) obtains following result:

In aminoacid sequence, there is following function motif:

(i) protein kinase C phosphorylation site (Protein kinase C phosphorylation site): 16-18,53-55,120-122,264-266

(ii) casein kinase i I phosphorylation site (Casein kinase IIphosphorylationsite): 10-13,54-57,120-123,253-256,

(iii) N-myristoylation site (N-myristoylation site): 34-39,45-50,58-63,99-104,115-120,134-139

And protein kinase C phosphorylation site, casein kinase i I phosphorylation site and N-myristoylation site are all relevant with posttranslational modification (post-translational modifications); and these sites play keying action in drug discovery process, can be used as the action target spot of treatment by the medicine of the microbial disease of Penicillium marneffei.

Embodiment 4

Spectrum is expressed in growing of Penicillium marneffei bacterium MDH gene

Electronics Northern express spectra.Press people's such as Ton C. method (Ton C et al., Biochem BiophysRes Commun 1997 Dec 18; 241 (2): 589-594; Hwang DM, et al., Circulation1997 Dec 16; 96 (12): 4146-4203), the BLAST retrieval will be done in the dbEST database of Penicillium marneffei bacterium MDH cDNA sequence in the GCG software package, in the EST that obtains, the EST of probable value＜10e-10, homogeny＞95%, can be considered the transcriptional expression of this gene in different growth and development processes originally, draw the spectrum of growing of expressing this gene thus, disclose it and in Penicillium marneffei bacterium different developmental phases, all bringing into play important effect.

Embodiment 5

Preparation and the purification of Penicillium marneffei bacterium MDH

In this embodiment, the Penicillium marneffei bacterium MDH encoding sequence of total length or segment are built into commercial protein merge among the expression vector, to express and purification of recombinant proteins.

(1) construction of prokaryotic expression vector, and transformed into escherichia coli

Complete encoding sequence (SEQ ID NO.1) according to Penicillium marneffei bacterium MDH, design amplifies complete coding and reads the primer of frame (corresponding respectively to about 20 above Nucleotide of encoding sequence 5 ' and 3 ' end), and (this is according to the pDEST that selects for use to introduce restriction endonuclease sites respectively on positive anti-primer ^TM17 carriers and decide), so that construction of expression vector.Penicillium marneffei bacterium MDH gene is being guaranteed to be cloned into pDEST under the correct prerequisite of reading frame ^TM17 carriers (Invitrogen, Carlsbad, CA).Identify that good expression vector utilizes electric shock transformation method to change e. coli bl21 over to, Screening and Identification obtains containing pDEST ^TMThe engineering bacteria BL21-pDEST of 17-MDH expression vector ^TM17-MDH.

(2) isolation identification of the engineering bacteria of expression GST-MDH recombinant protein

The BL21-pDEST of picking list bacterium colony ^TMThe 17-MDH engineering bacteria contains jolting overnight incubation in the LB substratum of 100 μ g/ml penbritins in 3ml, draws nutrient solution by 1: 100 concentration and cultivates about 3 hours in new LB substratum (containing 100 μ g/ml penbritins), to OD ₆₀₀After reaching 0.5, adding IPTG continues at 37 ℃ to final concentration 1mmol/L and cultivated respectively 0,1,2,3 hours.It is centrifugal to get the different 1ml bacterium liquid of incubation time, in the bacterial precipitation thing, add lysate (2 * SDS sample-loading buffer, 50 μ l, distilled water 45 μ l, 3-mercaptoethanol 5 μ l), the suspendible bacterial precipitation, boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of dyeing back observation expection molecular weight size increases with the IPTG induction time is the engineering bacteria of expressing the GST-MDH fusion rotein.

(3) the extraction purifying of GST-MDH fusion rotein

The proteic engineering bacteria BL21-pDEST of abduction delivering GST-MDH amalgamation and expression as stated above ^TM17-MDH.Bacterium centrifugation after inducing adds the resuspended bacterium of 20ml PBS, ultrasonication bacterium by every 400ml bacterium.The ultrasonic completely liquid of broken bacterium adds 50% saturated Triptide Sepharose 4B of PBS by every milliliter of amount that adds 20 microlitres, 37 ℃ of joltings were in conjunction with 30 minutes, 10000rpm precipitated the Triptide Sepharose 4B that combines GST-MDH in centrifugal 10 minutes, abandoned supernatant.Clean twice by the amount that every milliliter of ultrasonic liquid gained precipitation adds 100 μ l PBS, then add 10 μ l reduced glutathione elutriants by every milliliter of ultrasonic liquid gained precipitation, room temperature was put 10 minutes, centrifugal 10 minutes of 10000rpm, and supernatant is the fusion rotein of wash-out.Repeat twice of wash-out.The supernatant of wash-out is stored in-80 ℃, and carries out the SDS-PAGE electrophoresis, detects purification effect.Protein band at the 29.6kDa place is Penicillium marneffei bacterium MDH albumen.

Embodiment 6

Penicillium marneffei bacterium MDH carries out eukaryotic cell expression in yeast

1. the structure and the conversion of Penicillium marneffei bacterium MDH Yeast expression carrier

Complete encoding sequence (SEQ ID NO.1) according to Penicillium marneffei bacterium MDH, design amplifies the primer that complete coding is read frame, and on positive anti-primer, introduce restriction endonuclease sites (this is decided by the carrier of selecting for use) respectively, so that construction of expression vector.With the cDNA of Penicillium marneffei bacterium MDH under the prerequisite that guarantees reading frame, be cloned into the pYES2-DEST52 carrier (Invitrogen, Carlsbad, CA).Identify that good expression vector utilizes electric shock transformation method to change among the yeast cell DB3.1, utilizes the penbritin Screening and Identification to obtain containing the engineering bacteria DB3.1-pYES2-DEST52-MDH of pYES2-DEST52-MDH expression vector.

2. express the isolation identification of the engineering bacteria of MDH recombinant protein

The DB3.1-pYES2-DEST52-MDH engineering bacteria of picking list bacterium colony contains jolting overnight incubation in the LB substratum of 100 μ g/ml penbritins in 3ml, draws nutrient solution by 1: 100 concentration and be cultured to OD in new YPD substratum (containing 100 μ g/ml penbritins) ₆₀₀Reach 0.5.It is centrifugal to get 1ml bacterium liquid, adds lysate (2 * SDS sample-loading buffer, 50 μ l, distilled water 45 μ l in the bacterial sediment thing, 3-mercaptoethanol 5 μ l), the suspendible bacterial sediment boiled in the boiling water bath 5 minutes, centrifugal 1 minute of 10000rpm, supernatant adds electrophoresis in the 12%SDS-PAGE glue.The bacterial strain that the protein content of expection molecular weight size is observed in the dyeing back is the engineering bacteria of expressing Penicillium marneffei bacterium MDH fusion rotein.

Collect cell transformed and carry out the Western evaluation.The SDS-PAGE electrophoresis will be carried out behind the cellular lysate, glue behind the electrophoresis prints to protein transduction on the nitrocellulose membrane in the half-dried electrotransfer instrument of the Multiphor of Pharmacia II, nitrocellulose membrane is placed confining liquid sealing 1 hour, then in the antibody-solutions of anti-Penicillium marneffei bacterium MDH, sealed 1 hour, the jolting of TBS liquid is cleaned 5 minutes 2 times totally, then film is placed the anti-second antibody solution jolting of biotin labeled anti-Penicillium marneffei bacterium MDH one 1 hour, TBS cleans, adding avidin-alkaline phosphatase enzyme complex reacted 30 minutes, TBS cleans 2 times, adds freshly prepared colour developing liquid colour developing and observes protein band.

The clone of picking high expression level Penicillium marneffei bacterium MDH.

3. the extraction purifying of Penicillium marneffei bacterium MDH

The engineering bacteria DB3.1-pYES2-DEST52-MDH of abduction delivering MDH as stated above.Collect bacterial sediment, bacterial sediment adds the resuspended thalline of PBS, and the ultrasonication thalline is centrifugal, cleans several times, carries out the SDS-PAGE electrophoresis, detects purification effect.Protein band at the 29.6kDa place is Penicillium marneffei bacterium MDH.

Embodiment 7

The preparation of anti-Penicillium marneffei bacterium MDH antibody

It is standby that the Penicillium marneffei bacterium MDH that obtains among embodiment 5 and the embodiment 6 is separated the back with chromatography, also can separate with the SDS-PAGE gel electrophoresis, electrophoretic band is cut off from gel, and with isopyknic complete Freund ' s adjuvant emulsion.Get the female mouse of 6-8 week Balb/C in age, the albumen with 50-100 μ g/0.2ml emulsification carries out peritoneal injection to mouse.After 14 days, with the same antigen of non-complete Freund ' s adjuvant emulsion to mouse with the dosage of 50-100 μ g/0.2ml again booster immunization once be used for after 3-5 days merging.Prepare feeder cell then, carry out cytogamy again.

After cytogamy 10-15 days, need to check by the hole, in case find vigorous hybrid cell colony growth, just use the preliminary screening that Penicillium marneffei bacterium MDH does antibody activity, method commonly used has: immunofluorescent test, emission immunity test (RIA), enzyme linked immunosorbent assay (ELISA).After checking out the hole of antibody activity, clone cultivation at once, and isolate antibody.

Embodiment 8

Penicillium marneffei bacterium MDH gene is used to prepare biochip

Nucleotide sequence design primer with the Penicillium marneffei bacterium MDH gene that obtains amplifies the Penicillium marneffei bacterium MDH gene of total length or the part segment of this gene, as the sample of biochip point sample.

With the BioRobotics of Britain put automatically the film instrument with sample spot on 8 * 12cm nylon membrane, 4 points of each specimens point, every DNA amount is about 10ng.Positive control is the people β-actin gene of 4 point samples of repetition, and negative control is the lambda bacteriophage dna of 4 point samples of repetition.

The chip that contains Penicillium marneffei bacterium MDH gene can be used for infecting the diagnosis and the drug screening of Penicillium marneffei bacterial diseases.

Sequence table

＜110〉Research Center of Shanghai Human Genome

＜120〉a kind of new Penicillium marneffei MDH, its encoding sequence and application thereof

<130>NP-1274

<160>2

<170>PatentIn?version?3.2

<210>1

<211>1000

<212>DNA

＜213〉Penicillium marneffei bacterium (Penicillium marneffei)

<220>

<221>CDS

<222>(112)..(912)

<400>1

ctgtctcgcc?cttggtcttt?gcaatctcat?gcatccagca?tcaatacctc?ccgtacagag 60

ccctcaatac?cttactaaca?ctaaacaaaa?gagagagata?aaaagacaaa?c?atg?cct 117

Met?Pro

1

gtc?tcc?gtc?ccc?gcc?gca?gac?tcc?ctc?cac?gac?ctc?ttc?agc?ctc?aaa 165

Val?Ser?Val?Pro?Ala?Ala?Asp?Ser?Leu?His?Asp?Leu?Phe?Ser?Leu?Lys

5 10 15

ggc?aag?acg?atc?atc?atc?acc?ggc?gcc?tcc?ggc?cct?cgc?ggc?atg?ggc 213

Gly?Lys?Thr?Ile?Ile?Ile?Thr?Gly?Ala?Ser?Gly?Pro?Arg?Gly?Met?Gly

20 25 30

atc?gaa?gca?gca?cgc?ggc?tgc?gcc?gag?atg?ggc?gcc?aac?atc?gcg?ctc 261

Ile?Glu?Ala?Ala?Arg?Gly?Cys?Ala?Glu?Met?Gly?Ala?Asn?Ile?Ala?Leu

35 40 45 50

acc?tat?tct?tct?agg?ccc?gag?ggc?ggc?gaa?ata?aat?gcc?cgc?gac?ctg 309

Thr?Tyr?Ser?Ser?Arg?Pro?Glu?Gly?Gly?Glu?Ile?Asn?Ala?Arg?Asp?Leu

55 60 65

gct?aaa?cag?tac?ggc?gtc?aag?gct?aaa?gct?tac?aaa?tgc?aac?gtt?gcg 357

Ala?Lys?Gln?Tyr?Gly?Val?Lys?Ala?Lys?Ala?Tyr?Lys?Cys?Asn?Val?Ala

70 75 80

aaa?tgg?gag?gag?gtt?gag?aaa?ttg?gtg?gcg?gac?gtg?atc?aag?gag?ttt 405

Lys?Trp?Glu?Glu?Val?Glu?Lys?Leu?Val?Ala?Asp?Val?Ile?Lys?Glu?Phe

85 90 95

ggc?cag?atc?gat?ggg?ttc?gtg?gcc?aat?gcc?ggc?cga?aca?gct?gac?aag 453

Gly?Gln?Ile?Asp?Gly?Phe?Val?Ala?Asn?Ala?Gly?Arg?Thr?Ala?Asp?Lys

100 105 110

ggc?att?ttg?gaa?ggt?tcc?gtg?aag?gat?tgg?gag?gaa?gtc?att?cag?acc 501

Gly?Ile?Leu?Glu?Gly?Ser?Val?Lys?Asp?Trp?Glu?Glu?Val?Ile?Gln?Thr

115 120 125 130

gat?ctc?acg?ggt?acc?ttc?cac?tgt?gcc?aaa?gcc?gtg?ggc?ccc?cat?ttc 549

Asp?Leu?Thr?Gly?Thr?Phe?His?Cys?Ala?Lys?Ala?Val?Gly?Pro?His?Phe

135 140 145

aag?gag?aga?ggc?aaa?gga?agt?ttt?gtc?att?acc?gcc?agc?atg?tcc?ggc 597

Lys?Glu?Arg?Gly?Lys?Gly?Ser?Phe?Val?Ile?Thr?Ala?Ser?Met?Ser?Gly

150 155 160

cac?att?gca?aat?ttc?cct?cag?gaa?cag?act?tcg?tac?aac?gtc?gcg?aaa 645

His?Ile?Ala?Asn?Phe?Pro?Gln?Glu?Gln?Thr?Ser?Tyr?Asn?Val?Ala?Lys

165 170 175

gca?gga?tgt?atc?cac?ttt?gcg?cgg?tcg?ttg?gct?aac?gaa?tgg?cgc?gat 693

Ala?Gly?Cys?Ile?His?Phe?Ala?Arg?Ser?Leu?Ala?Asn?Glu?Trp?Arg?Asp

180 185 190

ttt?gcg?cgc?gtc?aac?tcc?att?tca?ccg?ggt?tat?att?gat?acg?ggt?ctg 741

Phe?Ala?Arg?Val?Asn?Ser?Ile?Ser?Pro?Gly?Tyr?Ile?Asp?Thr?Gly?Leu

195 200 205 210

tcg?gac?ttt?gtg?ccg?caa?aaa?att?cag?gat?ttg?tgg?aat?tcg?atg?atc 789

Ser?Asp?Phe?Val?Pro?Gln?Lys?Ile?Gln?Asp?Leu?Trp?Asn?Ser?Met?Ile

215 220 225

cct?ctg?gga?cgc?aac?gga?cat?gca?aag?gag?ctc?aag?ggg?gcg?tac?gtg 837

Pro?Leu?Gly?Arg?Asn?Gly?His?Ala?Lys?Glu?Leu?Lys?Gly?Ala?Tyr?Val

230 235 240

tac?ctg?ctc?agc?gat?gcg?agt?acg?tat?acc?acg?ggt?gcg?gat?att?gtg 885

Tyr?Leu?Leu?Ser?Asp?Ala?Ser?Thr?Tyr?Thr?Thr?Gly?Ala?Asp?Ile?Val

245 250 255

att?gat?gga?ggg?tat?act?gtg?cga?taa?gcctttattt?taactaatta 932

Ile?Asp?Gly?Gly?Tyr?Thr?Val?Arg

260 265

cggagtacta?cgaagtagca?taccttctat?aattctcgtg?atatgaaaaa?aaaaaaaaaa 992

aaaaaaaa 1000

<210>2

<211>266

<212>PRT

＜213〉Penicillium marneffei bacterium (Penicillium marneffei)

<400>2

Met?Pro?Val?Ser?Val?Pro?Ala?Ala?Asp?Ser?Leu?His?Asp?Leu?Phe?Ser

1 5 10 15

Leu?Lys?Gly?Lys?Thr?Ile?Ile?Ile?Thr?Gly?Ala?Ser?Gly?Pro?Arg?Gly

20 25 30

Met?Gly?Ile?Glu?Ala?Ala?Arg?Gly?Cys?Ala?Glu?Met?Gly?Ala?Asn?Ile

35 40 45

Ala?Leu?Thr?Tyr?Ser?Ser?Arg?Pro?Glu?Gly?Gly?Glu?Ile?Asn?Ala?Arg

50 55 60

Asp?Leu?Ala?Lys?Gln?Tyr?Gly?Val?Lys?Ala?Lys?Ala?Tyr?Lys?Cys?Asn

65 70 75 80

Val?Ala?Lys?Trp?Glu?Glu?Val?Glu?Lys?Leu?Val?Ala?Asp?Val?Ile?Lys

85 90 95

Glu?Phe?Gly?Gln?Ile?Asp?Gly?Phe?Val?Ala?Asn?Ala?Gly?Arg?Thr?Ala

100 105 110

Asp?Lys?Gly?Ile?Leu?Glu?Gly?Ser?Val?Lys?Asp?Trp?Glu?Glu?Val?Ile

115 120 125

Gln?Thr?Asp?Leu?Thr?Gly?Thr?Phe?His?Cys?Ala?Lys?Ala?Val?Gly?Pro

130 135 140

His?Phe?Lys?Glu?Arg?Gly?Lys?Gly?Ser?Phe?Val?Ile?Thr?Ala?Ser?Met

145 150 155 160

Ser?Gly?His?Ile?Ala?Asn?Phe?Pro?Gln?Glu?Gln?Thr?Ser?Tyr?Asn?Val

165 170 175

Ala?Lys?Ala?Gly?Cys?Ile?His?Phe?Ala?Arg?Ser?Leu?Ala?Asn?Glu?Trp

180 185 190

Arg?Asp?Phe?Ala?Arg?Val?Asn?Ser?Ile?Ser?Pro?Gly?Tyr?Ile?Asp?Thr

195 200 205

Gly?Leu?Ser?Asp?Phe?Val?Pro?Gln?Lys?Ile?Gln?Asp?Leu?Trp?Asn?Ser

210 215 220

Met?Ile?Pro?Leu?Gly?Arg?Asn?Gly?His?Ala?Lys?Glu?Leu?Lys?Gly?Ala

225 230 235 240

Tyr?Val?Tyr?Leu?Leu?Ser?Asp?Ala?Ser?Thr?Tyr?Thr?Thr?Gly?Ala?Asp

245 250 255

Ile?Val?Ile?Asp?Gly?Gly?Tyr?Thr?Val?Arg

260 265

Claims (16)

1. isolating polynucleotide from Penicillium marneffei bacterium (Penicillium marneffei), it contains the polynucleotide sequence of the Penicillium marneffei MDH of encoding, and described polynucleotide are selected from:

A) polynucleotide of polypeptide that contain the aminoacid sequence of SEQ ID NO.2 with coding have the polynucleotide of at least 70% homology,

B) coding contains the polynucleotide with the polypeptide of the aminoacid sequence of aminoacid sequence at least 70% homology of SEQ ID NO.2,

C) and a) or b) polynucleotide complementary polynucleotide, and

D) contain a), b) or the polynucleotide of at least 15 continuous bases of polynucleotide sequence c).

2. a kind of isolating polynucleotide as claimed in claim 1 is characterized in that, described polynucleotide encoding one polypeptide, and this polypeptide has the sequence shown in the SEQ ID NO.2.

3. a kind of isolating polynucleotide as claimed in claim 1 is characterized in that described polynucleotide contain

(i) as the nucleotide sequence of Nucleotide 112-912 position among the SEQ ID NO.1, or

(ii) in genetic code degeneracy scope corresponding at least one sequence of (i) sequence, or

(iii) be complementary to (i) or (ii) at least one sequence and randomly of the sequence hybridization of sequence

(iv) the justice that has of neutral function is suddenlyd change in (i).

4. a kind of isolating polynucleotide as claimed in claim 3 is characterized in that described polynucleotide contain the nucleotide sequence of Nucleotide 112-912 position among the SEQ ID NO.1.

5. an isolated polypeptide is characterized in that, described polypeptide has sequence or its fragment with aminoacid sequence at least 70% homology shown in the SEQ ID NO.2.

6. a kind of isolated polypeptide as claimed in claim 5 is characterized in that, described polypeptide is the polypeptide that contains aminoacid sequence shown in the SEQID NO.2.

7. a carrier is characterized in that, described carrier contains the described isolating polynucleotide of claim 1.

8. a genetically engineered host cell is characterized in that, described host cell is with the described carrier transformed host cells of claim 7.

9. a production has the method for the active polypeptide of Penicillium marneffei MDH, it is characterized in that this method comprises the steps:

(I) nucleotide sequence that coding is had an active polypeptide of Penicillium marneffei MDH operationally is connected in expression regulation sequence, form protein expression vector, show at least 70% homology from the nucleotides sequence of Nucleotide 112-912 position among described nucleotide sequence and the SEQ ID NO.1;

(II) change the expression vector in the step (I) over to host cell, form the Penicillium marneffei MDH reconstitution cell;

(III) be fit to express under the condition of Penicillium marneffei MDH polypeptide the reconstitution cell in the culturing step (II);

(IV) isolate and have the active polypeptide of Penicillium marneffei MDH.

10. a kind of production as claimed in claim 9 has the method for the active polypeptide of Penicillium marneffei MDH, it is characterized in that, described nucleotides sequence is classified among the SEQ ID NO.1 nucleotide sequence from Nucleotide 112-912 position as.

11. an antibody is characterized in that, described antibody be can with claim 5 or 6 described polypeptid specificity bonded antibody.

12. a probe molecule is characterized in that, described probe molecule contains 8-100 successive Nucleotide in the described polynucleotide of claim 1.

13. the polypeptide of claim 5 or 6 is treated at the application in the medicine of the microbial disease of Penicillium marneffei in preparation.

14. contain the pharmaceutical composition of the polypeptide of claim 5 or 6.

15. a test kit is characterized in that:

Described test kit comprises any polynucleotide sequence or its continuous fragment in the claim 1～4, perhaps

Described test kit comprises polypeptide or its continuous fragment of claim 5 or 6, perhaps

Described test kit comprises the antibody of claim 11.

16. a biochip is characterized in that:

Contain any polynucleotide sequence or its continuous fragment in the claim 1～4 on the carrier of described biochip, perhaps

The polypeptide or its continuous fragment that contain claim 5 or 6 on the carrier of described biochip, perhaps

The antibody that comprises claim 11 on the carrier of described biochip.