CN104561051A - Prawn Y-organ 7,8-dehydrogenase gene and application thereof - Google Patents
Prawn Y-organ 7,8-dehydrogenase gene and application thereof Download PDFInfo
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- CN104561051A CN104561051A CN201410735236.XA CN201410735236A CN104561051A CN 104561051 A CN104561051 A CN 104561051A CN 201410735236 A CN201410735236 A CN 201410735236A CN 104561051 A CN104561051 A CN 104561051A
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- expression plasmid
- desaturase
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Abstract
The invention belongs to the field of gene engineering, and particularly relates to a prawn Y-organ 7,8-dehydrogenase gene and application thereof. The invention provides a gene for coding 7,8-dehydrogenase, which is obtained from a constructed Metapenaeus ensis Y-organ cDNA (complementary deoxyribonucleic acid) library; and the nucleotide sequence of the gene is disclosed as SEQ ID NO.1. The application method comprises the following steps: cloning the 7,8-dehydrogenase coding gene, and connecting with an expression plasmid to construct a recombinant expression plasmid; after linearizing and purifying the recombinant expression plasmid, converting a receptor bacterium, and screening to obtain a gene engineering bacterium; and carrying out induction expression, purification and identification to obtain the 7,8-dehydrogenase of which the amino acid sequence is disclosed as SEQ ID NO.2. The 7,8-dehydrogenase can be used for converting cholesterol into 7-dehydrocholesterol by a one-step enzyme process, thereby enhancing the conversion efficiency, shortening the process route for producing vitamin D3, lowering the production cost and reducing the environmental pollution.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to a seed shrimp Y-organ 7,8-dehydrogenase gene and application thereof.
Background technology
Vitamins D
3that man and animal grows, grows, breeds, sustains life and keep fit requisite a kind of liposoluble vitamin.Its Main Function regulates alcium and phosphor metabolization, promotes Calcium and phosphorous absorption and sclerotin calcification in intestines, maintains the balance of blood calcium and serium inorganic phosphorus.In addition, vitamins D
3or a kind of novel immunomodulator, has Cell differentiation inducing activity, cell growth inhibiting, the formation of antagonism collagen and regulates the effects such as proto-oncogene is expressed in vain, Tumor suppression growth.Now, world population ages has become one of social concern highly visible, to the elderly's medication, all attaches great importance to both at home and abroad, vitamins D in the elderly's medication
3in occupation of consequence.In addition, along with the standard of living of people improves constantly, the amount of meat, egg, fowl is increased, vitamins D
3one of additive as fodder industry has had more wide market, vitamins D
3demand in ascendant trend year by year.
At present, vitamins D
3main employing photochemical method is produced, and namely for raw material, after uv irradiating, changes vitamins D into 7-DHC (7 one DHC)
3precursor (PreD
3), then thermal isomerization and obtaining.As the 7-DHC of main raw materials for production, multiple different raw material and operational path can be adopted, prepared by chemical synthesis.As being raw material with 6-alkene courage steroid beacon alcohol, first using 4-phenyl-1,2,4-triazoline-3,5-diketone to protect double bond on 6, then using NaHCO
3and Jone.s reagent is (by CrO
3and the vitriol oil is made into) oxidation, then use sodium borohydride reduction, obtain 7-DHC after hydrolysis.The people such as photographic chemistry institute of the Chinese Academy of Sciences of China Zhang Jiancheng then take to take cholesterol as basic raw material, obtain the operational path of 7-DHC through series reaction such as oxidation, additions.
Up to now, no matter adopt which kind of chemical synthesis raw materials 7-DHC, all have that technical process is long, step is many, by product removes that process is complicated, yield is low, cost is high and the shortcoming of easy contaminate environment, thus limit vitamins D
3production & marketing promote.Therefore, the production method developing the 7-DHC that a kind of operational path is short, cost is low becomes very urgent and necessary.
The research of mechanism of casting off a skin based on insect and arthropods, the gene (hereinafter referred to as maf) of a kind of new coding 7,8-desaturase is cloned from the new prawn of cutter volume (Metapenaeusensis) Y-organ cDNA library, and obtain 7 at eukaryotic expression, 8-desaturase, this 7, the carbon atom position dehydrogenation of 8-desaturase energy catalysis cholesterol 7,8, direct transformed cholesterol is 7-DHC.Such result, is extremely conducive to the desaturase manufacture method that next step studies the work of high enzyme, low cost, thus develops a kind of new biological process synthesis 7-DHC operational path, reduces 7-DHC and vitamins D
3production cost.
Summary of the invention
The object of the invention is the deficiency in order to overcome above-mentioned technology, providing a kind of 7 gene and application thereof of, 8-desaturase, developing the 7-DHC production line that a kind of operational path is short, cost is low.
The present invention deals with problems adopted technical scheme:
The invention provides a kind of coding 7,8-dehydrogenase gene, it obtains by the cutter volume new prawn Y-organ cDNA library built, and its nucleotide sequence is as shown in SEQ ID NO.1.
Present invention also offers one 7,8-desaturase, it is the expression product of coding 7,8-dehydrogenase gene, is made up of 428 amino acid, and its molecular weight is 49550.09Da, and aminoacid sequence is as shown in SEQ ID NO.2.
Present invention also offers a kind of recombinant expression plasmid, it is characterized in that, described recombinant expression plasmid is obtained by gene recombination described in expression plasmid and claim 1.
Preferably, described expression plasmid is eukaryon expression plasmid pPICZaA.
Present invention also offers described recombinant expression plasmid transform Host Strains and obtain genetic engineering bacterium X33-pPICZ α A-maf.
Present invention also offers 7, to be hydrolyzed cholesterol be application in 7-DHC to 8-desaturase, its enzyme digestion reaction is at 25 DEG C, reacts 12h under lucifuge condition.
Utilize coding 7,8-dehydrogenase gene prepare 7, a method for 8-desaturase, it is characterized in that, comprise following steps:
(1) gene of clones coding 7,8-desaturase;
(2) coding 7,8-dehydrogenase gene are connected with expression plasmid build recombinant expression plasmid;
(3) by recombinant expression plasmid linearizing and purifying;
(4) by the recombinant expression plasmid transformation receptor bacterium after purifying, after screening, genetic engineering bacterium is obtained;
(5) by genetic engineering bacterium abduction delivering, acquisition 7,8-desaturase after Purification.
Preferably, step 4) described in recipient bacterium be pichia pastoris X-33.
Preferably, step 5) described in genetic engineering bacterium X33-pPICZ α A-maf utilize methyl alcohol to carry out abduction delivering.
Preferably, the concentration 0.5% (v/v) of described methyl alcohol.
Compared with prior art, the beneficial effect that the present invention has is:
1, the present invention relates to this 7,8-dehydrogenase gene clone, the aminoacid sequence that corresponds and the expression in recipient bacterium pichia pastoris X-33 thereof, this expression enzyme can transformed cholesterol be 7-DHC, for biological process synthesis 7-DHC provides a kind of new method.
2, enzymolysis process that is of the present invention 7,8-desaturase is simply efficient, and realizing a step enzyme method transformed cholesterol is 7-DHC, has both improved its transformation efficiency, has shortened vitamins D
3operational path, reduce environmental pollution again; Due to the new prawn wide material sources of cutter volume, production cost can also be reduced.
Accompanying drawing explanation
Fig. 1 is recombinant plasmid PmlI, XbaI double digestion and PCR qualification figure, is be that template carries out pcr amplification with recombinant cloning vector, and recovery product and pPICZaA carry out double digestion respectively through PmlI and XbaI, and its result is detected by 0.8% agarose gel electrophoresis; Wherein M is Maker λ Hind III; 1-4 hole is Plasmid digested by PmlI/XbaI; 5th hole is MakerDL2000; 6-9 hole is PCRproduct of recombinant.
Fig. 2 is that recon expresses figure in pichia pastoris X-33 (P.pastoris X-33), is that fox extracting thallus protein after abduction delivering, analyzes through 12%SDS-PAGE with the recombinant bacterium containing empty pPICZaA carrier as blank; Wherein M is Protein molecular weight markers; 1st hole is Mediumsupernatant of negative control; 2-8 hole is Recombinant maf after 24,36,48,60,72,84and 96h induction
Fig. 3 is expression vector physical map, that recombination to be transformed into P.pastoris X-33 plasmid vector used be pPICZaA, this plasmid has following feature: with Zeocin selective marker, 3600bp, the gene be cloned is controlled by AOX1 promotor, with multiple clone site (ploylinker site).
Fig. 4 is the high-efficient liquid phase chromatogram of enzymatic reaction solution, is by after enzymatic reaction solution pre-treatment, and by reversed-phased high performace liquid chromatographic detection reaction result, testing conditions is: C
18post (5 μm, 250mm × 4.6mm), moving phase acetonitrile-Virahol (volume ratio 7: 3), column temperature 30 DEG C, flow velocity 1.0mL/min, UV determined wavelength 280nm.
Fig. 5 is the high-efficient liquid phase chromatogram of mark product 7-DHC; Wherein standard specimen 7-DHC Reversed phase high performance liquid chromatography method detected result, testing conditions is: C
18post (5 μm, 250mm × 4.6mm), moving phase acetonitrile-Virahol (volume ratio 7: 3), column temperature 30 DEG C, flow velocity 1.0mL/min, UV determined wavelength 280nm.
Embodiment
Below in conjunction with specific embodiment, elaboration detailed is further done to the present invention, but embodiments of the present invention are not limited to the scope that embodiment represents.These embodiments only for illustration of the present invention, but not for limiting the scope of the invention.In addition, after reading content of the present invention, those skilled in the art can do various amendment to the present invention, and these equivalent variations fall within appended claims limited range of the present invention equally.
embodiment:
1, source, library and plasmid
The new prawn of cutter volume (Metapenaeus ensis) Y-organ cDNA library and pPICZaA preserve by this laboratory.
2, main agents
Expressive host bacterium P.pastoris X-33 (wild-type), microbiotic Zeocin are all purchased from invitrogen company.Pm ll enzyme and XbaI enzyme are all purchased from TaKaRa bio-engineering corporation.
3, method
3.1, the choning and sequencing of gene
Carry out pcr amplification with the primer of designed, designed, upstream primer is 5-GCGCACGTGATGCTACTGGAACAGATTTGGG-3, and downstream primer is 5-GCGTCTAGAGCCCAGTCAAAAATTGATTTTGCTT-3.Adopt the reaction system of 25 μ L: 10 × buffer 2.5 μ L, dTNP 0.5 μ L, each 0.5 μ L of forward and reverse primer, cDNA template 0.5 μ L, RTaq enzyme 0.25 μ L, moisturizing to 25 μ L.PCR condition is: the first step: 95 DEG C, 5min; Second step: 94 DEG C, 50S; 3rd step: 52 DEG C, 45S; 4th step: 72 DEG C, 2min; 5th step: 72 DEG C, 10min; Wherein second step circulates 30 times to the 4th step.PCR primer detects through 0.8% agarose gel electrophoresis, is connected, obtains recombinant plasmid after reclaiming goal gene with pGM-T vector.Linked system is: pGM-T vector 1 μ L, PCR goal gene product 5 μ L, 10 × T4 ligase buffer 1 μ L, T4DNA ligase enzyme 1 μ L, distilled water 2 μ L, and 16 DEG C connect 12h and spend the night.100 μ L competence E.coli DH5 α are transformed by 10 μ L connecting fluids, EP pipe static 30min in ice bath of competent cell will be housed, then heat shock 90S in 42 DEG C of water-baths is moved to, quick subsequently EP pipe is transferred to ice bath 2min, finally adds the rear 37 DEG C of shaking tables concussion of 900 μ L LB substratum mixing and cultivate 45min (150r/min).Get dull and stereotyped upper 37 DEG C of the LB (0.5% yeast extract paste, 1% Tryptones, 1% sodium-chlor) that 100 μ L competent cells are coated on containing Amp (100 μ g/mL), IPTG (0.5mmol/L), X-gal (0.004%) and cultivate picking white colony after 16 hours, be inoculated in the LB substratum containing Amp, extracting plasmid carries out PCR and digestion verification, order-checking.
3.2, the structure of pPICZaA expression vector
Be that template carries out pcr amplification with recombinant cloning vector, reclaim product.Recovery product and pPICZaA are carried out PmlI and XbaI double digestion respectively, and double digestion system is as follows: PmlI and Xba I each 1.5 μ L, NEB Buffer2.1 3.0 μ L, 0.1%BSA 3.0 μ L, pPICZaB 18.0 μ L, ddH2O 3.0 μ L, cumulative volume 30.0 μ L.37 DEG C of reaction 12h, reclaim goal gene, have the pPICZaA fragment of identical sticky end with goal gene, both connected, ligation system is as follows: 10 × T4 DNA Ligase Buffer2.0 μ L, maf fragment 8.0 μ L, pPICZaB endonuclease bamhi 2.0 μ L, T4 ligase enzyme 2.0 μ L, ddH
2o6.0 μ L, Tota l20.0 μ L, 16 DEG C of reaction 12h, and Transformed E .coli DH5 α, Amp resistance screening, extracting recombinant plasmid carries out PCR, and enzyme cuts qualification, by the recombinant plasmid pPICZ aA-maf order-checking after qualification, verifies further.Double digestion and PCR the results are shown in Figure 1.
3.3, the linearizing of plasmid pPICZaA-maf and purifying
Linearizing: select Sac I as restriction enzyme site, reaction system is as follows: Sac I 10 μ L, 10 × L Buffer15 μ L, pPICZ α A-maf 110 μ L, ddH
2o 15 μ L, cumulative volume 150 μ L.16 DEG C of reaction 3h.The washing of phenol extraction conveniently, alcohol settling carries out plasmid purification, with sterilized water dissolution precipitation.Add isopyknic phenol/chloroform, the centrifugal 10min of 10000rpm after mixing, carefully get the clean EP pipe of supernatant liquor to, add the freezing dehydrated alcohol of two volumes, after mixing, the centrifugal 10min of 30min, 10000rpm placed by-20 DEG C, refrigerator, supernatant discarded, gained precipitation with 75% ethanol purge once.
3.4, pichia pastoris X-33 electricity transforms and recombination yeast screening
The expression plasmid drawing 10 μ L purifying mixes with 80 μ L X-33 competent cells, and under 1500V, electricity turns 5ms, then gets the YPD that 150 μ L bacterium liquid are spread evenly across containing 100 μ g/mL Zeocin dull and stereotyped.The bacterium colony that picking has Zeocin resistance carries out the screening of MD/MM speed spot.Expression plasmid expression of results in pichia pastoris X-33 is shown in Fig. 2.
3.5, positive transformant inducing culture
Picking positive transformant is inoculated in 25mL BMGY, and in 30 DEG C, collect thalline when the concussion of 200r/min shaking table is cultured to OD600=3, with BMMY re-suspended cell to OD600=1.0, adding 100% methyl alcohol to final concentration at interval of 24h is 0.5%, inducing culture.
3.6, recombination microzyme crude enzyme liquid extracts
Recombination microzyme X33-pPICZ α A-maf is through methanol induction, and centrifugal removing thalline, gets 20 μ L supernatant liquor ultrafiltration and concentration and obtain 10 μ L enzyme liquid.
3.7, be that substrate transforms with cholesterol
1g cholesterol is dissolved in the phosphate buffer solution containing 10mL1%Tween 80,0.01mol/L pH7.4, adds the mixing of 10mL enzyme liquid, 25 DEG C of lucifuge reaction 12h.Reaction solution petroleum ether extraction, sherwood oil and reaction solution volume ratio are 5:1, extraction temperature 40 DEG C, extraction time 60min.Collect reaction solution and adopt reversed-phased high performace liquid chromatographic detection reaction result, testing conditions is: C
18post (5 μm, 250mm × 4.6mm), moving phase acetonitrile-Virahol (volume ratio 7: 3), column temperature 30 DEG C, flow velocity 1.0mL/min, sample size is 20 μ L, UV determined wavelength 280nm.Detected result shows, and reaction solution contains 7-DHC, and its appearance time is consistent with mark product 7-DHC, the results are shown in accompanying drawing 4 and accompanying drawing 5.
The aforementioned description to concrete exemplary of the present invention is to illustrate and the object of illustration.These descriptions not want the present invention to be defined as disclosed precise forms, and obviously, according to above-mentioned instruction, can much change and change.The object selected exemplary embodiment and describe is to explain certain principles of the present invention and practical application thereof, thus those skilled in the art can be realized and utilize various different exemplary of the present invention and various different selection and change.Scope of the present invention is intended to limited by claims and equivalents thereof.
Claims (10)
1. coding 7, a 8-dehydrogenase gene, it obtains by the cutter volume new prawn Y-organ cDNA library built, and its nucleotide sequence is as shown in SEQ ID NO.1.
2. 7, a 8-desaturase, it is characterized in that, described 7,8-desaturase is the expression product of coding 7,8-dehydrogenase gene, and it is made up of 428 amino acid, and molecular weight is 49550.09Da, and aminoacid sequence is as shown in SEQ ID NO.2.
3. a recombinant expression plasmid, is characterized in that, described recombinant expression plasmid is obtained by gene recombination described in expression plasmid and claim 1.
4. recombinant expression plasmid according to claim 3, is characterized in that, described expression plasmid is eukaryon expression plasmid pPICZaA.
5. one kind as described in claim 3 or 4 recombinant expression plasmid transform Host Strains and genetic engineering bacterium X33-pPICZ α A-maf.
6. according to claim 27, the purposes of 8-desaturase in hydrolysis cholesterol, is characterized in that, product that is described 7,8-desaturase hydrolysis cholesterol is 7-DHC, and its enzymolysis is at 25 DEG C, reacts 12h under lucifuge condition.
7. utilize coding 7 as claimed in claim 1,8-dehydrogenase gene prepare 7, a method for 8-desaturase, it is characterized in that, described method comprises following steps:
(1) gene of clones coding 7,8-desaturase;
(2) coding 7,8-dehydrogenase gene are connected with expression plasmid build recombinant expression plasmid;
(3) by recombinant expression plasmid linearizing and purifying;
(4) by the recombinant expression plasmid transformation receptor bacterium after purifying, after screening, genetic engineering bacterium is obtained;
(5) by genetic engineering bacterium abduction delivering, acquisition 7,8-desaturase after Purification.
8. the method for preparation 7 according to claim 7,8-desaturase, is characterized in that, step 4) described in recipient bacterium be pichia pastoris X-33.
9. the method for preparation 7 according to claim 7,8-desaturase, is characterized in that, step 5) described in genetic engineering bacterium X33-pPICZ α A-maf utilize methyl alcohol to carry out abduction delivering.
10. the method for preparation 7 according to claim 9,8-desaturase, is characterized in that, the concentration 0.5% (v/v) of described methyl alcohol.
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| CN201410735236.XA CN104561051A (en) | 2014-12-05 | 2014-12-05 | Prawn Y-organ 7,8-dehydrogenase gene and application thereof |
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| CN201410735236.XA CN104561051A (en) | 2014-12-05 | 2014-12-05 | Prawn Y-organ 7,8-dehydrogenase gene and application thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101509004A (en) * | 2009-03-24 | 2009-08-19 | 广西大学 | Clone and expression of 7,8-desaturase gene |
| CN102392042A (en) * | 2011-12-13 | 2012-03-28 | 江南大学 | Method for preparing Metapenaeus ensis arginine kinase by pichia yeast |
| CN102559738A (en) * | 2011-12-13 | 2012-07-11 | 江南大学 | Method for preparing metapenaeus ensis tropomyosin by using pichia pastoris |
-
2014
- 2014-12-05 CN CN201410735236.XA patent/CN104561051A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101509004A (en) * | 2009-03-24 | 2009-08-19 | 广西大学 | Clone and expression of 7,8-desaturase gene |
| CN102392042A (en) * | 2011-12-13 | 2012-03-28 | 江南大学 | Method for preparing Metapenaeus ensis arginine kinase by pichia yeast |
| CN102559738A (en) * | 2011-12-13 | 2012-07-11 | 江南大学 | Method for preparing metapenaeus ensis tropomyosin by using pichia pastoris |
Non-Patent Citations (1)
| Title |
|---|
| 黄时海 等: "虾Y-器官7、8-脱氢酶转化胆固醇为7-脱氢胆固醇研究", 《安徽农业科学》 * |
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Application publication date: 20150429 |