CN102391999A - Homocysteine methyltransferase, nucleotide sequence thereof, recombinant vector, recombinant host cells, manufacturing method and kit - Google Patents
Homocysteine methyltransferase, nucleotide sequence thereof, recombinant vector, recombinant host cells, manufacturing method and kit Download PDFInfo
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- CN102391999A CN102391999A CN2011103436413A CN201110343641A CN102391999A CN 102391999 A CN102391999 A CN 102391999A CN 2011103436413 A CN2011103436413 A CN 2011103436413A CN 201110343641 A CN201110343641 A CN 201110343641A CN 102391999 A CN102391999 A CN 102391999A
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- nucleotide sequence
- seq
- homocysteine methyltransgerase
- homocysteine
- methyltransgerase
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Abstract
The invention discloses a homocysteine methyltransferase, the amino acid sequence of the homocysteine methyltransferase is the amino acid sequence as shown in (1) SEQ ID NO: 1 (sequence identity number: 1), or the amino acid sequence as shown in (2) SEQ ID NO: 3 or the (3) amino acid sequence which is obtained by performing deletion, addition and/or substitution on one or a plurality of amino acids in the amino acid sequence described in (1) or (2), but has the constant functions of the homocysteine methyltransferase. The invention further discloses a nucleotide sequence for encoding the homocysteine methyltransferase, a recombinant vector including the nucleotide sequence, recombinant host cells including the recombinant vector, a method for purifying the enzyme from the recombinant host cells and a kit including at least one of the homocysteine methyltransferase, the nucleotide sequence, the recombinant vector and the recombinant host cells. The homocysteine methyltransferase is good in heat resistance and very high in stability.
Description
Technical field
The present invention relates to a kind of enzyme; The encode nucleotide sequence of this enzyme; The recombinant vectors that comprises said nucleotide sequence; The recombinant host cell that comprises said recombinant vectors, the method for this enzyme of purifying from aforementioned recombinant host cell, and comprise at least a test kit in above-mentioned enzyme, nucleotide sequence, recombinant vectors and the recombinant host cell.More specifically; The present invention relates to a kind of homocysteine methyltransgerase; The nucleotide sequence of this homocysteine methyltransgerase of encoding comprises the recombinant vectors of said nucleotide sequence, comprises the recombinant host cell of said recombinant vectors; The method of this enzyme of purifying from aforementioned recombinant host cell, and comprise at least a test kit in above-mentioned homocysteine methyltransgerase, nucleotide sequence, recombinant vectors and the recombinant host cell.
Background technology
Methyltransgerase is the enzyme that plays crucial regulating effect in the vital movement, and it can be with the Methyl transporters on the S-adenosylmethionine (SAM) to the substrate molecule, generates the multiple important methyl physiologically active substance that contains, and reaches the purpose that function is regulated.Homocysteine methyltransgerase (EC 2.1.1.10) is a kind of in the methyltransgerase, can be used for the preparation of homocysteine (HCY) biochemical diagnosis reagent, is a kind of critical materials enzyme.
Existing homocysteine methyltransgerase is mainly extracted from yeast saccharomyces cerevisiae, exists the enzyme productive rate low, purification difficult, poor heat stability shortcomings such as (it are complete deactivation that storage temperature surpasses 40 ℃).
Summary of the invention
An object of the present invention is to overcome the shortcoming of existing homocysteine methyltransgerase poor heat resistance, the low purification difficult of productive rate, the homocysteine methyltransgerase of a kind of good heat resistance, the high and easy purifying of productive rate is provided.
Second purpose of the present invention provides the nucleotide sequence of the said homocysteine methyltransgerase of coding.
The 3rd purpose of the present invention provides the recombinant vectors that comprises said nucleotide sequence.
The 4th purpose of the present invention provides the recombinant host cell that comprises said recombinant vectors.
The 5th purpose of the present invention provides at least a test kit that comprises in above-mentioned homocysteine methyltransgerase, nucleotide sequence, recombinant vectors and the recombinant host cell.
Contriver of the present invention has paid creative work in large quantities; From Candida albicans (Candida albicans SC5314), obtain the homocysteine methyltransgerase that the present invention has the aminoacid sequence shown in the SEQ ID NO:1, and utilize the method for random mutation to obtain the homocysteine methyltransgerase that the present invention has the aminoacid sequence shown in the SEQ ID NO:3.And contriver of the present invention is surprised to find that two kinds of homocysteine methyltransgerase of gained all have good heat endurance, can in wide TR, keep advantages of higher stability.
The invention provides a kind of homocysteine methyltransgerase, wherein, the aminoacid sequence of said homocysteine methyltransgerase does
(1) aminoacid sequence shown in the SEQ ID NO:1, perhaps
(2) aminoacid sequence shown in the SEQ ID NO:3, perhaps
(3) (1) or (2) described aminoacid sequence is carried out one or several amino acid whose disappearance, interpolation and/or replacement, but the constant aminoacid sequence of the function of its homocysteine methyltransgerase.
The present invention also provides the nucleotide sequence of the homocysteine methyltransgerase according to the invention of encoding.
The present invention also provides the recombinant vectors that comprises nucleotide sequence according to the invention.
The present invention also provides the recombinant host cell that comprises recombinant vectors according to the invention.
The present invention also provides the method for this enzyme of purifying from aforementioned recombinant host cell.
The present invention also provides at least a test kit that comprises in homocysteine methyltransgerase of the present invention, nucleotide sequence, recombinant vectors and the recombinant host cell.
Homocysteine methyltransgerase provided by the invention has the advantage of good heat resistance.Can know referring to Fig. 3; Two kinds of homocysteine methyltransgerase of the present invention still all have the relative reactivity more than 60% after in the damping fluid of pH6.0, leaving standstill 15 minutes under 40 ℃; Wherein, the homocysteine methyltransgerase of the present invention with the aminoacid sequence shown in the SEQ ID NO:3 still all has the relative reactivity more than 50% after in the damping fluid of pH6.0, leaving standstill 15 minutes under 45 ℃.
Description of drawings
The isogenic sepharose of the homocysteine methyltransgerase of Fig. 1 embodiment 1 is identified photo;
The homocysteine methyltransgerase of Fig. 2 embodiment 1 induces the SDS-PAGE of back expression and purification to identify photo at IPTG; Wherein, the M swimming lane is a molecular weight marker, and the 1st swimming lane is not for receiving IPTG inductive protein expression; The 2nd swimming lane is the protein expression after IPTG induces; Can find out obviously that therefrom in the 2nd swimming lane, the target protein homocysteine methyltransgerase has obtained great expression;
Fig. 3 embodiment 1 and 2 homocysteine methyltransgerase thermostability figure.
Embodiment
The invention provides a kind of homocysteine methyltransgerase, wherein, the aminoacid sequence of said homocysteine methyltransgerase does
(1) aminoacid sequence shown in the SEQ ID NO:1, perhaps
(2) aminoacid sequence shown in the SEQ ID NO:3, perhaps
(3) (1) or (2) described aminoacid sequence is carried out one or several amino acid whose disappearance, interpolation and/or replacement, but the constant aminoacid sequence of the function of its homocysteine methyltransgerase.
Those skilled in the art are known, and 20 seed amino acid residues of constitutive protein matter can be divided into four types according to side chain polarity:
1, nonpolar amino acid: L-Ala (Ala), Xie Ansuan (Val), leucine (Leu), Isoleucine (Ile), methionine(Met) (Met), phenylalanine(Phe) (Phe), tryptophane (Trp) and proline(Pro) (Pro);
2, the uncharged amino acid of polarity: glycocoll (Gly), Serine (Ser), Threonine (Thr), halfcystine (Cys), l-asparagine (Asn), Stimulina (Gln) and tyrosine (Tyr);
3, positively charged amino acid: l-arginine (Arg), Methionin (Lys) and Histidine (His);
4, electronegative amino acid: aspartic acid (Asp) and L-glutamic acid (Glu) (referring to " biological chemistry " (second edition) first volume, Shen is with, Wang Jingyan, 82-83 page or leaf, Higher Education Publishing House, December nineteen ninety).If belonging to the amino-acid residue of a classification in the protein together replaces; For example replace Lys or replace Ile by Leu by Arg; Said residue role (such as positive charge being provided or forming the effect of hydrophobic capsule bag constructions) in protein domain does not change; Therefore can't exert an influence to proteinic three-dimensional arrangement, therefore still can realize proteic function.For example; As well known to those skilled in the art; Ala and Ser, Val and Ile, Asp and Glu, Ser and Thr, Ala and Gly, Ala and Thr, Ser and Asn, Ala and Val, Ser and Gly, Tyr and Phe, Ala and Pro, Lys and Arg, Asp and Asn, Leu and Ile, Leu and Val, Ala and Glu and Asp and Gly; Replace each other between any two, can not influence proteic three-dimensional arrangement and function.The said amino-acid residue replacement that belongs to a classification together can occur on any amino acid residue position on the homocysteine methyltransgerase.On the contrary, different classes of amino-acid residue replaces, and perhaps amino acid whose replacement does not meet the above-mentioned replacement rule of enumerating, and proteic structure is changed, and difference appears in function.
Homocysteine methyltransgerase provided by the invention can also be modified or suddenly change, and obtains deutero-protein." deutero-protein " according to the invention refers to have the difference on the aminoacid sequence with the homocysteine methyltransgerase with above-mentioned aminoacid sequence, and the difference on the modified forms that does not influence sequence is perhaps arranged, and perhaps haves both at the same time.These albumen comprise natural or the inductive genetic variant.Said induce variation body can obtain through various technology, like the random mutation that radiation or mutagenic compound etc. produce, and also can be through obtaining like fixed-point mutation method or the biological technology of other known moleculars.Said " deutero-protein " also comprises the analogue (like D type amino acid) with the amino acid whose residue of natural L type, and has non-natural analogue that exist or synthetic amino acid (like beta-amino acids, gamma-amino acid etc.).
(the not changing primary structure usually) form of modification comprises: the interior or external proteic chemically derived form of body, and like acetylize or carboxylated.Modify and also to comprise glycosylation, like those in proteic synthetic and processing or further carry out glycosylation modified and albumen that produce in the procedure of processing.This modification can be carried out glycosylated enzyme (like mammiferous glycosylase or deglycosylating enzyme) and accomplishes through albumen is exposed to.Modified forms also comprises have the phosphorylated amino acid residue sequence of (like Tyrosine O-phosphate, Serine O-phosphate, phosphothreonine).Thereby also comprise and modified the albumen that has improved its anti-proteolyze performance or optimized solubility property.
Under the preferable case, the aminoacid sequence of said homocysteine methyltransgerase is the aminoacid sequence shown in SEQ ID NO:1 or the SEQ ID NO:3.
The present invention also provides the nucleotide sequence of the homocysteine methyltransgerase according to the invention of encoding.
Known in this field, in 20 kinds of different amino acid of constitutive protein matter, except that Met (ATG) or Trp (TGG) are respectively single password coding; Other 18 seed amino acids are respectively by 2-6 codon coding (Sambrook etc., molecular cloning, press of cold spring harbor laboratory; New York, the U.S., second edition; 1989, see 950 pages of appendix D).Promptly because the degeneracy of genetic codon; Determine more than one mostly of an amino acid whose codon; The displacement of the 3rd Nucleotide often can not change amino acid whose composition in the triplet codon, and the nucleotide sequence of the gene of the same protein of therefore encoding can be different.Those skilled in the art are according to known password sublist; From aminoacid sequence disclosed by the invention; And the active constant aminoacid sequence of the homocysteine methyltransgerase that obtains by said aminoacid sequence; Can derive their nucleotide sequence of gene of to encode fully, obtain said nucleotide sequence through biological method (like PCR method, mutation method) or chemical synthesis process, so this partial nucleotide sequence should comprise within the scope of the present invention all.On the contrary, utilize the disclosed dna sequence dna of this paper, also can be by means commonly known in the art; For example method (molecular cloning, press of cold spring harbor laboratory, the New York of Sambrook etc.; The U.S., second edition, 1989) carry out; Through revising nucleotide sequence provided by the invention, obtain and the active consistent aminoacid sequence of homocysteine methyltransgerase according to the invention.
Under the preferable case, nucleotides sequence according to the invention is classified as
(1) nucleotide sequence shown in the SEQ ID NO:2; Perhaps
(2) nucleotide sequence shown in the SEQ ID NO:4; Perhaps
(3) nucleotide sequence shown in SEQ ID NO:2 or the SEQ ID NO:4 is carried out the nucleotide sequence that one or several Nucleotide replaces, lacks or increase and obtains, this nucleotide sequence coded homocysteine methyltransgerase function is constant.
Nucleotide sequence provided by the invention can use the method for pcr amplification method, recombination method or synthetic to obtain usually.For example, those skilled in the art can be easy to obtain template and primer according to nucleotide sequence provided by the present invention and recombinant bacterial strain, utilize PCR to increase and obtain relevant sequence.When sequence is longer, can carry out twice or pcr amplification repeatedly, then the gained fragment pressed the proper order splicing.In case obtained relevant nucleotide sequence, just can use the relevant aminoacid sequence of the large batch of acquisition of recombination method.Usually the gained nucleotide sequence is cloned into carrier, again in the transgene engineering bacteria, the host cell of the method through routine after the propagation separates and obtains relevant nucleotide sequence then.In addition, also the method for available known artificial chemosynthesis is synthesized relevant nucleotide sequence.
The present invention also provides the recombinant vectors that comprises nucleotide sequence according to the invention.Known in this field, said recombinant vectors generally comprises empty carrier and the goal gene that inserts this empty carrier, and said goal gene is the nucleotide sequence of homocysteine methyltransgerase of the present invention.
In the present invention; Various carrier known in the art can be selected for use in said " empty carrier " (or title " carrier "); Like commercially available various plasmids, clay, phage and retrovirus etc.; The carrier that the preferred said empty carrier of the present invention is the lac promotor, more preferably said empty carrier are selected from by in pET serial carrier (for example pET30a), the vehicle group that pUC19, pGEM and pBluescript formed.Said empty carrier can comprise multiple certification mark commonly used (for example reporter gene such as fluorescent mark, antibiotic marker) and restriction enzyme site.Construction of recombinant vector can adopt the various endonucleases (as using NcoI and XhoI etc. for pET30a) of the MCS of empty carrier own to carry out enzyme and cut the acquisition linear plasmid, is connected with the gene fragment that adopts the cutting of identical nucleic acid restriction endonuclease, obtains recombinant plasmid.
The present invention also provides the recombinant host cell that comprises recombinant vectors according to the invention.
Can said recombinant vectors be transformed, transduce perhaps transfection in host cell through the conventional method in this area, transform like Calcium Chloride Method chemical conversion, high-voltage electric shock, preferred electric shock transforms; Said host cell can be prokaryotic cell prokaryocyte or eukaryotic cell; Be preferably intestinal bacteria, Bacillus subtilus, yeast (like pichia spp) or various animal and plant cells; More preferably said host cell is this area genetic engineering bacterium commonly used, like intestinal bacteria, subtilis or pichia spp.Further preferred said host cell is e. coli bl21 (DE3), BL21, TOP10 or DH5a.Most preferably said host cell is e. coli bl21 (DE3).The substratum of said host cell and culture condition can adopt this area conventional substratum and culture condition.And colibacillary substratum preferably includes: the yeast powder (yeast extract) of the ammonium chloride (or sodium-chlor of 0.05-1%) of the Sodium phosphate, dibasic (or potassium hydrogenphosphate) of the potassium primary phosphate of 0.03-0.3% (or 0.03-1.5% SODIUM PHOSPHATE, MONOBASIC), 0.1-1%, 0.05-0.5%, the sal epsom of 0.01-0.2%, 0.5-5%, the peptone (for example Tryptones) of 0.1-1%, the glucose of 0.001-2%, the glycerine of 2.5%-10%, the lactose of 0.1%-0.8%, the oleic acid of 0.1-2%, the Yelkin TTS of 0.1-1%, and regulate pH to 6.8～7.2.Preferably; When the said host cell of cultivation is e. coli bl21 (DE3), in initial medium, insert the LB seed of OD600=2～4 of 5%～20% volume, in 37 ℃; Dissolved oxygen uses ammoniacal liquor, sulfuric acid to regulate pH6.5～pH7.3 greater than self-sow under 20% the condition.(when dissolved oxygen DO value rises to 40%) is cooled to 32 ℃ when nutritive ingredient exhausts in the initial substratum, adds feed supplement liquid 1 according to the ratio of 60～100ml/L and begins to induce, and add feed supplement liquid 2 according to the speed constant speed of 5～10ml/h/L, uses ammoniacal liquor to regulate pH.At induction period, every at a distance from 1 ± 0.1 ℃ of reduction in 3 ± 1 hours, reduce to 28 ± 0.5 ℃ after constant temperature to fermentation ends.Wherein, the culture medium prescription of using among the present invention is following successively:
Seed culture medium:
Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L.Sterilized 20 minutes for 121 ℃, sterilization cooling back adds sulphuric acid kanamycin to final concentration 50mg/L.
The fermentation initial medium:
Tryptones 10g/L, yeast extract 5g/L; Potassium hydrogenphosphate 6～12g/L, SODIUM PHOSPHATE, MONOBASIC 3～6g/L, Citric acid monohydrate Food grade 0.2～1g/L, sal epsom 0.2～2g/L, micro-mother liquor 1～10ml/L; Transfer pH to 6.8～7.2,121 ℃ sterilization 20 minutes.The glucose that adds sterilization before the inoculation uses ammoniacal liquor to transfer pH to 6.5～7.3 to final concentration 15～20g/L.Add sulphuric acid kanamycin to final concentration 50mg/L.
Phosphatic ratio is in initial medium: potassium hydrogenphosphate: SODIUM PHOSPHATE, MONOBASIC=2: 1
The prescription of trace element is:
10g FeSO
4.7H
2O, 2.25g ZnSO
4.7H
2O, 1g CuSO
4.5H
2O, 0.5gMnSO4.5H
2O, 0.23g Na
2B
4O
7.10H
2O, 2g CaCl
2.2H
2O, 0.1g (NH
4)
6Mo
7O
24, be dissolved in the 5M hydrochloric acid.
Feed supplement liquid 1:
Yeast extract 250 ± 50g/L, Tryptones 100 ± 20g/L, lactose 10 ± 2g/L, 121 ℃ of sterilization 20min.
Feed supplement liquid 2:
Glycerine 400 ± 50g/L, 121 ℃ of sterilization 20min.
Can use this area method commonly used from recombinant host cell, to separate and the purifying homocysteine methyltransgerase.For example, spinning substratum and recombinant host cell, cell debris, affinitive layer purification homocysteine methyltransgerase are removed in high-pressure homogenization smudge cells, centrifuging.For the product of the homocysteine methyltransgerase of separation and purification gained, can use this area method commonly used to carry out purity and identify.For example, Xylene Brilliant Cyanine G method, nitrogen determination, biuret method, lowry method, ultraviolet absorption method, affinity chromatography, enzymic activity, antigen-antibody method, electrophoretic analysis (for example sodium dodecyl sulfate-polyacrylamide gel electrophoresis), sedimetry, diffusion analysis, permanent solubility method, protein spectrum etc.When smudge cells, generally all can give birth to heat, need in smudge cells, lower the temperature usually, need heat radiation after the smudge cells process is accomplished, just can carry out next step purifying work then; Because homocysteine methyltransgerase good heat resistance of the present invention, the smudge cells process need not temperature-fall period, even if the temperature of smudge cells liquid can reach 40-45 ℃ behind the smudge cells, need not heat radiation, still can directly carry out next step purification step at once.For example the enchylema after fragmentation adds calcium chloride, centrifugal (, remove most of membranin and nucleic acid; In centrifugal gained supernatant, add the nickel ion protein precipitation again.The sedimentary albumen of gained can adopt following prescription to redissolve: the YD 30 (EDTA) of 10～50mmol/L pH7.0～8.0 phosphate buffered saline buffers, 2～20mmol/L Reduced nicotinamide-adenine dinucleotide (NAD) and 5～10mmol/L.Because therefore homocysteine methyltransgerase good heat resistance of the present invention can also adopt the broken liquid of reconstitution cell was heated 15-40 minute at 40-45 ℃, at the centrifugal 10-20min of 8000-15000rpm, remove heat labile foreign protein then in addition.Preferably, adopt the broken liquid of reconstitution cell is heated 30min at 45 ℃, the centrifugal 15min of 10000rpm removes most of heat labile foreign protein.More preferably, according to the following step purifying homocysteine methyltransgerase:
(1) fermented liquid is transferred pH to 7.0～8.0,600～900bar homogenate once, and the target protein homocysteine methyltransgerase is discharged.
(2) in homogenate, add the calcium chloride of 30～100mM, abundant stirring and evenly mixing, centrifugal 20 minutes of 5000g removes most nucleic acid, cell debris, and the part foreign protein.
(3) in centrifugal supernatant, add 1～5mM nickel ion (nickel ions of various salt forms), mixing left standstill under the room temperature 1 hour, the centrifugal 20min of 5000g, and most target proteins are in deposition.
(4) the redissolution damping fluid that in deposition, adds 1/5～1 times of volume of original volume stirred about 30 minutes.Centrifugal 20 minutes of 10000g.The buffer formulation of redissolving is 10～50mM pH7.0～8.0 phosphate buffered saline buffers (PB), 2～20mM NAD, 5～10mM EDTA.
(5) redissolution supernatant desalination behind 0.45 μ m membrane filtration.The desalination buffer formulation is 10～50mMpH7.0～8.0PB, 2～5mM EDTA.
The present invention also provides at least a test kit that comprises in homocysteine methyltransgerase of the present invention, nucleotide sequence, recombinant vectors and the recombinant host cell.Under the preferable case, test kit of the present invention comprises homocysteine methyltransgerase, and do not comprise nucleotide sequence, recombinant vectors and recombinant host cell.Test kit more preferably of the present invention comprises the dry powder of homocysteine methyltransgerase, and does not comprise nucleotide sequence, recombinant vectors and recombinant host cell.The dry powder that can prepare homocysteine methyltransgerase with this area method commonly used, as long as this method can access the dry powder of homocysteine methyltransgerase, and the activity of not destroying the dry powder of homocysteine methyltransgerase gets final product.For example use the vacuum decompression thickener to obtain spissated enzyme liquid, use the dry spissated enzyme liquid of freeze drier then; Perhaps use equipment such as vacuum decompression moisture eliminator.Be converted into liquid form in the buffer solution system (for example acetate buffer, phosphate buffered saline buffer, preferred pH is 5.5-7.0) that the dry powder of homocysteine methyltransgerase in use can be through being dissolved in pH5.0-8.0.
Test kit of the present invention can also comprise that this area is usually used in measuring the composition of the test kit of serum homocysteine.Corresponding to the homocysteine methyltransgerase in the test kit of the present invention can be liquid form or solid dry powder form, and other compositions in the test kit of the present invention also can be liquid form and/or solid dry powder form.For example; When test kit of the present invention is liquid form, can contain the homocysteine methyltransgerase of 0.01-0.5mol/L pH 8.0Tris-Cl salt buffer, 1000-10000U/L, the SAHH of 100-10000U/L, the adenosine deaminase of 100-10000U/L, the S-adenosylmethionine (SAM) of 1-100mmol/L, the a-ketoglutaric acid of 1-20mmol/L, the glutamate dehydrogenase of 1000-100000U/L, the reduced form nicotinamide adenine dinucleotide disodium (NADH) of 0.2-2mmol/L and three (2-propyloic) the phosphine hydrogenchloride (TCEP) of 1-20mmol/L in the reaction solution.Test kit of the present invention preferably contains 0.01-0.05mol/L pH 8.0 Tris-Cl salt buffers; The homocysteine methyltransgerase of 1000-10000U/L; The SAHH of 100-1000U/L; The adenosine deaminase of 1000-10000U/L; The S-adenosylmethionine of 1-15mmol/L (SAM); 1.2-10mmol/L a-ketoglutaric acid; The glutamate dehydrogenase of 1000-10000U/L; 0.3-1.5mmol/L reduced form nicotinamide adenine dinucleotide disodium and three (2-propyloic) the phosphine hydrogenchloride (TCEP) of 1-10mmol/L.The reagent constituents of liquid form of the present invention can be passed through Freeze Drying Technique, in conjunction with reduced pressure distillation technique, reverse osmosis technology and ultra-filtration technique etc., is prepared into reagent dry powder.Said dry powder can redissolve to original volume of said reagent with the solvent that is selected from deionized water, zero(ppm) water and distilled water before detecting sample to be measured.Test kit of the present invention can comprise the specification sheets that records above-mentioned various component method of use and consumption.Therefore, said solution also can be prepared according to prior art according to the record of test kit specification sheets before use by the user and get final product.
Being suitable for using the sample to be measured of test kit according to the invention can be under animal body (the comprising human body) state of health and the serum of separating out after the blood natural coagulation under the pathological state.
Further specify the present invention below in conjunction with embodiment, the used reagent of the present invention, substratum are the commercial goods unless stated otherwise.
A) acquisition of natural homocysteine Methyl transporters gene (promptly obtaining the nucleotide sequence shown in the sequence 1)
A-1) cultivation of Candida albicans (Candida albicans SC5314) (available from ATCC MYA-2876)
The solid medium that Candida albicans is cultivated is: 2% glucose, 1% peptone, 0.5% yeast extract paste and 2% agar, 121 ℃ of 20min sterilizations (like no specified otherwise, " % " refers to weight percent among this paper).The liquid nutrient medium that Candida albicans is cultivated is: 2% glucose, 1% peptone and 0.5% yeast extract paste, 121 ℃ of 20min sterilizations.
Candida albicans SC5314 activation on the above solid plate, reactivation process were promptly cultivated in 30 ℃ of incubators 36 hours, then with colony inoculation in above yeast liquid nutrient medium; Cultivated 24 hours under 30 ℃ of conditions.
A-2) extraction of candida albicans gene group DNA
Get the culture that step 1) obtains, at room temperature the centrifugal 5min of 10000rpm abandons supernatant; Deposition is suspended in (2% (v/v) Triton X-100,1% (m/v) SDS, 100mM NaCl in the 400 μ l lysis buffers again; 10mM Tris-Cl (pH8.0); 1mM EDTA (pH8.0)), add pickling glass pearl and the 400 μ l phenol/chloroforms that equate with cell volume.Ice bath, high vibration 5 * 30s.The centrifugal 10min of 10000rpm transfers to supernatant in the new EP pipe.Add 0.6 times of volume Virahol or 2 times of volume ethanol, put upside down mixing, room temperature is placed 10min.The centrifugal 10min of 10000rpm abandons supernatant.Add 500 μ l, 75% ethanol, vortex, the centrifugal 2min of 10000rpm abandons supernatant.To be deposited in air drying 10min, add 50 μ l TE (10mM Tris-Cl (pH8.0), 1mM EDTA (pH8.0)) dissolving.The RNase that adds the 10mg/ml of 1 μ l, 37 ℃ digest 30min down.Obtain genomic dna.
A-3) amplification homocysteine methyltransgerase gene
The homocysteine methyltransgerase gene primer of inventor's design is following:
Homocysteine methyltransgerase gene forward primer (SAMF) (SEQ ID NO:5):
ATCGCCATGGGACGTGTTCAGGATATTTTAG; Near the NcoI restriction enzyme site is arranged.
Homocysteine methyltransgerase gene reverse primer (SAMR) (SEQ ID NO:6):
ATCGCTCGAGCTATTGGTTGATCAATTGTGCAAC; Near the XhoI restriction enzyme site is arranged.
Amplification condition is: 20mM Tris-HCl (pH 8.8), 10mM KCl, 10mM (NH
4)
2SO
4, 2mM MgSO
4, 0.1%Triton X-100,50 μ M dATP, 50 μ M dGTP, 50 μ M dTTP, 50 μ M dCTP, 400nM primer SAMF, 400nM primer SAMR, 1.5U Pfu archaeal dna polymerase (Promega; USA); The 20ng genomic dna adds reaction system, transfers to reaction volume with sterilized water and reaches 50 μ L.
The amplified reaction program is: above-mentioned reaction system 95 ℃ of down reactions 5 minutes, is carried out 30 round-robin " 95 ℃ 30 seconds, 55 ℃ 30 seconds and 72 ℃ 2 minutes " then, kept 10 minutes down at 72 ℃ at last.The pcr amplification gene product that obtains is separated with 1% agarose gel electrophoresis, and reclaim the dna fragmentation of the single band in the 936bp left and right sides with QIAGEN rapid extraction gel reagents box.
B) structure of recombinant vectors
With steps A) the homocysteine methyltransgerase gene that obtains is through NcoI and XhoI double digestion 4h; With same pET30a carrier through NcoI and XhoI double digestion 4h; Through using the T4DNA ligase enzyme to connect the two, be built into the homocysteine methyltransgerase recombinant expression vector.
C) recombinant vectors is to the conversion of host cell and the cultivation of gained recombinant host cell
Picking is 37 ℃ of single bacterium colonies of cultivating 16 hours e. coli bl21 (DE3) on the LB solid medium, in liquid LB substratum, cultivate 16 hours at 37 ℃, the shaking table of 150rpm again.Get 1ml gained liquid culture and transfer in 100ml fresh liquid LB substratum, the rotating speed thermal agitation with 250-300rpm on 37 ℃ of shaking tables was cultivated 2.5 hours.Draw the cultured bacterium liquid of 1.5ml to the 1.5ml centrifuge tube, in cooled on ice after 10 minutes, under 4 ℃, 3000g centrifugal 5 minutes.Supernatant discarded adds 100 μ l at the CaCl2 of the 0.1mol/L of precooling on ice solution, inhales moving beating gently up and down with liquid-transfering gun and spares, and re-suspended cell was placed 20 minutes at ice.Under 4 ℃, 3000g centrifugal 5 minutes then, supernatant discarded added the CaCl of 100 μ l at the 0.1mol/L of precooling on ice
2Solution is inhaled moving beating gently up and down with liquid-transfering gun and is spared, and re-suspended cell obtains competent host cell.
Get the above-mentioned competent e. coli bl21s of 200 μ l (DE3) and place the 1.5ml centrifuge tube, the adding volume is that the pET30a solution (wherein pET30a content is 40ng) of 10 μ l shakes up gently, places on ice 30 minutes.Thermal shock 90 seconds in 42 ℃ of water-baths then placed cooled on ice rapidly 5 minutes then.In centrifuge tube, add 1ml LB liquid nutrient medium (not containing kantlex), 37 ℃ of shaking culture are 1 hour behind the mixing, make bacterium the restore normal growth state and the kantlex antibiotics resistance gene of expression plasmid coding.Get 100 μ l after above-mentioned bacterium liquid shaken up and coat on the LB screening flat board that contains Kan, face up and place half a hour, treat that bacterium liquid is absorbed the back by substratum fully and is inverted petridish, cultivated 20 hours for 37 ℃.On the screening flat board, colibacillary bacterium colony occurred, promptly pET30a has been converted in e. coli bl21 (DE3) competent cell, has obtained the recombinant host cell of present embodiment---e. coli bl21 (DE3) pET30a.
D) cultivation of recombinant host cell
With above-mentioned steps C) the corresponding e. coli bl21 (DE3) of the nucleotide sequence that has the homocysteine methyltransgerase of encode that obtains is cloned in and is cultured to the logarithmic phase (OD600=2) of growing in the LB substratum under 37 ℃; Be inoculated in the 3L initial medium; Promptly in initial medium, insert the LB seed liquor of the OD600=2 of 10% volume; In 37 ℃, dissolved oxygen uses ammoniacal liquor, sulfuric acid to regulate pH6.5～pH7.3 greater than self-sow under 20% the condition.(when dissolved oxygen DO value rises to 40%) is cooled to 32 ℃ when nutritive ingredient exhausts in the initial substratum, adds feed supplement liquid 1 according to the ratio of 60～100ml/L and begins to induce, and add feed supplement liquid 2 according to the speed constant speed of 5～10ml/h/L, uses ammoniacal liquor to regulate pH.At induction period; Whenever reduced by 1 ± 0.1 ℃ at a distance from 3 ± 1 hours; Reduce to constant temperature to fermentation ends after 28 ± 0.5 ℃ (the enzyme work that is homocysteine methyltransgerase in the fermented liquid is greater than 200KU/L, and the enzyme of wherein said homocysteine methyltransgerase is lived and measured with reference to the enzyme activity determination method of accompanying homocysteine methyltransgerase).Wherein, the culture medium prescription of using among the present invention is following successively:
Seed culture medium:
Tryptones 10g/L, yeast extract 5g/L, sodium-chlor 10g/L.Sterilized 20 minutes for 121 ℃, sterilization cooling back adds sulphuric acid kanamycin to final concentration 50mg/L.
The fermentation initial medium:
Tryptones 10g/L, yeast extract 5g/L, potassium hydrogenphosphate 8g/L, SODIUM PHOSPHATE, MONOBASIC 4g/L, Citric acid monohydrate Food grade 0.5g/L, sal epsom 1g/L, micro-mother liquor 5ml/L transfer pH to 7.0, sterilize 20 minutes for 121 ℃.The glucose that adds sterilization before the inoculation uses ammoniacal liquor to transfer pH to 7.0 to final concentration 18g/L.Add sulphuric acid kanamycin to final concentration 50mg/L.
Phosphatic ratio is in initial medium: potassium hydrogenphosphate: SODIUM PHOSPHATE, MONOBASIC=2: 1
The prescription of trace element is:
10g FeSO
4.7H
2O, 2.25g ZnSO
4.7H
2O, 1g CuSO
4.5H
2O, 0.5gMnSO
4.5H
2O, 0.23g Na
2B
4O
7.10H
2O, 2g CaCl
2.2H
2O and 0.1g (NH
4)
6Mo
7O
24, be dissolved in the 5M hydrochloric acid.
Feed supplement liquid 1:
Yeast extract 250g/L, Tryptones 100g/L, lactose 10g/L, 121 ℃ of sterilization 20min.
Feed supplement liquid 2:
Glycerine 400g/L, 121 ℃ of sterilization 20min.
E) separation of protein product is purified
With step D) fermented liquid that obtains adjustment pH to 7.0-8.0, with the resuspended cell of 800bar high-pressure homogenization crusher machine, earlier smudge cells liquid is heated 30min down at 45 ℃, target protein is discharged.The calcium chloride that in homogenate, adds 50mM, abundant stirring and evenly mixing, centrifugal 20 minutes of 5000g removes most nucleic acid, cell debris, and the part foreign protein.In centrifugal supernatant, add 4mM nickel ion (single nickel salt), mixing left standstill under the room temperature 1 hour, the centrifugal 20min of 5000g, and most target proteins are in deposition.The redissolution damping fluid that in deposition, adds 1 times of volume of original volume stirred about 30 minutes.Centrifugal 20 minutes of 10000g.The liquid formula that redissolves is 40mM pH7.5PB, 10mM NAD, 8mM EDTA.Redissolve supernatant through 0.45 μ m membrane filtration.
Gained filtrating is used sephadex G-25 chromatography column (Sephadex G-25) desalination again.Said desalination buffer formulation is the PB of 40mM pH7.5,4mM EDTA.With going up appearance on the good chromatography column of desalination damping fluid balance, wherein the flow velocity of sample solution is 5ml/min, uses desalination damping fluid 2 column volumes of flow velocity wash-out with 15ml/min then again.Totally 500 milliliters of liquid under the collection wash-out.
Collected liquid in general refrigerator-10 ℃ freezing 2 hours down; And then-40 ℃ of deep cooling refrigerator pre-freezes 8 hours; In the ALPHA 1-4LSC of German Ke Ruisite (CHRIST) type freeze drier, with the condition freeze-drying of vacuum tightness 0.04mbar, safe pressure 0.100mbar and condenser temperature-60 ℃ 25 hours.Waiting temperature of charge and Freeze Drying Equipment baffle temperature difference then is zero, and observes 15 seconds internal pressures and indicate when constant, finishes freeze-drying.The proteinic amount of freeze-drying gained is 12 grams.Lyophilized protein under freezing conditions seals preservation.The freeze-drying sample that takes a morsel records enzyme with reference to the enzyme activity determination method of accompanying homocysteine methyltransgerase and lives and be 100U/1mg.
Adopt " protein electrophorese experimental technique " (Guo Yaojun; 132-145 page or leaf, Science Press, 1999) the electrophoretic method of SDS-PAGE of the record proteinic molecular weight that determines present embodiment is 36KD; And according to " biological chemistry " (Wang Jingyan etc.; Higher Education Publishing House 2002, sees 168 pages) measuring method (dividing the peptide section to measure) of the prlmary structure of protein of record; The protein that records present embodiment has 330 amino-acid residues (referring to the aminoacid sequence shown in the SEQ ID NO:1), with consistent by the nucleotide sequence coded protein result shown in the SEQ ID NO:2.
Measure the activity of homocysteine methyltransgerase according to following method:
Reagent one: the TRIS of weighing 3g adds the ZnSO of 0.1435g with the deionized water dissolving of 400ml
4.7H
2O, with hydrochloric acid adjustment PH to 7.5, contain 1mM zine ion for 50mM Tris-HCl this moment then, in this solution, adds 100mM KCl.
Reaction reagent: in the solution of reagent one, add 5mM homocysteine (HCY) and 5mM S-adenosylmethionine (SAM) (at present joining existing usefulness) respectively.
Reaction principle:
Working method:
Reagent 3mL is answered in negate, and enzyme liquid/water 2mL mixes, and under 37 degree conditions, reacts 5min, and reaction system is carried out mass spectrometric detection.Wherein SAM has specific 399 spectrum peak, and the size of 399 peak areas determines whether to respond in contrast blank and the sample, thereby confirms methyl transferase activity.
To sum up; Contriver of the present invention has obtained a kind of homocysteine methyltransgerase; It derives from Candida albicans (Candida albicans SC5314), and molecular weight is 36KD, iso-electric point: 5.39, have the nucleotide sequence (like Fig. 1) shown in aminoacid sequence (like Fig. 2) shown in the SEQ ID NO:1 and the SEQ ID NO:2.And homocysteine methyltransgerase of the present invention still has the relative reactivity more than 60% at 40 ℃ of following 15min, and it is the homocysteine methyltransgerase of inactivation that its thermostability obviously is better than existing 40 ℃.Wherein, Those skilled in the art can know for example SEQ ID NO:2 of nucleotide sequence of the present invention, and can obtain said nucleotide sequence through chemical synthesis process through reading present embodiment; Therefore can be after obtaining having the nucleic acid of this nucleotide sequence; Need not to implement present embodiment A) step, but after obtaining the nucleotide sequence shown in the SEQ ID NO:2 of synthetic, directly implement present embodiment step B)-E) repeat present embodiment; Perhaps obtaining step D) directly implement present embodiment step D behind e. coli bl21 (DE3) bacterial strain of said homocysteine methyltransgerase) and E) repeat present embodiment.
With PCR method gene order is carried out random mutation, condition:
10 μ l 10 * sudden change PCR damping fluid (70mmol/L MgCl2; 500mM KCl; The 100mmol/L Tris-HCl of pH8.3; 0.1% (w/v) gelatin), and 50 μ M dATP, 50 μ M dGTP, 50 μ M dTTP, 50 μ M dCTP, 400nM primer SAMF, 400nM primer SAMR, 1.5U Pfu archaeal dna polymerase (Promega, USA); 20ng genomic dna (being the DNA with the nucleotide sequence shown in the SEQ ID NO:2 that embodiment 1 obtains) adds reaction system, transfers to reaction volume with sterilized water and reaches 100 μ L.The amplified reaction program is: above-mentioned reaction system 95 ℃ of down reactions 5 minutes, is carried out 30 round-robin " 95 ℃ 30 seconds, 55 ℃ 30 seconds and 72 ℃ 2 minutes " then, kept 10 minutes down at 72 ℃ at last.The pcr amplification gene product that obtains is separated with 1% agarose gel electrophoresis, and reclaim the dna fragmentation of the single band in the 936bp left and right sides with QIAGEN rapid extraction gel reagents box.
The PCR fragment that obtains through NcoI and XhoI double digestion 4h, through the pET30a carrier of NcoI and XhoI double digestion 4h, is connected the two through using the T4DNA ligase enzyme with equally, be built into recombinant expression vector, be converted in the e. coli bl21 (DE3).
The above-mentioned clone who obtains is inoculated into respectively in the LB substratum that contains kalamycin resistance; 37 ℃ are cultured to logarithmic phase; After inducing 4h with the IPTG of 1mM under 30 ℃ of conditions; Behind ultrasonic disruption cell (broken power 200w, broken time 4min), respectively at 40 ℃, 45 ℃, 50 ℃ following heating in water bath 15min.
The ultrasonic cell-break liquid of above-mentioned heating is measured enzyme according to preparation embodiment 1 said method lives.3 holes that have enzyme to live are arranged in 40 ℃ of enzyme liquid that heated, 1 hole that has enzyme to live is arranged, the hole that in 50 ℃ of enzyme liquid that heated, does not have enzyme to live in 45 ℃ of enzyme liquid that heated.The order-checking check is said behind 45 ℃ of heating ultrasonic cell-break liquid; Institute is corresponding in the hole that has enzyme to live clones, and obtains the encode nucleotide sequence (shown in SEQ ID No.:4) of heat-resisting sudden change homocysteine methyltransgerase and the e. coli bl21 (DE3) of expressing said heat-resisting sudden change homocysteine methyltransgerase.
According to preparing the method that embodiment 1 is put down in writing; Obtain heat-stable homocysteine methyltransgerase; It derives from Candida albicans (Candida albicans SC5314), and molecular weight is 36KD, iso-electric point: 5.39, have the nucleotide sequence shown in aminoacid sequence shown in the SEQ ID NO:3 and the SEQ ID NO:4.And present embodiment gained homocysteine methyltransgerase still all has the relative reactivity more than 50% after leaving standstill 15 minutes under 45 ℃; It is the homocysteine methyltransgerase of inactivation that its thermostability not only obviously is better than existing 40 ℃, and is better than the type halfcystine methyltransgerase of embodiment 1.
Test implementation example 1
Thermostability according to following method test preparation embodiment 1 homocysteine methyltransgerase of not suddenling change and the homocysteine methyltransgerase for preparing embodiment 2 sudden changes:
It is in 7.5 the 20mmol/L potassium phosphate buffer that the homocysteine methyltransgerase dry powder of 1KU is dissolved in the pH value, respectively 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, kept 15 minutes down.Measuring enzyme according to the method for preparing embodiment 1 lives.The ratio percentage ratio of living with the maximum institute enzyme of surveying work by the survey enzyme is that relative reactivity is an ordinate zou, and temperature is the X-coordinate curve plotting.The test result for preparing embodiment 1 and 2 is seen Fig. 3.
As can be seen from the figure; The thermostability of preparation embodiment 1 homocysteine methyltransgerase of suddenly change and the homocysteine methyltransgerase of preparation embodiment 2 sudden changes is all significantly greater than the thermostability (not shown on the figure, storage temperature is a complete deactivation above 40 ℃) of the homocysteine methyltransgerase of prior art.As shown in Figure 3; Two kinds of homocysteine methyltransgerase of preparation embodiment 1-2 still all have the relative reactivity more than 60% after leaving standstill 15 minutes under 40 ℃; Wherein, the homocysteine methyltransgerase of preparation embodiment 2 still all has the relative reactivity more than 50% after leaving standstill 15 minutes under 45 ℃.
Preparation embodiment 3-6
Formulated reagent one and reagent two according to table 1:
Table 1
The reagent one for preparing embodiment 3 and 4 is mixed with liquid form respectively with reagent two and gets final product.The reagent one and reagent two lyophilize under following condition respectively that prepare embodiment 5 and 6 :-10 ℃ were descended freezing 2 hours in general refrigerator; And then-40 ℃ of deep cooling refrigerator pre-freezes 8 hours; In the ALPHA 1-4LSC of German Ke Ruisite (CHRIST) type freeze drier, with the condition freeze-drying of vacuum tightness 0.04mbar, safe pressure 0.100mbar and condenser temperature-60 ℃ 25 hours.Waiting temperature of charge and Freeze Drying Equipment baffle temperature difference then is zero, and observes 15 seconds internal pressures and indicate when constant, finishes freeze-drying.The reagent dry powder that freeze-drying obtains can redissolve into the volume of primary liquid form before use with zero(ppm) water.The reagent dry powder that freeze-drying obtains, longer than the time that the reagent of liquid form is preserved, packing instructions are lower, and volume is littler, and it is easier to transport.
Preparation embodiment 3-6 has the calibration solution that homocysteine concentration is 34 μ mol/L.
Test implementation example 2
This test implementation example has been measured two samples to be measured: one is the homocysteine aqueous standard article of 9 μ mol/L of preparation; Another is 30 years old women's being in a good state of health a serum sample to be measured, extracts its 10ml blood, leaves standstill to the hemocyte precipitated and separated, gets upper serum 5ml and is used for the test kit of preparation embodiment 3-6 is tested.
The R1 that will prepare respectively among the embodiment 2-5 gets 240 μ l, adds sample 13 μ l, and sample and R1 add R2 reagent 65 μ l 37 ℃ of insulations 300 seconds.R1, R2 and sample mixed solution are incubated 60 seconds under 37 ℃ of conditions, record absorbancy numerical value A1 under the 340nm condition, and 180 seconds record absorbancy numerical value A2 are according to the variation of absorbancy numerical value.Test result is seen table 2.
Table 2
Can find out by table 2; No matter be the serum specimen that detects the preparation sample of concentration known or detect complicated component, the standard deviation of test kit detected result of the present invention is all very little, and is all very approaching with actual value; Therefore they have all proved test kit stable performance of the present invention, measure accurately.
Claims (11)
1. a homocysteine methyltransgerase is characterized in that, the aminoacid sequence of said homocysteine methyltransgerase does
(1) aminoacid sequence shown in the SEQ ID NO:1, perhaps
(2) aminoacid sequence shown in the SEQ ID NO:3, perhaps
(3) (1) or (2) described aminoacid sequence is carried out one or several amino acid whose disappearance, interpolation and/or replacement, but the constant aminoacid sequence of the function of its homocysteine methyltransgerase.
2. homocysteine methyltransgerase according to claim 1, wherein, the aminoacid sequence of said homocysteine methyltransgerase is the aminoacid sequence shown in SEQ ID NO:1 or the SEQ ID NO:3.
3. the nucleotide sequence of the homocysteine methyltransgerase of encoding is characterized in that, said nucleotides sequence is classified the nucleotide sequence of coding claim 1 or 2 described homocysteine methyltransgerase as.
4. nucleotide sequence according to claim 3, wherein, said nucleotides sequence is classified as
(1) nucleotide sequence shown in the SEQ ID NO:2; Perhaps
(2) nucleotide sequence shown in the SEQ ID NO:4; Perhaps
(3) nucleotide sequence shown in SEQ ID NO:2 or the SEQ ID NO:4 is carried out the nucleotide sequence that one or several Nucleotide replaces, lacks or increase and obtains, this nucleotide sequence coded homocysteine methyltransgerase function is constant.
5. nucleotide sequence according to claim 3, wherein, said nucleotides sequence is classified the nucleotide sequence shown in SEQ ID NO:2 or the SEQ ID NO:4 as.
6. a recombinant vectors is characterized in that, said recombinant vectors is made up of empty carrier and the goal gene that inserts this empty carrier, and said goal gene is the nucleotide sequence of any described coding homocysteine methyltransgerase among the claim 3-5.
7. recombinant vectors according to claim 6 is characterized in that said empty carrier is selected from the vehicle group of being made up of pET serial carrier, pUC19, pGEM and pBluescript.
8. a recombinant host cell is characterized in that, said recombinant host cell contains claim 6 or 7 described recombinant vectorss.
9. recombinant host cell according to claim 8 is characterized in that, said host cell is e. coli bl21 (DE3), BL21, TOP10 or DH5a.
10. an Accessory Right requires the method for purifying homocysteine methyltransgerase in the 8 or 9 described recombinant host cells; It is characterized in that; Said method adopts the broken liquid with reconstitution cell to heat 15-40 minute at 40-45 ℃; Then at the centrifugal 10-20min of 8000-15000rpm, to remove heat labile foreign protein.
11. a test kit that detects the serum homocysteine is characterized in that, said test kit comprises at least a in following (a)-(d):
(a) claim 1 or 2 described homocysteine methyltransgerase;
(b) nucleotide sequence of any described homocysteine methyltransgerase among the claim 4-6;
(c) claim 6 or 7 described recombinant vectorss;
(d) claim 8 or 9 described recombinant host cells.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108588047A (en) * | 2018-04-11 | 2018-09-28 | 苏州汇桢生物技术有限公司 | Homocysteine methyltransgerase mutant and its application and nucleic acid, expression vector, host cell, reagent |
| CN111394347A (en) * | 2020-03-26 | 2020-07-10 | 中国人民解放军联勤保障部队第九八〇医院 | Preparation method of candida-free nucleic acid sample suitable for PCR (polymerase chain reaction) experiment |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588047A (en) * | 2018-04-11 | 2018-09-28 | 苏州汇桢生物技术有限公司 | Homocysteine methyltransgerase mutant and its application and nucleic acid, expression vector, host cell, reagent |
| CN108588047B (en) * | 2018-04-11 | 2021-09-03 | 苏州汇桢生物技术有限公司 | Homocysteine methyltransferase mutant and application thereof, nucleic acid, expression vector, host cell and reagent |
| CN111394347A (en) * | 2020-03-26 | 2020-07-10 | 中国人民解放军联勤保障部队第九八〇医院 | Preparation method of candida-free nucleic acid sample suitable for PCR (polymerase chain reaction) experiment |
| CN111394347B (en) * | 2020-03-26 | 2022-03-01 | 中国人民解放军联勤保障部队第九八〇医院 | Preparation method of candida-free nucleic acid sample suitable for PCR (polymerase chain reaction) experiment |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102391999B (en) | 2013-04-24 |
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