CN105063081A - Recombinant vector used in Kluyveromyces marxianus auxotrophic strain - Google Patents
Recombinant vector used in Kluyveromyces marxianus auxotrophic strain Download PDFInfo
- Publication number
- CN105063081A CN105063081A CN201510562564.9A CN201510562564A CN105063081A CN 105063081 A CN105063081 A CN 105063081A CN 201510562564 A CN201510562564 A CN 201510562564A CN 105063081 A CN105063081 A CN 105063081A
- Authority
- CN
- China
- Prior art keywords
- fragment
- amplification
- obtains
- recombinant vectors
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a recombinant vector used in a Kluyveromyces marxianus auxotrophic strain, and a preparation method and application thereof. The recombinant vector sequentially comprises a PKD1 vector, an inulase promoter, an inulase signal peptide, a polyclone site, an inulase terminator, a nutrient gene promoter and a nutrient gene open reading frame. The constructed recombinant vector and preparation method thereof can be used for constructing a transformant, thereby implementing expression of the exogenous gene.
Description
Technical field
The present invention relates to a kind of recombinant vectors, particularly relate to a kind of recombinant expression vector used in Kluyveromyces marxianus auxotrophic strain.
Background technology
Yeast is single celled eukaryote, and it has characteristic and the Eukaryotic albumen synthesis system of processing of microorganism concurrently.Therefore, it is widely used in and expresses multiple external source eukaryotic protein.The yeast expression system of current main flow comprises pichia spp and Saccharomyces Serevisiae Expression System.But the protein expression of pichia spp needs, with methanol induction, not to be suitable for the production of food protein.The easy producing and ethanol of yeast saccharomyces cerevisiae, stand density is low, and expression level is on the low side.For this series of problems, the yeast expression system that security is high and output is high of Development of Novel has very high industrial value.
Kluyveromyces marxianus (Kluyveromycesmarxianus) have passed GRAS and the QPS safety certification of US and European, himself is not only considered to safe microorganism strains, also be considered to the safe microorganisms bacterial strain that can be used for preparing food grade recombinant protein (enzyme), be a kind of yeast of the food grade approved by European Union simultaneously.It has nutritional requirement extremely simple, grow the features such as vigorous, biomass large, growth temperature wide accommodation.Meanwhile, it also has the feature of efficient secretion albumen.Therefore, Kluyveromyces marxianus has high expression and eats the great potential with feed protein.
Expression system is made up of Host Strains and expression vector.For other yeast expression system of aliment security level, Host Strains selects nutrient defect type mark usually.
Summary of the invention
The invention provides a kind of recombinant expression vector using in Kluyveromyces marxianus auxotrophic strain, carry out exogenous gene expression.
The present invention first aspect is to provide a kind of recombinant vectors used in Kluyveromyces marxianus auxotrophic strain, comprises PKD1 carrier, inulinase promotor, inulinase signal peptide, multiple clone site, inulinase terminator, vegetative gene promotor, vegetative gene open reading frame (ORF) in order.
Wherein, described vegetative gene is preferably the vegetative gene of Marx's Crewe dimension bacterial strain, as any one or a few in URA3 gene, HIS3 gene, ADE2 gene, is more preferably URA3 gene.
Wherein, described multiple clone site can be the sequence comprising one or more sites restriction enzyme site, described restriction enzyme site is as any one or a few in SmaI, XmaI, SpeI, NotI, be more preferably in SpeI, NotI and SmaI and/or XmaI any one or a few, as SmaI/XmaI, SpeI, NotI.
Wherein, described multiple clone site length is preferably 15-25bp, is more preferably 18-23bp, is more preferably 19-21bp.
In one preferred embodiment of the invention, the DNA sequence dna of described recombinant vectors is as shown in SEQIDNo.1.
The present invention second aspect is to provide a kind of method building above-mentioned recombinant vectors and comprises:
Amplification pUC19 plasmid, PKD1 carrier, obtain the fragment I that pUC19 and PKD1 connects;
Amplification Kluyveromyces marxianus genome, obtains the fragment II comprising vegetative gene promotor or ORF;
Amplification Kluyveromyces marxianus genome, obtains the fragment III comprising inulinase gene promoter, inulinase signal peptide and part multiple clone site;
Amplification Kluyveromyces marxianus genome, obtains the fragment IV comprising inulinase terminator and part multiple clone site,
Fragment I, II, III, IV are connected, obtains the first recombinant vectors;
Step 2,
Increase the first recombinant vectors, obtains containing inulinase promotor, multiple clone site, inulinase terminator, the Segment A of vegetative gene promotor and vegetative gene ORF;
Increase the first recombinant vectors, obtains the fragment B comprising PKD1 sequence;
Step 3,
Segment A is connected with fragment B, transforms auxotrophic strain, obtain transformant;
Extracting the transformant genome that increases, isolate the described recombinant vectors containing Segment A and fragment B.
Wherein, in step 2, the amplimer obtained in Segment A process is:
5'-CCGTGCCGATTCGCACGCTGCAACG-3；
5'-TGATGCATGCCGAGCTCGGTACCCCTCTTAAGCGG-3'。
Wherein, in step 2, the amplimer obtained in fragment B process is:
5'-ACCGAGCTCGGCATGCATCACTAATGAAAAGCATACG-3；
5'-CGTTGCAGCGTGCGAATCGGCACGG-3'。
Wherein, in step 3, the amplimer obtained in fragment C process is:
5'-ACAAAGGTGATGCTTTGGGACA-3；
5'-GCTTCTTACGTTACGGTTATGGAGC-3'。
Wherein, the primer of amplification pUC19 plasmid is preferably:
Forward primer:
5'-GAGGGGTACCGAGCTCGAATTAGCTCGAATTCGTAATCATGTCATAGCTGTTTCCT-3'；
Reverse primer:
5'-TACAATTTTATGGTGCACTCTCAGTACAATCTGCT-3'。
Wherein, the primer of amplification PKD1 plasmid is preferably:
Forward primer:
5'-CCAAGCTTGCATGCATCACTAATGAAAAGCATACGACGCC-3'；
Reverse primer:
5'-AATTCGAGCTCGGTACCCCTCCGTTGCAGCGTGCGAATCGGCACGG-3'。
Wherein, in an advantageous embodiment, the fragment I method obtaining pUC19 and PKD1 connection is as follows: amplification pUC19 plasmid obtains A fragment, the pcYGW of amplification Gateway systemic vectors obtains B fragment, amplification PKD1 carrier obtains C fragment, A, B are connected with C fragment, increase connecting the plasmid obtained, obtaining the fragment I comprising pUC19 and PKD1.
Wherein, the pcYGW the primer that increases is preferably:
Forward primer: 5'-GAGTGCACCATAAAATTGTAAACGTTAATATTTTG-3';
Reverse primer: 5'-GCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCG-3'.
Wherein, the primer of amplification plasmid is preferably:
Forward primer: 5'-GATCCGCTTAAGAGGGGTACCGAGCTCGAATT-3';
Reverse primer:
5'-ACCTTTTCGGATCGGCATGCCGTTGCAGCGTGCGAATCGGCACGG-3'。
Wherein, amplification obtains the primer of fragment II preferably:
Forward primer: 5'-CTAGGATCGGTCGAATTCTGATTGGAAAGACCATT-3';
Reverse primer: 5'-GTACCCCTCTTAAGCGGATCTGCCTACTCTCTTCA-3'.
Wherein, amplification obtains the primer of fragment III preferably:
Forward primer: 5'-GCATGCCGATCCGAAAAGGTAAACAGACACAAAAAC-3';
Reverse primer:
5'-GTCCCGGGGTCACCGTCTCTCTTGTAATTGATCACTGAAG-3'。
Wherein, amplification obtains the primer of fragment IV preferably:
Forward primer:
5'-ACGGTGACCCCGGGACTAGTGCGGCCGCTTAAGGCCGCAAGCTTTGATCTG-3'；
Reverse primer: 5'-CAGAATTCGACCGATCCTAGAATGTTGGTCAGATGTG-3'.
In a kind of preferred embodiment of the present invention, described method also comprises:
Amplification obtains the fragment containing yeast alpha factor signal peptide, suddenlys change as primer pair gained recombinant vectors.
Wherein, preferably, the PPIC9K carrier that increases obtains the fragment containing described yeast alpha factor signal peptide.
Wherein, the PPIC9K the primer that increases is preferably:
Forward primer:
5’-CCCATAAGTGACACATTTAATTTTTTTTTTGTTAGATATGAGATTTCCTTCAATTTTTACTGC-3’；
Reverse primer:
5’-CTAGTCCCGGGGTCACCGTCGTAAGCTTCAGCCTCTCTTTTCTCG-3’。
Third aspect of the present invention is to provide a kind of transformant, is inserted into by foreign gene in the multiple clone site of described recombinant vectors, obtains the plasmid containing foreign gene, is proceeded in vegetative gene deficient strain by described plasmid, obtain described transformant.
In an advantageous embodiment, method foreign gene being inserted into the multiple clone site of recombinant vectors described in claim 1 comprises:
Increase described recombinant vectors, obtains fragment D, and amplimer is:
---forward primer 5'-GGCCGCAAGCTTTGATCTGATCTGCTTAC-3';
---reverse primer 5'-CTTGTAATTGATCACTGAAGCACTGACTCC-3'
5 ' end of the forward primer of amplification foreign gene ORF adds the sequence of 5'-CTTCAGTGATCAATTACAAG-3'; The sequence of 5'-TCAGATCAAAGCTTGCGGCC-3' is added at 5 ' end of the reverse primer of amplification foreign gene ORF; Amplification obtains fragment E;
Fragment D is connected with E.
Wherein, described vegetative gene deficient strain is preferably vegetative gene defective type Marx Crewe dimension bacterial strain.
Described vegetative gene defect preferably comprise in URA3 gene, HIS3 gene, ADE2 genetic flaw any one or a few, be more preferably URA3 genetic flaw.
The recombinant vectors for foreign gene secreting, expressing that the present invention builds, and preparation method thereof, can be used for building transformant, realize the expression of foreign gene.
Accompanying drawing explanation
Fig. 1 is constructed recombinant vectors schematic diagram in the embodiment of the present invention 1.
Embodiment
Step 1, builds the first recombinant vectors
1.1, increase pUC19 plasmid
Forward primer:
5'-GAGGGGTACCGAGCTCGAATTAGCTCGAATTCGTAATCATGTCATAGCTGTTTCCT-3'
Reverse primer: 5'-TACAATTTTATGGTGCACTCTCAGTACAATCTGCT-3'
Utilize described primer amplification pUC19 plasmid.Pcr amplification reaction carries out according to the working instructions of the PhantaSuperFidelityDNAPolymerase of Vazyme company, and annealing temperature is 58 DEG C, and the extension time is 3 minutes, 30 circulations.PCR primer called after A1 fragment.
The pcYGW of 1.2, amplification Gateway systemic vectors
Forward primer: 5'-GAGTGCACCATAAAATTGTAAACGTTAATATTTTG-3'
Reverse primer: 5'-GCAAGCTTGGCACTGGCCGTCGTTTTACAACGTCG-3'
Utilize the pcYGW of described primer amplification Gateway systemic vectors.Pcr amplification conditional synchronization rapid 1, except the extension time is 1 minute.PCR primer called after B1 fragment.
1.3, amplification PKD1 carriers
Forward primer: 5'-CCAAGCTTGCATGCATCACTAATGAAAAGCATACGACGCC-3' reverse primer:
5'-AATTCGAGCTCGGTACCCCTCCGTTGCAGCGTGCGAATCGGCACGG-3'
Utilize described primer amplification PKD1 carrier.Pcr amplification conditional synchronization rapid 1, except the extension time is 5 minutes.PCR primer called after C1 fragment.
1.4, junction fragment A1, B1, C1
Operate according to the working instructions of NEB company GibsonAssemblyMasterMix, Segment A 1, B1, C1 are connected, transformation of E. coli, the plasmid called after PUC19-PKD1 of acquisition.
1.5, amplification PUC19-PKD1 plasmids
Forward primer: 5'-GATCCGCTTAAGAGGGGTACCGAGCTCGAATT-3'
Reverse primer:
5'-ACCTTTTCGGATCGGCATGCCGTTGCAGCGTGCGAATCGGCACGG-3'
Utilize described primer amplification PUC19-PKD1 plasmid.Pcr amplification conditional synchronization rapid 1, except the extension time is 8 minutes.PCR primer called after D1 fragment.Part PUC19 sequence and PKD1 sequence is included in D1 fragment.
1.6, amplification URA3 gene promoter and ORF fragment
Forward primer: 5'-CTAGGATCGGTCGAATTCTGATTGGAAAGACCATT-3'
Reverse primer: 5'-GTACCCCTCTTAAGCGGATCTGCCTACTCTCTTCA-3'
Extract the specification sheets operation of test kit (DP307) according to Yeast genome, utilize the genome of described primer amplification kluyveromyces marxianus.Pcr amplification conditional synchronization rapid 1, except the extension time is 1.5 minutes.PCR primer called after E1 fragment.Promotor and the ORF of URA3 gene is contained in E1 fragment.
1.7, the promotor of amplification inulinase gene and signal peptide fragment
Forward primer: 5'-GCATGCCGATCCGAAAAGGTAAACAGACACAAAAAC-3'
Reverse primer: 5'-GTCCCGGGGTCACCGTCTCTCTTGTAATTGATCACTGAAG-3'
Utilize the genome of described primer amplification kluyveromyces marxianus.Pcr amplification conditional synchronization rapid 1, except the extension time is 1.5 minutes.PCR primer called after F1 fragment.The promotor of inulinase gene, signal peptide and part multiple clone site is contained in F1 fragment.
1.8, the terminator fragment of amplification inulinase gene
Forward primer:
5'-ACGGTGACCCCGGGACTAGTGCGGCCGCTTAAGGCCGCAAGCTTTGATCTG-3'
Reverse primer: 5'-CAGAATTCGACCGATCCTAGAATGTTGGTCAGATGTG-3'
Utilize the genome of described primer amplification kluyveromyces marxianus.Pcr amplification conditional synchronization rapid 1, except the extension time is 1 minute.PCR primer called after G1 fragment.Terminator and the part multiple clone site of inulinase gene is contained in G1 fragment.
1.9, connect D1, E1, F1, G1 fragment
Operate according to the working instructions of NEB company GibsonAssemblyMasterMix, fragment D1, E1, F1, G1 are connected, transformation of E. coli, the plasmid called after PUKDN112 of acquisition.
Step 2, amplification PUKDN112 carrier
2.1, amplified fragments A
Forward primer: 5'-CCGTGCCGATTCGCACGCTGCAACG-3'
Reverse primer: 5'-TGATGCATGCCGAGCTCGGTACCCCTCTTAAGCGG-3'
With described primer amplification PUKDN112 carrier.When the condition of pcr amplification and PUKD112 plasmid construction, step 1.1 is consistent, except the extension time is 4 minutes.Reclaim the specification sheets operation of test kit according to the glue of Simgen, reclaim PCR primer, this product is named as A fragment.Inulinase promotor is contained, multiple clone site, inulinase terminator, URA3 promotor and URA3ORF in A fragment.2.2, amplified fragments B
Forward primer: 5'-ACCGAGCTCGGCATGCATCACTAATGAAAAGCATACG-3'
Reverse primer: 5'-CGTTGCAGCGTGCGAATCGGCACGG-3'
With described primer amplification PUKDN112 carrier.When the condition of pcr amplification and PUKD112 plasmid construction, step 1.1 is consistent, except the extension time is 5 minutes.Reclaim the specification sheets operation of test kit according to the glue of Simgen, reclaim PCR primer, this product is named as B fragment.PKD1 sequence is contained in B fragment.
Step 3, obtains transformant
Operate according to the working instructions of NEB company GibsonAssemblyMasterMix, Segment A is connected with B, by connecting in the bacterial strain of the Kluyveromycesmarxianus of product conversion URA3 transgenation, the substratum lacking uracil screens transformant.
The specification sheets operation of test kit (DP307) is extracted, extracting URA3 according to the Yeast genome of sky root
+the genome of transformant.By forward primer (5'-ACAAAGGTGATGCTTTGGGACA-3') and reverse primer (5'-GCTTCTTACGTTACGGTTATGGAGC-3') amplification gene group.When pcr amplification condition and PUKD112 plasmid construction, step 1.1 condition is consistent, except the extension time is 1 minute.Reclaim the operation of test kit specification sheets according to the glue of Simgen, reclaim PCR primer, this product is named as C fragment.The connection portion of A fragment and B fragment is contained in this fragment.By Jie Li company forward primer (5'-ACAAAGGTGATGCTTTGGGACA-3'), fragment C is checked order, checking A fragment and B fragment whether exact connect ion.
With reference to Fig. 1, sequencing result is as follows:
Total length: 8357bp
1-4759:PKD1 carrier sequence
4768-5983: inulinase promotor
5984-6051:alpha signal peptide
6062-6081: multiple clone site, comprises SmaI/XmaI, SpeI, NotI tri-sites.
6086-6950: inulinase terminator
6951-7536:KmURA3 promotor
7537-8340：KmURA3ORF
PUKD118 complete sequence is as shown in SEQIDNo.1.
Operate according to the specification sheets of the ZymoprepYeastPlasmidMiniprepII test kit of Zymoresearch company, the transformant containing exact connect ion product is carried out to the extracting of yeast plasmid, acquisition plasmid is PUKDN118.
Purposes:
With forward primer 5'-GGCCGCAAGCTTTGATCTGATCTGCTTAC-3' and reverse primer 5'-CTTGTAATTGATCACTGAAGCACTGACTCC-3' amplification PUKD118 plasmid.When pcr amplification condition and PUKD112 plasmid construction, step 1.1 condition is consistent, except the extension time is 8 minutes.The PCR primer obtained is named as D fragment.
Add the sequence of 5'-CTTCAGTGATCAATTACAAG-3' at 5 ' end of the forward primer of amplification foreign gene ORF, add the sequence of 5'-TCAGATCAAAGCTTGCGGCC-3' at 5 ' end of the reverse primer of amplification foreign gene ORF.Utilize this to primer amplification foreign gene.When pcr amplification condition and PUKD112 plasmid construction, step 1.1 condition is consistent, and the extension time is per minute/1K.The PCR primer obtained is named as E fragment.
Operate according to the working instructions of NEB company GibsonAssemblyMasterMix, fragment D is connected with E, by connecting in the bacterial strain of the Kluyveromycesmarxianus of product conversion URA3 transgenation, the substratum lacking uracil screens transformant.
According to the specification sheets of the ZymoprepYeastPlasmidMiniprepII test kit of Zymoresearch company, to URA3
+transformant carries out the extracting of yeast plasmid.To increase plasmid with forward primer (5'-CAGCAATTAAATCCGGGGTAAG-3') and reverse primer (5'-TATAAAATGTCGCTGTGACCAGGC-3').When pcr amplification condition and PUKD112 plasmid construction, step 1 condition is consistent, except the extension time is per minute/1K.
Reclaim test kit according to the glue of Simgen to reclaim PCR primer, this product is named as F fragment.Checked order to fragment F by Jie Li company forward primer (5'-CAGCAATTAAATCCGGGGTAAG-3') and reverse primer (5'-TATAAAATGTCGCTGTGACCAGGC-3'), whether checking foreign gene is connected correctly with PUKD118.
Select containing exact connect ion the transformant of the PUKD118 of foreign gene, carry out the secreting, expressing of foreign gene, expression and the method detected are carried out (ApplMicrobiolBiotechnol (2005) 67:364 – 369) according to reference.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.
Claims (10)
1. the recombinant vectors used in Kluyveromyces marxianus auxotrophic strain, it is characterized in that, comprise PKD1 carrier, inulinase promotor, inulinase signal peptide, multiple clone site, inulinase terminator, vegetative gene promotor, vegetative gene open reading frame in order.
2. recombinant vectors according to claim 1, is characterized in that, described vegetative gene is preferably Marx's Crewe dimension bacterial strain URA3 gene.
3. recombinant vectors according to claim 1, is characterized in that, the DNA sequence dna of described recombinant vectors is SEQIDNo.1.
4. recombinant vectors according to claim 1, is characterized in that, described multiple clone site be selected from restriction enzyme site SmaI, XmaI, SpeI, NotI any one or a few.
5. build a method for recombinant vectors described in claim 1, it is characterized in that, comprising:
Step 1,
Amplification pUC19 plasmid, PKD1 carrier, obtain the fragment I that pUC19 and PKD1 connects;
Amplification Kluyveromyces marxianus genome, obtains the fragment II comprising vegetative gene promotor or ORF;
Amplification Kluyveromyces marxianus genome, obtains the fragment III comprising inulinase gene promoter, inulinase signal peptide and part multiple clone site;
Amplification Kluyveromyces marxianus genome, obtains the fragment IV comprising inulinase terminator and part multiple clone site,
Fragment I, II, III, IV are connected, obtains the first recombinant vectors;
Step 2,
Increase the first recombinant vectors, obtains containing inulinase promotor, multiple clone site, inulinase terminator, the Segment A of vegetative gene promotor and vegetative gene ORF;
Increase the first recombinant vectors, obtains the fragment B comprising PKD1 sequence;
Step 3,
Segment A is connected with fragment B, transforms auxotrophic strain, obtain transformant;
Extracting the transformant genome that increases, isolate the described recombinant vectors containing Segment A and fragment B.
6. method according to claim 5, is characterized in that,
In step 2, the amplimer obtained in Segment A process is:
5'-CCGTGCCGATTCGCACGCTGCAACG-3；
5'-TGATGCATGCCGAGCTCGGTACCCCTCTTAAGCGG-3'。
In step 2, the amplimer obtained in fragment B process is:
5'-ACCGAGCTCGGCATGCATCACTAATGAAAAGCATACG-3；
5'-CGTTGCAGCGTGCGAATCGGCACGG-3'。
7. method according to claim 5, is characterized in that, in step 3, the amplimer obtained in fragment C process is:
5'-ACAAAGGTGATGCTTTGGGACA-3；
5'-GCTTCTTACGTTACGGTTATGGAGC-3'。
8. method according to claim 5, is characterized in that, the fragment I method obtaining pUC19 and PKD1 connection is as follows:
Amplification pUC19 plasmid obtains A fragment, and the pcYGW of amplification Gateway systemic vectors obtains B fragment, and amplification PKD1 carrier obtains C fragment, A, B is connected with C fragment, increasing, obtaining the fragment I comprising pUC19 and PKD1 by connecting the plasmid obtained.
9. a transformant, is characterized in that, is inserted into by foreign gene in the multiple clone site of recombinant vectors described in claim 1, obtains the plasmid containing foreign gene, is proceeded in vegetative gene deficient strain by described plasmid, obtain described transformant.
10. transformant according to claim 9, is characterized in that, method foreign gene being inserted into the multiple clone site of recombinant vectors described in claim 1 comprises:
Increase described recombinant vectors, obtains fragment D, and amplimer is:
---forward primer 5'-GGCCGCAAGCTTTGATCTGATCTGCTTAC-3';
---reverse primer 5'-CTTGTAATTGATCACTGAAGCACTGACTCC-3'
5 ' end of the forward primer of amplification foreign gene ORF adds the sequence of 5'-CTTCAGTGATCAATTACAAG-3'; The sequence of 5'-TCAGATCAAAGCTTGCGGCC-3' is added at 5 ' end of the reverse primer of amplification foreign gene ORF; Amplification obtains fragment E;
Fragment D is connected with E.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510562564.9A CN105063081A (en) | 2015-09-07 | 2015-09-07 | Recombinant vector used in Kluyveromyces marxianus auxotrophic strain |
| PCT/CN2016/094868 WO2017036294A1 (en) | 2015-08-28 | 2016-08-12 | Kluyveromyces marxianus and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510562564.9A CN105063081A (en) | 2015-09-07 | 2015-09-07 | Recombinant vector used in Kluyveromyces marxianus auxotrophic strain |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105063081A true CN105063081A (en) | 2015-11-18 |
Family
ID=54492591
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510562564.9A Pending CN105063081A (en) | 2015-08-28 | 2015-09-07 | Recombinant vector used in Kluyveromyces marxianus auxotrophic strain |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105063081A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105524923A (en) * | 2015-12-28 | 2016-04-27 | 大连理工大学 | Kluyveromyces marxianus derived high-expression promoter and expression system thereof |
| CN106399138A (en) * | 2016-09-07 | 2017-02-15 | 复旦大学 | Oral porcine circovirus II-like particle vaccine, and preparation method and application thereof |
| CN106399350A (en) * | 2016-09-07 | 2017-02-15 | 复旦大学 | Porcine circovirus type II virus-like particle vaccine and preparation method thereof |
| WO2017036294A1 (en) * | 2015-08-28 | 2017-03-09 | 复旦大学 | Kluyveromyces marxianus and use thereof |
| CN108004263A (en) * | 2016-11-02 | 2018-05-08 | 清华大学 | A kind of Kluyveromyces marxianus plasmid vector and its application |
| CN108118006A (en) * | 2016-11-29 | 2018-06-05 | 复旦大学 | A kind of heatproof zytase kluyveromyces marxianus engineered strain and its application |
| CN108273055A (en) * | 2018-01-03 | 2018-07-13 | 复旦大学 | Canine parvovirus vaccine composition and the preparation method and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101157935A (en) * | 2007-09-20 | 2008-04-09 | 复旦大学 | Novel expression enzyme yeast gene engineering system |
-
2015
- 2015-09-07 CN CN201510562564.9A patent/CN105063081A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101157935A (en) * | 2007-09-20 | 2008-04-09 | 复旦大学 | Novel expression enzyme yeast gene engineering system |
Non-Patent Citations (3)
| Title |
|---|
| DANGUOLE BARTKEVICIUTE ET AL.: "Heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPG1) using versatile autonomously replicating vector for a wide range of host", 《ENZYME AND MICROBIAL TECHNOLOGY》 * |
| RONALD J. M. BERGKAMP ET AL: "Expression of an a-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus", 《APPL MICROBIOL BIOTECHNOL》 * |
| 方自安: "分泌信号肽介导异源蛋白在酵母中分泌表达的研究及K1HAP1基因在乳酸克鲁维酵母中的功能研究", 《中国博士论文全文数据库(电子期刊)基础科学辑》 * |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017036294A1 (en) * | 2015-08-28 | 2017-03-09 | 复旦大学 | Kluyveromyces marxianus and use thereof |
| CN105524923A (en) * | 2015-12-28 | 2016-04-27 | 大连理工大学 | Kluyveromyces marxianus derived high-expression promoter and expression system thereof |
| CN105524923B (en) * | 2015-12-28 | 2019-01-15 | 大连理工大学 | A kind of High-expression promoter and its expression system from kluyveromyces marxianus |
| CN106399138A (en) * | 2016-09-07 | 2017-02-15 | 复旦大学 | Oral porcine circovirus II-like particle vaccine, and preparation method and application thereof |
| CN106399350A (en) * | 2016-09-07 | 2017-02-15 | 复旦大学 | Porcine circovirus type II virus-like particle vaccine and preparation method thereof |
| CN106399138B (en) * | 2016-09-07 | 2019-11-12 | 复旦大学 | Porcine Circovirus takes orally virus sample particle vaccines and its preparation method and application |
| CN108004263A (en) * | 2016-11-02 | 2018-05-08 | 清华大学 | A kind of Kluyveromyces marxianus plasmid vector and its application |
| CN108118006A (en) * | 2016-11-29 | 2018-06-05 | 复旦大学 | A kind of heatproof zytase kluyveromyces marxianus engineered strain and its application |
| CN108273055A (en) * | 2018-01-03 | 2018-07-13 | 复旦大学 | Canine parvovirus vaccine composition and the preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105063081A (en) | Recombinant vector used in Kluyveromyces marxianus auxotrophic strain | |
| CN105063082A (en) | Recombinant vector used for carrying out foreign gene secretory expression in auxotrophic kluyveromyces marxianus strain | |
| CN105063080A (en) | Signal peptide-free recombinant vector for exogenous gene expression in Kluyveromyces marxianus nutritional deficient strain | |
| CN101319225B (en) | Saccharomyces cerevisiae polygene expression vector, construction method and application thereof | |
| Rubio‐Texeira et al. | Highly efficient assimilation of lactose by a metabolically engineered strain of Saccharomyces cerevisiae | |
| Wang et al. | Advances allowing feasible pyrG gene editing by a CRISPR-Cas9 system for the edible mushroom Pleurotus eryngii | |
| Han et al. | An efficient Agrobacterium-mediated transformation method for aflatoxin generation fungus Aspergillus flavus | |
| CN107779466B (en) | Methods for gene disruption in higher fungi | |
| CN105132452A (en) | Recombinant vector for expressing histidine-tag-fused foreign gene in Kluyveromyces marxianus nutritional deficient strain | |
| CN105483034B (en) | A method of conversion yeast mating type | |
| CN108660085B (en) | Nucleic acid-producing saccharomyces cerevisiae engineering bacterium and construction method and application thereof | |
| CN105087632B (en) | saccharomyces cerevisiae chromosome and construction method and application thereof | |
| Punt et al. | Protein targeting and secretion in filamentous fungi: a progress report | |
| CN108949602B (en) | High-yield xylanase saccharomyces cerevisiae and application thereof | |
| Dueñas-Santero et al. | A new toolkit for gene tagging in Candida albicans containing recyclable markers | |
| CN104004760A (en) | Foreign protein expression device used for secretory expression in Aspergillus oryzae cells and Aspergillus oryzae genetically engineered bacteria | |
| CN107723253B (en) | Double-plasmid co-transformed exogenous gene high-expression genetic engineering bacterium | |
| CN106591351A (en) | Recombinant saccharomyces cerevisiae utilizing starch and secreting antibacterial peptide | |
| CN115786384A (en) | Integration site of fusarium venenatum TB01 and application thereof | |
| CN103497969B (en) | A kind of Escherichia coli-yeast-agrobacteshuttle shuttle expression vector and application thereof | |
| EP3290519A1 (en) | Novel gene targeting method | |
| CN107523568B (en) | Constitutive promoter of pichia pastoris and application thereof | |
| CN111057712A (en) | Recombinant yeast strain and construction method and application thereof | |
| CN116555269B (en) | Bumblebee candida utilis inducible promoter and application thereof | |
| CN110305891B (en) | Method for verifying feasibility of knocking out candida utilis target gene by CRISPR-Cas9 system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20151118 |